WO2025054500A2 - Protéines bifonctionnelles et leurs utilisations - Google Patents

Protéines bifonctionnelles et leurs utilisations Download PDF

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WO2025054500A2
WO2025054500A2 PCT/US2024/045653 US2024045653W WO2025054500A2 WO 2025054500 A2 WO2025054500 A2 WO 2025054500A2 US 2024045653 W US2024045653 W US 2024045653W WO 2025054500 A2 WO2025054500 A2 WO 2025054500A2
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seq
amino acid
acid sequence
binding moiety
bifunctional protein
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WO2025054500A3 (fr
WO2025054500A9 (fr
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Yunxiang Zhu
Yangde Chen
Yanfeng ZHOU
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Mlab Biosciences Inc
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Mlab Biosciences Inc
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Publication of WO2025054500A3 publication Critical patent/WO2025054500A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/42—Notch; Delta; Jagged; Serrate

Definitions

  • the present invention relates to bifunctional protein constructs comprising a first binding moiety that specifically binds to a muscle-specific molecule and a second binding moiety that specifically binds to a Notch receptor and activates the Notch receptor.
  • the present invention also relates to methods for making such bifunctional protein constructs and the use of such bifunctional protein constructs for treating muscle-related diseases (e.g., muscular dystrophies and sarcopenia).
  • muscle-related diseases e.g., muscular dystrophies and sarcopenia
  • Also provided are engineered DLL4 extracellular domains (ECDs) and protein constructs comprising thereof.
  • ECDs engineered DLL4 extracellular domains
  • BACKGROUND Muscular dystrophy (MD) is a diverse group of muscle diseases of many subtypes that present with muscle weakness and degeneration at different levels of severity.
  • An MD is commonly linked to a defect(s) in a gene encoding a protein that is important for muscle integrity or function.
  • Degenerative MDs are typically caused by defects in structural proteins that are directly or indirectly associated with the dystrophin-associated glycoprotein complex (DGC), which is essential to maintaining muscle cell membrane integrity.
  • DGC dystrophin-associated glycoprotein complex
  • a direct link between MD and Notch signaling in human was established with limb-girdle muscular dystrophy (LGMD R21), which is caused by Protein O- glucosyltransferase 1 (POGLUT1) biallelic mutations, reduced Notch signaling, and satellite cell loss (Servián-Morilla et al., 2016, EMBO Mol Med.
  • the present application in one aspect provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor.
  • the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina.
  • the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M- cadherin (CDH15). In some embodiments, the target antigen is a component of the dystrophin-associated glycoprotein complex (DGC).
  • DGC dystrophin-associated glycoprotein complex
  • the target antigen is selected from the group consisting of ⁇ -dystroglycan ( ⁇ -DG), ⁇ -DG, laminin-211, perlecan, collagen, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, biglycan, sarcospan, and matriglycan on ⁇ -DG.
  • the target antigen is a laminin subunit alpha-2 (LAMA2), CDH15, ⁇ -DG, or matriglycan on ⁇ -DG.
  • the first binding moiety comprises an antibody moiety specifically binding to the muscle-specific molecule.
  • the antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a F(ab’)2, an scFv, and an sdAb.
  • the antibody moiety specifically binds to LAMA2 (anti-LAMA2 antibody moiety).
  • the anti-LAMA2 antibody moiety comprises: (i) an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR1 2 sf-6122278 Attorney Docket No.: 340742000140 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • the anti-LAMA2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49, and a VL comprising the amino acid sequence of SEQ ID NO: 50.
  • the anti-LAMA2 antibody moiety is an anti-LAMA2 scFv.
  • the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 51.
  • the anti-LAMA2 antibody moiety is an anti-LAMA2 Fab.
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 52, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 53.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12.
  • the anti-LAMA2 antibody moiety comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 65; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 66; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 67; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 68; (v) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 69; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ
  • the anti-LAMA2 antibody moiety is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO:145. [0015] In some embodiments according to any of the bifunctional protein constructs described above, the anti-LAMA2 antibody moiety is an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the antibody moiety specifically binds to matriglycan on ⁇ -DG (anti- matriglycan antibody moiety).
  • the anti-matriglycan antibody moiety comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (ii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC
  • the anti- matriglycan antibody moiety comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98.
  • the anti-matriglycan antibody moiety comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 89, and a VL comprising the amino acid sequence of SEQ ID NO: 94; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) a VH comprising the amino acid sequence
  • the anti-matriglycan antibody moiety is an anti-matriglycan scFv.
  • the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 99.
  • the anti-matriglycan antibody moiety is an anti-matriglycan Fab.
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.
  • the antibody moiety specifically binds to CDH15 (anti-CDH15 antibody moiety).
  • the anti-CDH15 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
  • the anti-CDH15 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 108, and a VL comprising the amino acid sequence of SEQ ID NO: 109.
  • the anti-CDH15 antibody moiety is an anti-CDH15 scFv.
  • the anti-CDH15 scFv comprises the amino acid sequence of SEQ ID NO: 110.
  • the anti-CDH15 antibody moiety is an anti-CDH15 Fab.
  • the anti-CDH15 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 111, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 112.
  • the first binding moiety comprises a non-antibody moiety specifically binding to the muscle-specific molecule.
  • the non-antibody moiety comprises a protein domain selected from the group consisting of a laminin G-like domain (LG domain) of laminin, an LG domain of agrin, an LG domain of nidogen, an LG domain of perlecan, the laminin coiled-coil binding domain of agrin, and the laminin ⁇ binding domain of nidogen.
  • LG domain laminin G-like domain
  • the non-antibody moiety comprises an LG 18 sf-6122278 Attorney Docket No.: 340742000140 domain of laminin.
  • the non-antibody moiety comprises an LG domain of laminin subunit ⁇ -1 (LAMA1), LAMA2, laminin subunit ⁇ -3 (LAMA3), laminin subunit ⁇ -4 (LAMA4), or laminin subunit ⁇ -5 (LAMA5).
  • the non-antibody moiety comprises the amino acid sequence of any of SEQ ID NOs: 113-117. [0025] In some embodiments according to any of the bifunctional protein constructs described above, the non-antibody moiety comprises an LG domain of LAMA2. In some embodiments, the non-antibody moiety comprises LG4-5 domains of LAMA2 (LAMA2 LG4-5).
  • the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119.
  • the non-antibody moiety comprises an LG domain of perlecan. In some embodiments, the non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 120. [0027] In some embodiments according to any of the bifunctional protein constructs described above, the non-antibody moiety comprises an LG domain of agrin. In some embodiments, the non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 121.
  • the non-antibody moiety comprises a laminin coiled-coil binding domain of agrin. In some embodiments, the laminin coiled-coil binding domain of agrin comprises the amino acid sequence of SEQ ID NO: 122. [0029] In some embodiments according to any of the bifunctional protein constructs described above, the non-antibody moiety comprises a laminin ⁇ binding domain of nidogen. In some embodiments, the laminin ⁇ binding domain of nidogen comprises the amino acid sequence of SEQ ID NO: 123 or 124.
  • the second binding moiety comprises an extracellular domain (ECD) or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.
  • ECD extracellular domain
  • the second binding moiety comprises a DLL4 ECD or a variant thereof.
  • the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125.
  • the second binding moiety comprises a variant DLL4 ECD
  • the variant 19 sf-6122278 Attorney Docket No.: 340742000140 DLL4 ECD comprises MNNL domain, DSL domain, and EGF1-5 domains of DLL4, or a sequence having at least about 90% sequence identity to the sequence of MNNL domain, DSL domain, and EGF1-5 domains of DLL4.
  • the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the second binding moiety comprises a DLL1 ECD or a variant thereof.
  • the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the second binding moiety comprises a DLL3 ECD or a variant thereof.
  • the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the second binding moiety comprises a Jag1 ECD or a variant thereof.
  • the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the second binding moiety is an anti-Notch antibody moiety that activates the Notch receptor.
  • the anti-Notch agonist antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a F(ab’)2, an scFv, and an sdAb.
  • the first binding moiety is fused to the second binding moiety via an optional linker.
  • the first binding moiety comprises two or more non-antibody moieties connected in tandem.
  • the first binding moiety comprises an LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.
  • the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the bifunctional protein construct comprises the amino acid sequence of any of SEQ ID NOs: 136-139.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 or 143.
  • the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144.
  • the first binding moiety comprises an anti-LAMA2 scFv.
  • the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of any of SEQ ID NOs: 146-154.
  • the first binding moiety comprises an anti-matriglycan scFv.
  • the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of any of SEQ ID NOs: 156-164.
  • the first binding moiety comprises a Fab specifically binding to a muscle- specific molecule.
  • the second binding moiety is fused to the N- terminus of the VL of the Fab via an optional linker.
  • the first binding moiety comprises an anti-LAMA2 Fab.
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti- LAMA2 Fab, and wherein the fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 167-175.
  • the first binding moiety comprises an anti-matriglycan Fab.
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, and wherein the fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 178-186.
  • the first binding moiety is fused to the second binding moiety via a carrier protein.
  • the carrier protein is selected from the group consisting of an human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of an Fc domain.
  • the first binding moiety is fused to the N-terminus of the carrier protein via an optional first linker
  • the second binding moiety is fused to the C-terminus of the carrier protein via an optional second linker.
  • the first binding 22 sf-6122278 Attorney Docket No.: 340742000140 moiety is fused to the C-terminus of the carrier protein via an optional first linker
  • the second binding moiety is fused to the N-terminus of the carrier protein via an optional second linker.
  • the bifunctional protein construct comprises (i) the second binding moiety comprising an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) the first binding moiety comprising: (a) an antibody moiety, wherein the antibody moiety is an scFv, a Fab, or an sdAb specifically binding to a muscle-specific molecule; or (b) a non-antibody moiety, wherein the non-antibody moiety comprises a protein domain selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.
  • the first binding moiety comprises two or more non-antibody moieties connected in tandem.
  • the bifunctional protein construct further comprises an Fc domain comprising a first subunit and a second subunit.
  • the Fc domain is derived from human IgG1, human IgG2, or human IgG4.
  • the Fc domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187.
  • each subunit of the Fc domain comprises a mutation that reduces or abolishes the effector function.
  • each subunit of the Fc domain comprises an L234A/L235A mutation (EU numbering).
  • either the first subunit of the Fc domain or the second subunit of the Fc domain comprises an H435R/Y436F mutation (EU numbering).
  • the Fc domain comprises knob-into-hole mutations, and wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • the Fc domain comprises charged-pair mutations, and wherein: i) an amino acid residue in the first subunit of the Fc domain is replaced with a positively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced with a negatively charged residue; or ii) an amino acid residue in the first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced with a positively charged residue.
  • the Fc domain comprises i) an amino acid residue at D399 (EU numbering) in 23 sf-6122278 Attorney Docket No.: 340742000140 the first subunit of the Fc domain is replaced with a positively charged residue, and an amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue; or ii) an amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue at D399 (EU numbering) in the second subunit of the Fc domain is replaced with a positively charged residue.
  • each subunit of the Fc domain comprises a mutation that reduces half-life.
  • each subunit of the Fc domain comprises an H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the bifunctional protein construct comprises: i) a first unit of a second binding moiety specifically binding to a first Notch receptor, and ii) a second unit of a second binding moiety specifically binding to a second Notch receptor, and wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 125-129.
  • the first unit of the second binding moiety and the second unit of the second binding moiety are the same.
  • the first unit of the second binding moiety and the second unit of the second binding moiety are different.
  • the bifunctional protein construct comprises: i) a first unit of a first binding moiety specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule.
  • the first unit of the first binding moiety and the second unit of the first binding moiety are the same. In some embodiments, the first unit of the first binding moiety and the second unit of the first binding moiety are different. In some embodiments, the first unit of the first binding moiety comprises a first antibody moiety specifically binding to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second antibody moiety specifically binding to the second muscle- specific molecule, and wherein the first antibody moiety and the second antibody moiety are each independently selected from the group consisting of a Fab, an scFv, and an sdAb.
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – VH1-(H1-CH1); ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – VH2-(H2-CH1); iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 165
  • the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of any of SEQ ID NOs: 189-192
  • the first unit of the second binding moiety and/or the second unit of the second 25 sf-6122278 Attorney Docket No.: 340742000140 binding moiety comprises a DLL1 ECD
  • the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 165
  • the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 193.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD
  • the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 165
  • the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 194.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD
  • the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 165
  • the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 195 or 196.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD
  • the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 165
  • the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 197.
  • the bifunctional protein construct comprises Fab1 and Fab2 both specifically bind to matriglycan.
  • the anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 176, and wherein: (i) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of any of SEQ ID NOs: 198-201; (ii) the first polypeptide and the second polypeptide of the bifunctional protein construct each 26 sf-6122278 Attorney Docket No.: 340742000140 comprises the amino acid sequence of SEQ ID NO: 202; (iii) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 203; (iv) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 204 or 205; or (v) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprises the amino acid sequence of SEQ ID NO: 206.
  • the first antibody moiety is a first sdAb (sdAb1)
  • the second antibody moiety is a second sdAb (sdAb2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – sdAb1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – sdAb2.
  • the first antibody moiety is a first scFv (scFv1)
  • the second antibody moiety is a second scFv (scFv2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – scFv1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – scFv2.
  • the bifunctional protein construct comprises scFv1 and scFv2 both specifically bind to LAMA2.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the linker comprises an amino acid sequence of any one of SEQ ID NOs: 207-222.
  • the first subunit of the Fc domain and the second subunit of the Fc domain each comprises the amino acid sequence of SEQ ID NO: 188.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of any of SEQ ID NOs: 223-226.
  • the first unit of the second binding moiety and/or the second unit of the second binding 27 sf-6122278 Attorney Docket No.: 340742000140 moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 227.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 228.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 229 or 230.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 231. [0054] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2 both specifically bind to matriglycan.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising: (i) the amino acid sequence of any one of SEQ ID NOs: 232-235; (ii) the amino acid sequence of SEQ ID NO: 236; (iii) the amino acid sequence of SEQ ID NO: 237; (iv) the amino acid sequence of SEQ ID NO: 238 or 239; or (v) the amino acid sequence of SEQ ID NO: 240.
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and iv) a fourth polypeptide 28 sf-6122278 Attorney Dock
  • the first antibody moiety is a first sdAb (sdAb1)
  • the second antibody moiety is a second sdAb (sdAb2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: sdAb1 – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: sdAb2– optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.
  • the first antibody moiety is a first scFv (scFv1)
  • the second antibody moiety is a second scFv (scFv2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: scFv1– optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: scFv2– optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL) – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and iv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL) – optional linker – a second unit of a second binding moiety specifically binding to a first Notch receptor; and iv) a fourth polypeptide compris
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional 29 sf-6122278 Attorney Docket No.: 340742000140 linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: a second unit of a second unit of a second
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: V
  • the bifunctional protein construct further comprises a third unit of a second binding moiety specifically binding to a third Notch receptor, and a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor, and wherein the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.
  • the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof of DLL4.
  • the third unit of the second binding moiety and/or the fourth unit of the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125.
  • the third unit of the second binding moiety and/or the fourth unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises MNNL domain, DSL domain, and EGF1-5 domains of DLL4.
  • the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267.
  • the first antibody moiety is a first Fab (Fab1)
  • the second antibody moiety is a second Fab (Fab2)
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – VH2-(H2-CH1) – optional linker
  • the first unit of the first binding moiety comprises a first non-antibody moiety specifically binding to the first muscle-specific molecule
  • the second unit of the first binding moiety comprises a second non-antibody moiety specifically binding to the second muscle-specific molecule
  • the first non-antibody moiety and the second non- antibody moiety are each independently selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.
  • the first unit of the first binding moiety comprises two or more first non-antibody moieties connected in tandem
  • the second unit of the first binding moiety comprises two or more second non-antibody moieties connected in tandem
  • the first non-antibody moiety and/or the second non-antibody moiety comprises LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the first binding moiety – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: the second unit of the first binding moiety – optional linker – the second subunit of 31 sf-6122278 Attorney Docket No.: 340742000140 the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – the first unit of the first binding moiety; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the first binding moiety.
  • the first unit of the first binding moiety comprises two LAMA2 LG4-5 connected in tandem
  • the second unit of the first binding moiety comprises two LAMA2 LG4-5 connected in tandem
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD
  • the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of any one of SEQ ID NOs: 241-244.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 245.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 246.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 247 or 248.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises 32 sf-6122278 Attorney Docket No.: 340742000140 the amino acid sequence of SEQ ID NO: 135.
  • the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 249.
  • the first binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N- terminus of the second subunit of the Fc domain via an optional second linker; or ii) the first binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional second linker.
  • the first binding moiety is a Fab specifically binding to the muscle-specific molecule, and wherein the C-terminus of the CH1 of the Fab is fused to the N-terminus of the first subunit of the Fc domain or the second subunit of the Fc domain via the optional first linker.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.
  • the bifunctional protein construct comprises: i) a first unit of a second binding moiety specifically binding to a first Notch receptor, and ii) a second unit of a second binding moiety specifically binding to a second Notch receptor; wherein the first unit of the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and the second unit of the second binding moiety is fused to the N- terminus of the second subunit of the Fc domain via an optional second linker; and wherein the first binding moiety is fused to the C-terminus of either the first subunit of the Fc domain or the second subunit of the Fc domain via an optional third linker.
  • the bifunctional protein construct comprises: i) a first unit of a first binding moiety specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule; and wherein the first unit of the first binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the C-terminus of the second subunit of the Fc domain via an optional fourth linker.
  • the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- 33 sf-6122278 Attorney Docket No.: 340742000140 specific molecules; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the C-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C- terminus of the second subunit of the Fc domain.
  • the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the C- terminus of the second subunit of the Fc domain.
  • the bifunctional protein construct comprises: i) a first unit of a second binding moiety specifically binding to a first Notch receptor, and ii) a second unit of a second binding moiety specifically binding to a second Notch receptor; wherein the first unit of the second binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional first linker, and the second unit of the second binding moiety is fused to the C- terminus of the second subunit of the Fc domain via an optional second linker; and wherein the first binding moiety is fused to the N-terminus of either the first subunit of the Fc domain or the second subunit of the Fc domain via an optional third linker.
  • the bifunctional protein construct comprises: i) a first unit of a first binding moiety specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule; and wherein the first unit of the first binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the N- terminus of the second subunit of the Fc domain via an optional fourth linker.
  • the second binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of 34 sf-6122278 Attorney Docket No.: 340742000140 the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C- terminus of the second subunit of the Fc domain.
  • the second binding moiety is fused to the C-terminus of the second subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the N- terminus of the second subunit of the Fc domain.
  • the one or more units of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) the one or more units of the first binding moiety each independently comprises an antibody moiety specifically binding to a muscle- specific molecule, and wherein the antibody moiety is independently selected from the group consisting of an scFv, a Fab, and an sdAb.
  • the two units of the second binding moiety are the same; and/or ii) the two or more units of the first binding moiety are the same.
  • the two units of the second binding moiety are different; and/or ii) at least one of the two or more units of the first binding moiety is different from the other(s).
  • the Fc domain comprises knob-into-hole mutations and/or charged-pair mutations.
  • the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255;
  • the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348;
  • the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257;
  • the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256;
  • the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO:
  • provided herein is one or more isolated nucleic acids encoding any of the bifunctional protein construct described above.
  • one or more vectors comprising one or more isolated nucleic acids described above.
  • the one or more vectors is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.
  • AAV adeno-associated virus
  • a host cell expressing any of the bifunctional protein constructs described above, the one or more isolated nucleic acids described above, or the one or more vectors described above.
  • the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.
  • pharmaceutical compositions comprising: i) any of the bifunctional protein constructs described above, the one or more isolated nucleic acids described above, or the one or more vectors described above; and ii) a pharmaceutically acceptable excipient.
  • a method of producing a bifunctional protein construct comprising: i) culturing a host cell comprising the one or more isolated nucleic acids described above or the one or more vectors described above, or the host cell described above, under a condition suitable for the expression of the bifunctional protein construct; and ii) recovering the expressed bifunctional protein construct from the cultured host cell.
  • the method further comprises introducing the one or more isolated nucleic acids or the one or more vectors into the host cell.
  • a method of treating a muscle-related disease in an individual comprising administering to the individual an effective amount of any of the bifunctional protein constructs described above or the pharmaceutical composition described above.
  • the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy.
  • the muscle disease is muscular dystrophy.
  • the muscular dystrophy comprises one or more of: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), 36 sf-6122278 Attorney Docket No.: 340742000140 oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disorder, and Emery-Dreifuss muscular dystrophy (EDMD).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • LGMD limb-girdle muscular dystrophy
  • CMD congenital muscular dystrophy
  • FSHD facioscapulohumeral muscular dystrophy
  • DM myotonic dystrophy
  • OPMD oculopharyngeal muscular
  • the bifunctional protein construct or the pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly.
  • the individual is a human.
  • the present inventions provides an antibody construct comprising an antibody moiety specifically recognizing matriglycan on ⁇ -DG (anti- matriglycan antibody moiety), wherein the anti-matriglycan antibody moiety comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (ii) an HC-CDR1, HC- CDR2, and
  • the anti-matriglycan antibody moiety comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98.
  • the anti-matriglycan antibody moiety comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 89, and a VL comprising the amino acid sequence of SEQ ID NO: 94; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 91, and a VL comprising
  • the anti-matriglycan antibody moiety is selected from the group consisting of a full- length antibody, a Fab, a Fab’, a F(ab’)2, and an scFv.
  • the anti- matriglycan antibody moiety is an anti-matriglycan scFv.
  • the anti- matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 99.
  • the anti-matriglycan antibody moiety is an anti-matriglycan Fab.
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.
  • the anti-matriglycan antibody moiety is an anti-matriglycan Fab.
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.
  • the anti-matriglycan antibody moiety is an anti-matriglycan full-length antibody.
  • the anti-matriglycan full-length antibody comprises an Fc domain derived from human IgG1, human IgG2, or human IgG4.
  • the Fc 39 sf-6122278 Attorney Docket No.: 340742000140 domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187.
  • each subunit of the Fc domain comprises an L234A/L235A mutation (EU numbering).
  • each subunit of the Fc domain comprises an H435A mutation (EU numbering).
  • each subunit of the Fc domain comprises an L234A/L235A/P329G mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the antibody construct further comprises a second binding moiety specifically binding to a second target molecule.
  • the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.
  • the present invention provides an engineered DLL4 extracellular domain (ECD), wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N260.
  • the further mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D.
  • the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) G2S, E14H, T52N, F81L, R66T, P80L, T135N, H168Y, and N231D; and (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N231D;
  • the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264.
  • the present invention provides a protein construct comprising the engineered DLL4 ECD.
  • the protein construct further comprises a binding moiety that specifically binds to a muscle-specific molecule.
  • FIG. 1A-1M depict different configurations of bifunctional proteins comprising an anti-muscle target protein and a Notch ligand or agonist.
  • FIG. 1A depicts a monovalent Notch ligand with an anti-muscle protein antibody (Ab) in “Knob-into-Hole” format.
  • FIGs. 1B and 1C depict additional formats and configurations of bifunctional proteins, of which R1, R2, R3, and R4 can be an anti-muscle target protein or a Notch ligand/agonist.
  • FIG. 1D depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the Fc and an anti-muscle protein antibody Fab fused to the C-terminus of the Fc.
  • FIG. 1A depicts a monovalent Notch ligand with an anti-muscle protein antibody (Ab) in “Knob-into-Hole” format.
  • FIGs. 1B and 1C depict additional formats and configurations of bifunctional proteins, of which R
  • FIG. 1E depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the Fc and an anti- muscle protein antibody scFv fused to the C-terminus of the Fc.
  • FIG. 1F depicts a bivalent fusion protein Notch ligands fused to the N-terminus of the Fc and an anti-muscle target protein antibody single chain Ab (VHH or nanobody) fused to the C-terminus of the Fc.
  • FIG. 1G depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the Fc and an anti-muscle protein antibody Fab fused to the N-terminus of the Fc.
  • FIG. 1E depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the Fc and an anti- muscle protein antibody scFv fused to the C-terminus of the Fc.
  • FIG. 1F depicts a bivalent fusion protein Not
  • FIG. 1H depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the Fc and an anti- muscle protein scFv fused to the N-terminus of the Fc.
  • FIG. 1I depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of Fc and an anti-muscle protein VHH/nanobody fused to the N-terminus of Fc.
  • FIG. 1J depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the antibody light chains.
  • FIG. 1K depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the antibody light chains.
  • FIG. 1L depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the antibody heavy chains.
  • FIG. 1M depicts a tetravalent fusion protein with Notch ligands fused to the N-terminus of the antibody light and heavy chains.
  • FIGs. 2A-2J depict different configurations of bifunctional proteins comprising a non-antibody moiety (e.g., an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan) and a Notch ligand or agonist.
  • a non-antibody moiety e.g., an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan
  • FIG. 2A depicts a monovalent protein with a Notch ligand fused to the N-terminus of a laminin G-like domain (LG domain) monomer, or alternatively, a scFv, sdAb, or Fab to a muscle specific protein.
  • LG domain laminin G-like domain
  • FIG. 2B depicts a monovalent protein with a Notch ligand fused to the N-terminus of an LG domain dimer, or alternatively, a scFv, sdAb, or Fab specifically recognizing a muscle specific protein.
  • FIG. 2C depicts a monovalent protein with a Notch ligand fused to the N- terminus of an LG domain trimer or multimers in tandem, or alternatively, a scFv, sdAb, or Fab to a muscle specific protein.
  • FIG. 2D depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain monomer.
  • FIG. 2E depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain dimer.
  • FIG. 2F depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain trimer or multimers in tandem.
  • FIG. 3A depicts an SDS-PAGE of the reduced and non-reduced DLL4v-Fc-LG4-5 construct.
  • FIG. 3B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4v-Fc-LG4-5 construct (lower profile).
  • FIG. 4A depicts an SDS-PAGE of the reduced and non-reduced DLL4wt-Fc-LG4- 5 construct.
  • FIG. 4B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4wt-Fc-LG4-5 construct (lower profile).
  • FIG. 5A depicts an SDS-PAGE of the reduced and non-reduced DLL4v-Fc- LAMA2scFv construct.
  • FIG. 5B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4v-Fc-LAMA2scFv construct (lower profile).
  • FIG. 6 depicts binding of bifunctional proteins DLL4v-Fc-LG4-5, DLL4wt-Fc- LG4-5, and DLL4v-Fc-LAMA2scFv to Notch-1 using a sandwich ELISA.
  • the DLL4v-Fc- LG4-5 and DLL4v-Fc-LAMA2scFv constructs have higher affinity (or avidity) to Notch 1, compared to the DLL4wt-Fc-LG4-5 construct.
  • FIG. 7A depicts a kinetic analysis of the DLL4v-Fc-LG4-5 construct binding to different concentrations of purified matriglycan using Biacore system.
  • the y-axis represents the absolute response (RU), and the x-axis represents the time in seconds.
  • the arrows indicate the injection points of purified matriglycan.
  • FIG. 7B depicts Western blot results of a laminin-overlay assay that shows DLL4v-Fc-LG4-5 binds to matriglycan. 42 sf-6122278 Attorney Docket No.: 340742000140 [0098] FIG.
  • FIG. 8 depicts a kinetic analysis of the DLL4v-Fc-LAMA2scFv construct binding to different concentrations of recombinant human LG4-5 using Biacore system, confirming that LAMA2scFv binds to LG4-5.
  • the arrows indicate the injection points of recombinant human LG4-5.
  • TNP irrelevant antigen trinitrophenol
  • FIG. 9B depicts the forelimb grip strength of WT, DLL4v-Fc-LAMA2scFv- treated, and isotype control-treated D2.mdx mice over 7 months.
  • FIG. 9C depicts the bodyweight of WT, DLL4v-Fc-LAMA2scFv-treated, and isotype control-treated D2.mdx mice over 7 months.
  • FIG. 10A depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile), huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG- LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS.
  • FIG. 10B depicts a table outlining the yield and percentage of main peak as determined by analytical size exclusion chromatography (aSEC). [0101] FIG.
  • FIG. 11A depicts binding affinity of huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS to a murine Notch1 receptor, compared to that of the huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS (parental) and mud4v constructs as determined by an ELISA assay.
  • FIG. 11B depicts a table outlining the binding affinity (K D ) of each construct measured in nM.
  • the present application provides bifunctional protein constructs that specifically target and delivered to muscle tissues with Notch activation in a spatiotemporally controlled manner. Binding of the bifunctional protein construct to muscle tissue, either via a protein on the muscle (cell) sarcolemma or a protein in the extracellular matrix (basal lamina), anchors the bifunctional protein construct and Notch activating moiety comprised therein (e.g., a Notch ligand or agonistic antibody specifically recognizing Notch 1-4), which is a 43 sf-6122278 Attorney Docket No.: 340742000140 requirement for Notch signaling.
  • Notch activating moiety e.g., a Notch ligand or agonistic antibody specifically recognizing Notch 1-4
  • the bifunctional protein constructs disclosed in this disclosure fulfill the three requirements for specific Notch signaling in muscles: 1) muscle targeting to ensure tissue specificity; 2) Notch ligand anchoring required for trans- activation; and 3) timely control of Notch activation for satellite cells renewal while permitting myoblast differentiation during muscle repair.
  • the present application thus in one aspect provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor.
  • a method of making the bifunctional protein constructs described herein is provided.
  • Isolated nucleic acids, vectors, and host cells encoding any of the bifunctional protein constructs are also provided. Also provided are methods of treating a muscle-related disease in an individual (e.g., human), by administering to the individual an effective amount of the bifunctional protein construct described herein or pharmaceutical composition thereof.
  • an engineered DLL4 extracellular domain (ECD) comprising a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • protein constructs comprising the DLL4 ECD described herein. I.
  • antibody herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • antibody moiety refers to a full-length antibody or an antigen-binding fragment thereof.
  • an “antibody” may refer to an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen (including the basic 4- chain antibody unit).
  • Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antigen-binding fragments (such as Fv, Fab, Fab’, F(ab) 2 and F(ab’) 2 ), as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC- CDR2, and HC-CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al- Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • Several of the major antibody classes are divided into subclasses such as lgG1 ( ⁇ 1 heavy chain), lgG2 ( ⁇ 2 heavy chain), lgG3 ( ⁇ 3 heavy chain), lgG4 ( ⁇ 4 heavy chain), lgA1 ( ⁇ 1 heavy chain), or lgA2 ( ⁇ 2 heavy chain).
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv’), a disulfide 45 sf-6122278
  • 340742000140 stabilized diabody ds diabody
  • scFv single-chain Fv
  • scFv dimer bivalent diabody
  • a multispecific antibody formed from a portion of an antibody comprising one or more CDRs a single domain antibody (sdAb) (e.g., a camelized single domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fv is the minimum antibody fragment, which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association.
  • Single-chain Fv also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4- chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N- terminus, a variable domain (VH) followed by three constant domains (CH) for each of the ⁇ 46 sf-6122278 Attorney Docket No.: 340742000140 and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen- binding site.
  • L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
  • the “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains.
  • HVRs hypervariable regions
  • FR framework regions
  • variable domains of 47 sf-6122278 Attorney Docket No.: 340742000140 native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
  • Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences see, e.g., WO 1998/24893; WO 1996/34096; 48 sf-6122278 Attorney Docket No.: 340742000140 WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S.
  • full-length antibody “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment.
  • full-length 4-chain antibodies include those with heavy and light chains including an Fc region.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen- binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
  • Chimeric antibodies of interest herein include PRIMATTZFACTOR D® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • “humanized antibody” is used as a subset of “chimeric antibodies.”
  • 49 sf-6122278 Attorney Docket No.: 340742000140 [0121]
  • the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J.
  • the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or hypervariable region (HVR) of the variable domain.
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
  • “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a suitable human acceptor antibody may be one selected from a 51 sf-6122278 Attorney Docket No.: 340742000140 conventional database, e.g., the KABAT database, Los Alamos database, the AbM, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • a “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.
  • donor antibody refers to an antibody (monoclonal, and/or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to 52 sf-6122278 Attorney Docket No.: 340742000140 provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
  • acceptor antibody refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
  • a human antibody is the acceptor antibody.
  • attach refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that, for example, where a first polypeptide is directly bound to a second polypeptide or material, and, for example, where one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are disposed between the first polypeptide and the second polypeptide or material.
  • intermediate compounds e.g., amino acids, peptides, polypeptides, etc.
  • Percent (%) amino acid sequence identity or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two 53 sf-6122278 Attorney Docket No.: 340742000140 sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the C H 1, C H 2 and C H 3 domains (collectively, C H ) of the heavy chain and the CHL (or CL) domain of the light chain.
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“ ⁇ ”) and lambda (“ ⁇ ”), based on the amino acid sequences of their constant domains.
  • CH1 domain (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).
  • Hinge region is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.
  • the “CH2 domain” of a human IgG Fc domain usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • the “CH3 domain” (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).
  • Fc domain or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc domains and variant Fc domains.
  • the human IgG heavy-chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU 54 sf-6122278 Attorney Docket No.: 340742000140 numbering system) of the Fc domain may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc domains for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • Fc receptor or “FcR” describes a receptor that binds the Fc domain of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors, Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody or antibody moiety that specifically recognizes a target is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a 55 sf-6122278 Attorney Docket No.: 340742000140 radioimmunoassay (RIA).
  • an antibody that specifically binds a target has a dissociation constant (KD) of ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -9 M, ⁇ 10 -10 M, ⁇ 10 -11 M, ⁇ 10 -12 , ⁇ 10 -13 , or ⁇ 10 -14 M.
  • KD dissociation constant
  • an antibody specifically binds an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art.
  • Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -tests and peptide scans.
  • specificity refers to selective recognition of an antigen binding protein or antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • multispecific denotes that an antigen binding protein or an antibody has two or more antigen-binding sites of which at least two bind a different antigen or a different epitope of the same antigen.
  • Bispecific as used herein denotes that an antigen binding protein or an antibody has two different antigen-binding specificities.
  • Effector cells are leukocytes which express one or more FcRs and perform effector functions. In one aspect, the effector cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells and neutrophils.
  • the effector cells may be isolated from a native source, e.g., blood.
  • Effector cells generally are lymphocytes associated with the effector phase, and function to produce cytokines (helper T cells), killing cells in infected with pathogens (cytotoxic T cells) or secreting antibodies (differentiated B cells).
  • cytokines helper T cells
  • cytotoxic T cells cytotoxic T cells
  • secreting antibodies secreting antibodies
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).
  • Kd dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • An “on-rate,” “rate of association,” “association rate,” or “kon” as used herein can also be determined as described above using methods such as biolayer interferometry and surface plasmon resonance (SPR).
  • An “isolated” polypeptide is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide’s natural environment will not be present. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.
  • An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen- binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • the isolated nucleic acid 57 sf-6122278 Attorney Docket No.: 340742000140 is free of association with all components associated with the production environment.
  • the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature.
  • Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • hybridoma refers to a cell resulting from the fusion of a B-lymphocyte and a fusion partner such as a myeloma cell.
  • a hybridoma can be cloned and maintained indefinitely in cell culture and is able to produce monoclonal antibodies.
  • a hybridoma can also be considered to be a hybrid cell.
  • nucleic acid molecule refers to a polymer of nucleotides.
  • nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
  • isolated nucleic acid refers to a 58 sf-6122278 Attorney Docket No.: 340742000140 nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • “Complementary” as used herein to refer to a nucleic acid refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • a vector may be a viral vector, plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may 59 sf-6122278 Attorney Docket No.: 340742000140 be an adeno-associated virus (AAV) vector or a lentiviral vector.
  • a vector may be a DNA or RNA vector.
  • a vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • polypeptide also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • conjuggated refers to covalent attachment of one molecule to a second molecule.
  • “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically 60 sf-6122278 Attorney Docket No.: 340742000140 limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
  • the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.
  • regulating can mean any method of altering the level or activity of a substrate.
  • Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and/or translation), affecting folding, affecting degradation or protein turnover, and affecting localization of a protein.
  • Non-limiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity.
  • “Regulator” refers to a molecule whose activity includes affecting the level or activity of a substrate.
  • a regulator can be direct or indirect.
  • a regulator can function to activate or inhibit or otherwise modulate its substrate.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non- toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are 61 sf-6122278 Attorney Docket No.: 340742000140 unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.
  • a “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
  • a “reconstituted” formulation is one which has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout.
  • the reconstituted formulation is suitable for administration (e.g. subcutaneous administration) to a patient to be treated with the protein of interest and, in certain embodiments, may be one which is suitable for parenteral or intravenous administration.
  • An “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm.
  • the term “hypotonic” describes a formulation with an osmotic pressure below that of human blood.
  • hypoertonic is used to describe a formulation with an osmotic pressure above that of human blood.
  • Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • the formulations of the present application can be hypertonic as a result of the addition of salt and/or buffer.
  • transfected or transformed or transduced refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • treatment or “treating” is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), 62 sf-6122278 Attorney Docket No.: 340742000140 preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • alleviating one or more symptoms resulting from the disease diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), 62 sf-6122278 Attorney Docket No
  • the methods of the application contemplate any one or more of these aspects of treatment.
  • the terms “effective amount” and “pharmaceutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
  • the terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • to “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to that of a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%).
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
  • the terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is a human.
  • An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, 63 sf-6122278 Attorney Docket No.: 340742000140 compared to a parent antibody that does not possess those alteration(s).
  • an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.
  • the term “about X-Y” used herein has the same meaning as “about X to about Y.” [0179] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. II. Bifunctional Protein Constructs [0180] The present application in one aspect provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor.
  • the term “bifunctional protein construct” used here refers to a protein construct which comprises binding functions to both a muscle-specific molecule and a Notch receptor, and can encompass constructs having additional functionalities.
  • the bifunctional protein construct is multispecific.
  • the bifunctional protein construct comprises a first binding moiety and a second binding moiety, wherein the first binding moiety comprises a means for binding to a muscle-specific molecule, and wherein the second binding moiety comprises a means for binding to a Notch receptor and activates the Notch receptor.
  • the first binding moiety comprising an antibody moiety, which includes, but is not limited to, full length antibody, scFv, Fab, or sdAb. In some embodiments, the first binding moiety comprises a non-antibody moiety.
  • Exemplary first binding moieties include, but are not limited to, anti-LAMA2 antibody moieties (e.g., any of the anti-human LAMA2 antibody moieties described herein), anti-matriglycan antibody moieties (e.g., any of the anti-human ADG41 antibody moieties described herein), and anti- CDH15 antibodies (e.g., any of the anti-human CDH15 antibody moieties described herein).
  • anti-LAMA2 antibody moieties e.g., any of the anti-human LAMA2 antibody moieties described herein
  • anti-matriglycan antibody moieties e.g., any of the anti-human ADG41 antibody moieties described herein
  • anti- CDH15 antibodies e.g., any of the anti-human CDH15 antibody moieties described herein.
  • the first binding moiety comprises a non-antibody binding moiety specifically binding to the muscle-specific molecule, for example, a protein domain (e.g., a laminin G-like domain (LG domain)) of an extracellular matrix (ECM) protein that can bind to matriglycan.
  • the non-antibody moiety comprises a protein domain selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.
  • the first binding moiety comprises two or more non-antibody moieties connected in tandem.
  • Means for binding to a muscle-specific molecule described herein can be any of the non- antibody binding moieties described herein and functional equivalents thereof.
  • the second binding moiety described herein specifically binds to a Notch receptor and activates the Notch receptor.
  • the second binding moiety comprises an extracellular domain (ECD) or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130.
  • the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 131.
  • the second binding moiety comprises a DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 125, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 125.
  • the second binding moiety comprises a variant DLL4 ECD comprising 65 sf-6122278 Attorney Docket No.: 340742000140 the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 126-129 and 260-267.
  • the second binding moiety comprises a Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 132, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 132.
  • the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 133 or 134.
  • the second binding moiety comprises a Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 135.
  • Means for binding to a Notch receptor (and optionally activates the Notch receptor) described herein can be any of the non-antibody moieties described herein and functional equivalents thereof.
  • the second binding moiety is an anti-Notch antibody moiety that activates the Notch receptor.
  • Means for binding to a Notch receptor (and optionally activates the Notch receptor) described herein can be any of the anti-Notch antibody moieties described herein and functional equivalents thereof.
  • the first binding moiety and the second binding moiety described herein may be directly or indirectly fused (i.e., covalently linked) to each other.
  • the first binding moiety is fused to the second binding moiety via a carrier protein (e.g., an albumin, an anti-human serum albumin antibody, or a monomeric Fc domain).
  • the first binding moiety is fused to the second binding moiety via an optional peptide linker (e.g., any of the peptide linkers described under the “Linkers” subsection), for example a peptide linker that is no more than about 30 (such as no more than about any one of 25, 20, or 15) amino acids long.
  • the bifunctional protein construct provided herein comprises an Fc domain comprising a first subunit and a second subunit.
  • the bifunctional protein constructs provided herein can be used with any one of a variety of bispecific or multispecific antibody construct formats known in the art. Numerous formats have been developed in the art to address therapeutic opportunities afforded by molecules with multiple binding specificities.
  • the first binding moiety and the second binding moiety can be directly or indirectly linked (i.e., via a peptide linker or a carrier protein, such as a monomeric carrier protein) to each other.
  • the first binding moiety can be fused to the second binding moiety via an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • the first binding moiety and the second binding moiety may also be fused directly to each other.
  • the first binding moiety may be fused at the N-terminus or the C-terminus of the second binding moiety, such as at either the N-terminus or the C-terminus of any one of the polypeptide(s) of the second binding moiety.
  • the first binding moiety and the second binding moiety are linked to each other via a carrier protein.
  • the carrier protein is selected from the group consisting of a human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of an Fc domain.
  • Anti-HSA antibody moieties are known in the art, including, for example, Mandrup et al., Commun Biol., 4(1):310 (2021) and Benjamin et al., Hybridoma, 6(2):183-90 (1987).
  • the first binding moiety is fused to 67 sf-6122278 Attorney Docket No.: 340742000140 the N-terminus of the carrier protein via an optional first linker
  • the second binding moiety is fused to the C-terminus of the carrier protein via an optional second linker.
  • the first binding moiety is fused to the C-terminus of the carrier protein via an optional first linker
  • the second binding moiety is fused to the N-terminus of the carrier protein via an optional second linker.
  • the carrier protein e.g., HSA
  • extends the half-life of the bifunctional protein construct e.g., extending at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or longer).
  • the first binding moiety comprises an scFv comprising a VH and a VL specifically binding to a muscle-specific molecule, wherein the scFv is fused to a second binding moiety specifically binding to a Notch receptor.
  • the second binding moiety can be fused to the N-terminus or C-terminus of the scFv specifically recognizing a muscle- specific molecule via an optional linker.
  • the optional linker between the second binding moiety and the scFv comprises a carrier protein, such as HSA, an anti- HSA antibody moiety, and a subunit of an Fc domain.
  • the optional linker between the second binding moiety and the scFv is a peptide linker.
  • the bifunctional protein construct comprises from the N’ to the C’: [VH – first optional linker (e.g., a peptide linker) – VL] forming the scFv – second optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor.
  • the bifunctional protein construct comprises from the N’ to the C’: [VL – first optional linker (e.g., a peptide linker) – VH] forming the scFv – second optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor.
  • first optional linker e.g., a peptide linker
  • VH forming the scFv – second optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor.
  • the bifunctional protein construct comprises from the N’ to the C’: a second binding moiety specifically binding to a Notch receptor – first optional linker (e.g., a peptide linker or carrier protein) – [VH – second optional linker (e.g., a peptide linker) – VL] forming the scFv.
  • the bifunctional protein construct comprises from the N’ to the C’: a second binding moiety specifically binding to a Notch receptor – first optional linker (e.g., a peptide linker or carrier protein) – [VL – second optional linker (e.g., a peptide linker) – VH] forming the scFv.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the first binding moiety comprises an anti-matriglycan scFv (e.g., SEQ ID NO: 155).
  • the first binding moiety comprises an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, 68 sf-6122278 Attorney Docket No.: 340742000140 and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the first binding moiety comprises more than one (e.g., two or three) scFvs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • a peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • the first binding moiety comprises a Fab comprising a first polypeptide chain comprising VH and CH1 and a second polypeptide chain comprising VL and CL, wherein the Fab specifically binds to a muscle-specific molecule and is fused to a second binding moiety specifically binding to a Notch receptor via an optional linker.
  • the second binding moiety is fused to the N-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the N-terminus of the second polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the second polypeptide chain of the Fab.
  • the bifunctional protein construct comprises: i) a fusion polypeptide comprising from N’ to C’: VH-CH1 – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor; and ii) a second polypeptide comprising from N’ to C’: VL-CL; wherein VH- CH1 and VL–CL form an Fab that specifically binds to a muscle-specific molecule.
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH-CH1; and ii) a fusion polypeptide comprising from N’ to C’: VL-CL – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor; wherein VH-CH1 and VL–CL form an Fab that specially binds to a muscle-specific molecule.
  • the bifunctional protein construct comprises: i) a fusion polypeptide comprising from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – VH-CH1; ii) a second polypeptide comprising from N’ to C’: VL-CL; wherein VH-CH1 and VL-CL form an Fab that specifically binds to a muscle-specific molecule.
  • a fusion polypeptide comprising from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – VH-CH1; ii) a second polypeptide comprising from N’ to C’: VL-CL; wherein VH-CH1 and VL-CL form an Fab that specifically binds to a muscle-specific molecule.
  • the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH-CH1; and ii) a fusion polypeptide comprising from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – VL-CL; wherein VH-CH1 and VL–CL form a Fab that specially binds to a muscle-specific molecule.
  • a Notch receptor e.g., a peptide linker or a carrier protein
  • the optional linker between the second binding moiety and the Fab comprises a carrier protein, 69 sf-6122278 Attorney Docket No.: 340742000140 such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain.
  • the optional linker between the second binding moiety and the Fab is a peptide linker.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the first binding moiety comprises an anti-matriglycan Fab, such as an anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the first binding moiety comprises an anti-LAMA2 Fab, such as an anti- LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the first binding moiety comprises more than one (e.g., two or three) Fabs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • the first binding moiety comprises an sdAb comprising a VHH domain specifically binding to a muscle-specific molecule, wherein the sdAb is fused to a second binding moiety specifically binding to a Notch receptor.
  • the second binding moiety can be fused to the N-terminus or C-terminus of the sdAb.
  • the bifunctional protein construct comprises from N’ to C’: sdAb (e.g., VHH) specifically binding to a muscle-specific molecule – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor.
  • the bifunctional protein construct comprises from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – sdAb (e.g., VHH) specifically binding to a muscle-specific molecule.
  • the optional linker between the second binding moiety and the sdAb comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain.
  • the optional linker between the second binding moiety and the sdAb is a peptide linker.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and 70 sf-6122278 Attorney Docket No.: 340742000140 Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the first binding moiety comprises more than one (e.g., two or three) sdAbs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • the first binding moiety comprises a non-antibody moiety specifically binding to a muscle-specific molecule, wherein the non-antibody moiety comprises an LG domain, e.g., selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.
  • the bifunctional protein construct comprises from N’ to C’: a non- antibody moiety specifically binding to a muscle-specific molecule – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor.
  • the bifunctional protein construct comprises from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – a non-antibody moiety specifically binding to a muscle- specific molecule.
  • the optional linker between the second binding moiety and the non-antibody moiety comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain.
  • the optional linker between the second binding moiety and the non-antibody moiety is a peptide linker.
  • the first binding moiety comprises an LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the first binding moiety comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) non-antibody moieties (such a LG domain) linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • non-antibody moieties such as LG domain
  • a peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., any of SEQ ID NOs: 126-135 and 260-267); an optional peptide linker (e.g., 71 sf-6122278 Attorney Docket No.: 340742000140 any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5).
  • a DLL4 ECD or a variant thereof e.g., any of SEQ ID NOs: 126-135 and 260-267
  • an optional peptide linker e.g., 71 sf-6122278 Attorney Docket No.: 340742000140 any of SEQ ID NOs: 211, 212, 337, and 338
  • a first binding moiety comprising an LG domain of LAMA2 (
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 136 (hereinafter denoted as “DLL4wt-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 137 (hereinafter denoted as “DLL4v-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 variant ECD, wherein the DLL4 variant ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 138 (hereinafter denoted as “DLL4max-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 139 (hereinafter denoted as “DLL4deimmune (DLL4di)-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5).
  • a bifunctional 72 sf-6122278 Attorney Docket No.: 340742000140 protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 140 (hereinafter denoted as “DLL1wt-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker ; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 141 (hereinafter denoted as “DLL3wt-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5) (e.g., SEQ ID NO: 119).
  • a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 (hereinafter denoted as “Jag1wt-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • 73 sf-6122278 Attorney Docket No.: 340742000140
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 143 (hereinafter denoted as “Jag1v-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144 (hereinafter denoted as “Jag2wt-LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).
  • a DLL4 ECD or a variant thereof such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 146 (hereinafter denoted as “DLL4wt-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 147 (hereinafter denoted as “DLL4v-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, 74 sf-6122278 Attorney Docket No.: 340742000140 wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 148 (hereinafter denoted as “DLL4max-LG21scFv”).
  • DLL4max-LG21scFv amino acid sequence of SEQ ID NO: 148
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 149 (hereinafter denoted as “DLL4di-LG21scFv”). [0210] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti- LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 150 (hereinafter denoted as “DLL1wt-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti- LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 151 (hereinafter denoted as “DLL3wt-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant 75 sf-6122278 Attorney Docket No.: 340742000140 thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).
  • a second binding moiety comprising a Jag1 ECD or a variant 75 sf-6122278 Attorney Docket No.: 340742000140 thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211,
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti- LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 152 (hereinafter denoted as “Jag1wt-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti- LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 153 (hereinafter denoted as “Jag1v-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti- LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 154 (hereinafter denoted as “Jag2wt-LG21scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matriglycan scFv (e.g., SEQ ID NO: 155).
  • a DLL4 ECD or a variant thereof such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 156 (hereinafter denoted as “DLL4wt-ADG41scFv”).
  • DLL4wt-ADG41scFv amino acid sequence of SEQ ID NO: 156
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 157 (hereinafter denoted as “DLL4v-ADG41scFv”).
  • DLL4v-ADG41scFv amino acid sequence of SEQ ID NO: 157
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 158 (hereinafter denoted as “DLL4max-ADG41scFv”).
  • DLL4max-ADG41scFv amino acid sequence of SEQ ID NO: 158
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 159 (hereinafter denoted as “DLL4di-ADG41scFv”). [0221] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-matriglycan scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ 77 sf-6122278 Attorney Docket No.: 340742000140 ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130
  • an optional peptide linker e.g., SEQ 77 sf-6122278 Attorney Docket No.: 340742000140 ID NO: 211
  • a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-mat
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 160 (hereinafter denoted as “DLL1wt-ADG41scFv”). [0222] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-matriglycan scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 161 (hereinafter denoted as “DLL3wt-ADG41scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matriglycan scFv (e.g., SEQ ID NO: 155).
  • a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matriglycan scFv (e.g., SEQ ID NO:
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti- matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 162 (hereinafter denoted as “Jag1wt-ADG41scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti- matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 163 (hereinafter denoted as “Jag1v-ADG41scFv”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matriglycan scFv.
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 164 (hereinafter denoted as “Jag2wt- ADG41scFv”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 167 (hereinafter denoted as “DLL4wt- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the 79 sf-6122278 Attorney Docket No.: 340742000140 second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127
  • the anti-LAMA2 Fab comprises a first polypeptide compris
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 168 (hereinafter denoted as “DLL4v- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 169 (hereinafter denoted as “DLL4max- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide 80 sf-6122278 Attorney Docket No.: 340742000140 comprising the amino acid sequence of SEQ ID NO: 170 (hereinafter denoted as “DLL4di- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 171 (hereinafter denoted as “DLL1wt-LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion 81 sf-6122278 Attorney Docket No.: 340742000140 polypeptide comprising the amino acid sequence of SEQ ID NO: 172 (hereinafter denoted as “DLL3wt-LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 173 (hereinafter denoted as “Jag1wt- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide 82 sf-6122278 Attorney Docket No.: 340742000140 comprising the amino acid sequence of SEQ ID NO: 174 (hereinafter denoted as “Jag1v- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- LAMA2 Fab and a second binding moiety comprising a Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 175 (hereinafter denoted as “Jag2wt- LG21-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab (e.g., SEQ ID NO: 176) and a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab (e.g., SEQ ID NO: 176) via an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a 83 sf-6122278 Attorney Docket No.: 340742000140 second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (hereinafter denoted as “DLL4wt-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 179 (hereinafter denoted as “DLL4v-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 (hereinafter denoted as “DLL4max-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- 84 sf-6122278 Attorney Docket No.: 340742000140 matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first binding moiety comprising an anti- 84 sf-6122278 Attorney Docket No.: 340742000140 matriglycan Fab and a second binding moiety comprising a variant D
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (hereinafter denoted as “DLL4di-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti- matriglycan Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti-matriglycan Fab and a second binding moiety comprising a DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and wherein the anti- matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the anti- matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182 (hereinafter denoted as “DLL1wt-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti- matriglycan Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first 85 sf-6122278 Attorney Docket No.: 340742000140 binding moiety comprising an anti-matriglycan Fab and a second binding moiety comprising a DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131, and wherein the anti- matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first 85 sf-6122278 Attorney Docket No.: 340742000140 binding moiety comprising an anti-matriglycan Fab and a second binding moiety comprising a DLL3 ECD,
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 183 (hereinafter denoted as “DLL3wt-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab (e.g., SEQ ID NO: 176) and a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab (e.g., SEQ ID NO: 176) via an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional peptide linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptid
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 184 (hereinafter denoted as “Jag1wt-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the 86 sf-6122278 Attorney Docket No.: 340742000140 second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the 86 sf-6122278 Attorney Docket No.:
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 185 (hereinafter denoted as “Jag1v-ADG41-Fab”).
  • a bifunctional protein construct comprising a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti- matriglycan Fab via an optional peptide linker.
  • a bifunctional protein construct comprising a first binding moiety comprising an anti-matriglycan Fab and a second binding moiety comprising a Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein the anti- matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • an optional peptide linker e.g., SEQ ID NO: 211
  • the anti- matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 186 (hereinafter denoted as “Jag2wt-ADG41-Fab”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, 87 sf-6122278 Attorney Docket No.: 340742000140 wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 319 (hereinafter denoted as “DLL4(EGF1-6)-GSlinker-laminin-a2 LG4-5”).
  • DLL4(EGF1-6)-GSlinker-laminin-a2 LG4-5 the amino acid sequence of SEQ ID NO: 319
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339
  • an optional peptide linker e.g., SEQ ID NO: 337
  • a carrier protein e.g., SEQ ID NO: 344
  • an optional peptide linker e
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 320 (hereinafter denoted as “DLL4(EGF1-6)-HSA-laminin-a2 LG4-5”).
  • DLL4(EGF1-6)-HSA-laminin-a2 LG4-5 the amino acid sequence of SEQ ID NO: 320 (hereinafter denoted as “DLL4(EGF1-6)-HSA-laminin-a2 LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO:
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • a second binding moiety comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 323 (hereinafter denoted as “DLL1(EGF1-6)-HSA-laminin-a2 LG4-5”). 88 sf-6122278 Attorney Docket No.: 340742000140 [0255]
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 325 (hereinafter denoted as “Jagged1(EGF1-6)-laminin- a2 LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a second binding moiety comprising a Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 326 (hereinafter denoted as “Jagged1(EGF1-6)-HSA- laminin-a2 LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag2 ECD, wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 89 sf-6122278 Attorney Docket No.: 340742000140 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 328 (hereinafter denoted as “Jagged2(EGF1-6)-laminin- a2 LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag2 ECD, wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • a second binding moiety comprising a variant Jag2 ECD, wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 329 (hereinafter denoted as “Jagged2(EGF1-6)-HSA- laminin-a2 LG4-5”).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).
  • a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343.
  • the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 322 (hereinafter denoted as “DLL1(EGF1-6)-laminin-a2 LG4-5”).
  • the bifunctional protein construct comprises an Fc domain comprising a first subunit and a second subunit.
  • the Fc domain can be a third moiety 90 sf-6122278 Attorney Docket No.: 340742000140 connected to the first binding moiety and the second binding moiety.
  • the Fc domain when the first binding moiety or second binding moiety is a full-length antibody, the Fc domain can is part of the full-length antibody.
  • the Fc domain is a variant Fc domain possessing minimal or no effector functions.
  • the variant Fc domain is derived from IgG1 Fc or IgG4 Fc.
  • the Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the Fc domain possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • the Fc domain comprises one or more mutations that increase the half-life of the antibody moiety in vivo. In some embodiments, the variant Fc domain has increased FcRn binding at pH 6.0. In some embodiments, the variant Fc domain comprises an M252Y/S254T/T256E (“YTE”) mutation (EU numbering). See, e.g., Dall’Acqua et al., J Immunol., 169:5171–5180 (2002) and Wang et al., Protein & Cell, 9(1):63-73 (2016). In some embodiments, the variant Fc domain comprises an M428L/N434S mutation.
  • Fc receptor (FcR) binding assays can be conducted to determine whether the antibody possesses Fc ⁇ R binding (hence likely ADCC activity), and/or retains FcRn binding ability.
  • FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. Et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. Et al., J. Exp. Med.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry 91 sf-6122278 Attorney Docket No.: 340742000140 (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al.
  • Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include a variant Fc with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • the Fc domain of the bifunctional protein construct comprises one or more of these mutations.
  • the variant Fc domain comprises an N297A, N297Q, or N297G mutation.
  • the variant Fc domain comprises a L235E mutation.
  • the Fc domain is derived from human IgG1, human IgG2, or human IgG4.
  • the Fc domain is derived from an IgG1 Fc domain (e.g., human IgG1 Fc domain).
  • the human IgG1 Fc comprises the amino acid sequence of SEQ ID NO: 187.
  • each subunit of the Fc domain comprises a mutation that reduces or abolishes the effector function.
  • effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “effector-less mutation.”
  • the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region (EU 92 sf-6122278 Attorney Docket No.: 340742000140 numbering).
  • effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli.) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).
  • each subunit of the Fc domain comprises an L234A/L235A “LALA” mutation (EU numbering).
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the Fc domain comprises a mutation that reduces half-life of the bifunctional protein construct in blood circulation, such as an H435A mutation (EU numbering).
  • each subunit of the Fc domain e.g., human IgG1 Fc
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the Fc domain is an IgG4 (e.g., human IgG4) Fc domain.
  • the Fc domain comprises a mutation that reduces Fab exchange, such as an S228P mutation.
  • the IgG4 Fc domain comprises an F234A/L235A mutations. See, e.g., Xu et al., Cell Immunol., 200:16–26 (2000) and Wang et al., Protein & Cell, 9(1):63-73 (2016).
  • the Fc domain can comprise one or more mutations that facilitate assembly of the heterodimeric protein.
  • knob-into- hole is a heterodimerization technology for the CH3 domain of an antibody.
  • knobs-into-holes technology has been applied to the production of human full-length bispecific antibodies with a single common light chain (LC) (Merchant et al.
  • the bifunctional protein construct comprises an Fc domain comprising a first CH3 domain and a second CH3 domain 93 sf-6122278 Attorney Docket No.: 340742000140 comprising knob-into-hole (KIH) residues.
  • the first CH3 domain is altered so that within the CH3/CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the first CH3 domain that interacts with the second CH3 domain; and the second CH3 domain is altered so that within the CH3/CH3 interface, one or more amino acid residues are replaced amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the second CH3 domain that interacts with the first CH3 domain.
  • the protuberance is a knob.
  • the alteration to generate the knob is T366W.
  • the cavity is a hole.
  • the alterations to generate the hole are T366S/L368A/Y407V.
  • the Fc domain described herein comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering). Unless indicated otherwise, all amino acid positions within the Fc domain are numbered according to EU numbering system.
  • the bifunctional protein construct comprises an Fc domain comprising a mutation allowing for purification of asymmetric Fc domains.
  • either the first subunit of the Fc domain or the second subunit of the Fc domain comprises an H435R/Y436F mutation (EU numbering).
  • each subunit of the Fc domain comprises an H435R/Y436F mutation (EU numbering).
  • heterodimerization domains having a strong preference for forming heterodimers over homodimers can be incorporated into the instant bifunctional protein constructs.
  • Illustrative examples include but are not limited to, for example, WO2007147901 (Kj ⁇ rgaard et al.– Novo Nordisk: describing ionic interactions); WO2009/089004 (Kannan et al.– Amgen: describing electrostatic steering effects); WO2010/034605 (Christensen et al. – Genentech; describing coiled coils). See also, for example, Pack, P.
  • the bifunctional protein construct comprises one or more heterodimerization domains.
  • the Fc domain when the bifunctional protein construct is a heterodimeric protein comprising two different polypeptide chains, can comprise one or more charged-pair mutations that facilitate assembly of the heterodimeric protein.
  • an amino acid residue in the first subunit of the Fc domain is replaced with a positively charged residue (e.g., R, H, or K), and an amino acid residue in the second subunit of the Fc domain is replaced with a negatively charged residue (e.g., D or E).
  • a positively charged residue in the first subunit of the Fc domain is replaced with a negatively charged residue
  • an amino acid residue in the second subunit of the Fc domain is replaced with a positively charged residue.
  • an amino acid residue at D399 (EU numbering) in the first subunit of the Fc domain is replaced with a positively charged residue
  • an amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue
  • an amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue
  • an amino acid residue at D399 (EU numbering) in the second subunit of the Fc domain is replaced with a positively charged residue.
  • the Fc domain used in the bifunctional protein construct described herein comprises the amino acid sequence of any one of SEQ ID NO: 188, 254, and 277.
  • a bifunctional protein construct comprising: i) one or more units of a first binding moiety specifically binding to a muscle- specific molecule; ii) a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; iii) a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; optionally wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the first unit of the second binding moiety and the second unit of the second binding moiety are the same. In some embodiments, the first unit of the second binding moiety and the second unit of the second binding moiety are different.
  • the one or 95 sf-6122278 Attorney Docket No.: 340742000140 more units of the first binding moiety can be the same or different; and/or can bind to the same or different muscle-specific molecules.
  • the bifunctional protein construct further comprises a third unit of a second binding moiety specifically binding to a third Notch receptor, and a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor.
  • the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the first, second, third, and/or fourth unit of the second binding moiety can be the same or different; and/or can bind to the same or different muscle- specific molecules.
  • all four units of the second binding moiety are the same.
  • at least one of the four units of the second binding moiety is different from the others.
  • the one or more units of the first binding moiety can comprise antibody moiety or non-antibody moiety.
  • the various binding moieties can independently fuse to the N and/or C terminus of the Fc domain.
  • the bifunctional protein construct can be homodimeric or heterodimeric.
  • a bifunctional protein construct comprising: i) a first unit of a first binding moiety specifically binding to a first muscle- specific molecule; ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule; iii) one or more units of a second binding moiety specifically binding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit.
  • a bifunctional protein construct comprising: i) a first unit of a first binding moiety comprising a first antibody moiety specifically binding to a first muscle-specific molecule, ii) a second unit of a first binding moiety comprising a second antibody moiety specifically binding to a second muscle-specific molecule, iii) one or more units of a second binding moiety specifically binding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first antibody moiety and the second antibody moiety are each independently selected from the group consisting of a Fab, an scFv, and an sdAb.
  • a bifunctional protein construct comprising: i) a first unit of a first binding moiety comprising a first non-antibody moiety specifically binding to a first muscle-specific molecule, ii) a second unit of a first binding moiety comprising a second non-antibody moiety specifically binding to a second muscle-specific molecule, iii) one or more units of a second binding moiety specifically 96 sf-6122278 Attorney Docket No.: 340742000140 binding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first non-antibody moiety and the second non-antibody moiety are each independently selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.
  • the first unit of the first binding moiety comprises two or more first non-antibody moieties connected in tandem, and the second unit of the first binding moiety comprises two or more second non-antibody moieties connected in tandem.
  • the first unit of the first binding moiety and the second unit of the first binding moiety are the same.
  • the first unit of the first binding moiety and the second unit of the first binding moiety are different.
  • the one or more units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the various binding moieties can independently fuse to the N and/or C terminus of the Fc domain.
  • the bifunctional protein construct can be homodimeric or heterodimeric.
  • the one or more units of the first binding moiety in the bifunctional protein construct comprises (or is) a Fab, wherein the Fab can be connected to the C-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2- CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain –
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2- CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 98 sf-6122278 Attorney Docket No.: 340742000140 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv can be connected to the C-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • a bifunctional protein construct comprising: a first scFv (scFv1) specifically binding to a first muscle- specific molecule comprising VH (VH1) and VL (VL1); a second scFv (scFv2) specifically binding to a second muscle-specific molecule comprising a VH (VH2) and VL (VL2); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – scFv1 (VH1 – optional linker
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • scFv1 and scFv2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, scFv1 and scFv2 both specifically bind to matriglycan.
  • anti-matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, scFv1 and scFv2 both specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • DLL1 Delta-like 1
  • DLL3 DLL4, Jag1, and Jag2
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moieties comprises (or is) an sdAb, wherein the sdAb can be connected to the C-terminus of a subunit of the Fc domain, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first sdAb (sdAb1, e.g., VHH1) specifically binding to a first muscle- specific molecule; a second sdAb (sdAb2, e.g., VHH2) specifically binding to a second muscle-specific molecule; a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – sdAb1; ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding mo
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • sdAb1 and sdAb2 can be the same or different; and/or can bind to the same or different muscle-specific molecules.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moieties comprises (or is) a Fab, wherein the Fab can be connected to the N-terminus of a subunit of the Fc domain via CH1 or CL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific 100 sf-6122278 Attorney Docket No.: 340742000140 molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1)
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VL1-(L1-CL) – optional linker – the first subunit of the Fc domain – optional link
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the 101 sf-6122278 Attorney Docket No.: 340742000140 same or different muscle-specific molecules.
  • Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: i) a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule, wherein the full-length antibody comprises an Fc domain comprising a first subunit and a second subunit; ii) a first unit of a second binding moiety fused (directly or via an optional linker) to the C-terminus of the first subunit of the Fc domain; and iii) a second unit of a second binding moiety fused (directly or via an optional linker) to the C-terminus of the second subunit of the Fc domain; wherein the first unit of the second binding moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety specifically binds to a second Notch receptor and activates the second Notch receptor.
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ - DG, and matriglycan on ⁇ -DG.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • DLL1 Delta-like 1
  • DLL3 DLL4, Jag1, and Jag2
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv can be connected to the N-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first scFv (scFv1) specifically binding to a first muscle-specific molecule comprising VH (VH1) and VL (VL1); a second scFv (scFv2) specifically binding to a second muscle-specific molecule comprising a VH (VH2) and VL (VL2); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: scFv1 (VH1 -optional linker – VL1 or VL1 -optional linker – VH1) -optional linker– the first subunit of the F
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • scFv1 and scFv2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, scFv1 and scFv2 both specifically bind to matriglycan.
  • anti-matriglycan scFv1 and/or anti- matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, scFv1 and scFv2 both specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • DLL1 Delta-like 1
  • DLL3 DLL4, Jag1, and Jag2
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moieties comprises (or is) an sdAb, wherein the sdAb can be connected to the N-terminus of a subunit of the Fc domain, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first sdAb (sdAb1, e.g., VHH1) specifically binding to a first muscle-specific molecule; a second sdAb (sdAb2, e.g., VHH2) specifically binding to a second muscle- specific molecule; a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: sdAb1 – optional linker– the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; and ii) a second polypeptide comprising from N’ to C’: sdAb2 – optional linker
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • sdAb1 and sdAb2 can be the same or different; and/or can bind to the same or different muscle-specific molecules.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically 104 sf-6122278 Attorney Docket No.: 340742000140 binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: i) a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule; ii) a first unit of a second binding moiety fused (directly or via an optional linker) to the C-terminus of a first light chain of the full-length antibody; and iii) a 105 sf-6122278 Attorney Docket No.: 340742000140 second unit of a second binding moiety fused (directly or via an optional linker) to the C- terminus of a second light chain of the full-length antibody; wherein the first unit of the second binding moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety specifically binds to a second Notch receptor and activates the second Notch receptor.
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ - DG, and matriglycan on ⁇ -DG.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle- 106 sf-6122278 Attorney Docket No.: 340742000140 specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule, a first unit of a second binding moiety fused (directly or via an optional linker) to the N-terminus of a first light chain of the full-length antibody, and a second unit of a second binding moiety fused (directly or via an optional linker) to the N- terminus of a second light chain of the full-length antibody; wherein the first unit of the second binding moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety specifically binds to a second Notch receptor and activates the second Notch receptor.
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ - DG, and matriglycan on ⁇ -DG.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the 107 sf-6122278 Attorney Docket No.: 340742000140 engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – VH1-(H1-CH1)
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected 108 sf-6122278 Attorney Docket No.: 340742000140 from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule, a first unit of a second binding moiety fused (directly or via an optional linker) to the N-terminus of a first heavy chain of the full-length antibody, and a second unit of a second binding moiety fused (directly or via an optional linker) to the N- terminus of a second heavy chain of the full-length antibody; wherein the first unit of the second binding moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety specifically binds to a second Notch receptor and activates the second Notch receptor.
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ - DG, and matriglycan on ⁇ -DG.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; a third unit of a second binding moiety specifically binding to a third Notch receptor and activating the third Notch receptor; a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor and activating the fourth Notch 109 sf-6122278
  • Fab1 specifically binding to a first muscle-specific molecule comprising
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • Fab1 and Fab2 can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab.
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Fab1 and Fab2 both specifically bind to LAMA2.
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the first, second, third, and/or fourth units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the others. In some embodiments, the second binding moiety (one or more units, or all units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the first, second, third, and/or fourth units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the others.
  • the second binding moiety (one or more units, or all units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or more units, or all units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the one or more units of the first binding moiety comprises a non-antibody moiety specifically binding to a muscle-specific molecule, wherein the non- antibody moiety can be connected to the C-terminus or N-terminus of a subunit of an Fc domain, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • an optional linker e.g., any of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety specifically binding to a first muscle-specific molecule, a second non-antibody moiety specifically binding to a second muscle-specific molecule, a first unit of a second binding moiety specifically binding to a first Notch receptor 111 sf-6122278 Attorney Docket No.: 340742000140 and activating the first Notch receptor, a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding mo
  • a bifunctional protein construct comprising: a first non-antibody moiety specifically binding to a first muscle-specific molecule, a second non-antibody moiety specifically binding to a second muscle-specific molecule, a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor, a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first non-antibody moiety -optional linker– the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; and ii) a second polypeptide comprising from N’ to C’: the second non-antibody moiety – optional linker – the second subunit of the Fc domain – optional linker –
  • the first and/or second non-antibody moiety comprises a protein domain independently selected from the group consisting of a laminin G-like domain (LG domain) of laminin, an LG domain of agrin, an LG domain of nidogen, an LG domain of perlecan, the laminin coiled-coil binding domain of agrin, and the laminin ⁇ binding domain of nidogen.
  • the first and/or second non-antibody moiety comprises two or more protein domains (e.g., LG domain) connected in tandem, such as via an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • the first non-antibody moiety and/or the second non-antibody moiety comprises LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.
  • the first non-antibody moiety comprises two LAMA2 LG4-5 connected in tandem, and the second non-antibody moiety comprises two LAMA2 LG4-5 connected in tandem.
  • the non-antibody moiety comprises a laminin G-like domain (LG domain).
  • the bifunctional protein construct comprises more than one (e.g., two or three) non-antibody moieties (such as LG domains) linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).
  • the first and second non-antibody 112 sf-6122278 Attorney Docket No.: 340742000140 moieties can be the same or different; and/or can bind to the same or different muscle- specific molecules.
  • the optional peptide linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct.
  • the first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptors.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • a bifunctional protein construct comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti- LAMA2 Fab2); a first unit of second binding moiety comprising a DLL4 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • Anti-LAMA2 Fab1 and anti-LAMA2 Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same 113 sf-6122278 Attorney Docket No.: 340742000140 or different Notch receptor.
  • each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • a bifunctional protein construct comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of any of SEQ ID NOs: 189, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “DLL4wt-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 190, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “DLL4v-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 191, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “DLL4max-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 192, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “DLL4di-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • a bifunctional protein construct comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti- LAMA2 Fab2); a first unit of second binding moiety comprising a DLL1 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker –
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • Anti-LAMA2 Fab1 and anti-LAMA2 Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 193, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “DLL1wt-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti- LAMA2 Fab2); a first unit of second binding moiety comprising a DLL3 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL3 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – 115 sf-6122278 Attorney Docket No.: 340742000140 optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker .
  • anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • Anti-LAMA2 Fab1 and anti-LAMA2 Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti- LAMA2 Fab2); a first unit of second binding moiety comprising a Jag1 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – VL2-(L2-CL
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 195, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “Jag1wt-Fc-LG21-Fab”).
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 196, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “Jag1v-Fc-LG21-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti- LAMA2 Fab2); a first unit of second binding moiety comprising a Jag2 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second
  • anti-LAMA2 Fab1 and/or 117 sf-6122278 Attorney Docket No.: 340742000140 anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • Anti-LAMA2 Fab1 and anti-LAMA2 Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 197, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter denoted as “Jag2wt-Fc-LG21-Fab”).
  • a bifunctional protein construct comprising: a first anti-matriglycan Fab (anti-matriglycan Fab1); a second anti-matriglycan Fab (anti-matriglycan Fab2); a first unit of second binding moiety comprising a DLL4 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – V
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Anti-matriglycan Fab1 and anti-matriglycan Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor. [0308] In some embodiments, the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 118 sf-6122278 Attorney Docket No.: 340742000140 126.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 198, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL4wt-Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 199, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL4v-Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 200, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL4max-Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 201, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL4di-Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan Fab (anti-matriglycan Fab1); a second anti-matriglycan Fab (anti-matriglycan Fab2); a first unit of second binding moiety comprising a DLL1 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Anti-matriglycan Fab1 and anti-matriglycan Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 202, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL1wt- Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan Fab (anti-matriglycan Fab1); a second anti-matriglycan Fab (anti-matriglycan Fab2); a first unit of second binding moiety comprising a DLL3 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL3 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc 120 sf-6122278 Attorney Docket No.: 340742000140 domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional link
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Anti-matriglycan Fab1 and anti-matriglycan Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 203, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “DLL3wt- Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan Fab (anti-matriglycan Fab1); a second anti-matriglycan Fab (anti-matriglycan Fab2); a first unit of second binding moiety comprising a Jag1 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – VL2-
  • anti-matriglycan Fab1 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid 121 sf-6122278 Attorney Docket No.: 340742000140 sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Anti-matriglycan Fab1 and anti-matriglycan Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 204, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “Jag1wt-Fc- ADG41-Fab”).
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 205, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “Jag1v-Fc-ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan Fab (anti-matriglycan Fab1); a second anti-matriglycan Fab (anti-matriglycan Fab2); a first unit of second binding moiety comprising a Jag2 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C
  • anti-matriglycan Fab1 122 sf-6122278 Attorney Docket No.: 340742000140 and/or anti-matriglycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • Anti-matriglycan Fab1 and anti-matriglycan Fab2 can be the same or different.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 206, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176 (hereinafter denoted as “Jag2wt-Fc- ADG41-Fab”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit of second binding moiety comprising a DLL4 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-LAMA2 scFv1; and ii)
  • Anti-LAMA2 scFv1 and anti- LAMA2 scFv2 can be the same or different.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • each subunit of the Fc domain comprises the amino acid sequence SEQ ID NOs: 188 or 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • 123 sf-6122278 Attorney Docket No.: 340742000140 [0317]
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 223 (hereinafter denoted as “DLL4wt-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 224 (hereinafter denoted as “DLL4v-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 225 (hereinafter denoted as “DLL4max-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 226 (hereinafter denoted as “DLL4di-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 124 sf-6122278 Attorney Docket No.: 340742000140 260.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 269 (hereinafter denoted as “huD4v_G2S_F81L_H168Y -Fc(AAG)-LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 261.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 270 (hereinafter denoted as “huD4v11_G2S_T52N_F81L_T135N_H168Y- Fc(AAG)-LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 262.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 271 (hereinafter denoted as “huD4v12_G2S_R66S_F81L_H168Y-Fc(AAG)- LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 263.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 272 (hereinafter denoted as “huD4v13_G2S_E14H_T52N_R66T_P76L_F81L_T135N_H168Y_N231D-Fc(AAG)- LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 264.
  • a bifunctional protein construct comprising a 125 sf-6122278 Attorney Docket No.: 340742000140 first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 273 (hereinafter denoted as “huD4v14_G2S_T52N_R66T_P76L_F81L_T135N_H168Y_Q220H_N260D-Fc(AAG)- LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 265.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 274 (hereinafter denoted as “huD4v22_G2S_ R66S_F81L_H168Y-Fc(AAG)- LG21scFvDS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 266.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 275 (hereinafter denoted as “huD4v23_G2S_E14H_R66T_P76L_F81L_H168Y_N231D-FcAAG-LG21DS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 267.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 276 (hereinafter denoted as “huD4v24_G2S_R66T_P76L_F81L_H168Y_Q220H_N260D-FcAAG-LG21DS”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv 126 sf-6122278 Attorney Docket No.: 340742000140 (anti-LAMA2 scFv2); a first unit of second binding moiety comprising a DLL1 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional
  • Anti-LAMA2 scFv1 and anti- LAMA2 scFv2 can be the same or different.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 227 (hereinafter denoted as “DLL1wt-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit of second binding moiety comprising a DLL3 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL3 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-LAMA2 scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit of the F
  • Anti-LAMA2 scFv1 and anti- LAMA2 scFv2 can be the same or different.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the 127 sf-6122278 Attorney Docket No.: 340742000140 amino acid sequence of SEQ ID NO: 131.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 228 (hereinafter denoted as “DLL3wt-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit of second binding moiety comprising a Jag1 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-LAMA2 scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit of the Fc domain
  • Anti-LAMA2 scFv1 and anti-LAMA2 scFv2 can be the same or different.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 229 (hereinafter denoted as “Jag1wt-Fc-LG21scFv”).
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 230 (hereinafter denoted as “Jag1v-Fc-LG21scFv”).
  • each subunit of Fc domain 128 sf-6122278 Attorney Docket No.: 340742000140 comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit of second binding moiety comprising a Jag2 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-LAMA2 scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit of the Fc domain
  • Anti-LAMA2 scFv1 and anti-LAMA2 scFv2 can be the same or different.
  • anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 231 (hereinafter denoted as “Jag2wt-Fc-LG21scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan scFv (anti-matriglycan scFv1); a second anti-matriglycan scFv (anti-matriglycan scFv2); a first unit of second binding moiety comprising a DLL4 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-matriglycan scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second
  • Anti-matriglycan scFv1 and anti-matriglycan scFv2 can be the same or different.
  • anti- matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 232 (hereinafter denoted as “DLL4wt-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 233 (hereinafter denoted as “DLL4v-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 234 (hereinafter denoted as “DLL4max-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 235 (hereinafter denoted as “DLL4di-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan scFv (anti-matriglycan scFv1); a second anti-matriglycan scFv (anti-matriglycan scFv2); a first unit of second binding moiety comprising a DLL1 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-matriglycan scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second
  • Anti-matriglycan scFv1 and anti-matriglycan scFv2 can be the same or different.
  • anti- matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 236 (hereinafter denoted as “DLL1wt-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan scFv (anti-matriglycan scFv1); a second anti-matriglycan 131 sf-6122278 Attorney Docket No.: 340742000140 scFv (anti-matriglycan scFv2); a first unit of second binding moiety comprising a DLL3 ECD or a variant thereof; a second unit of a second binding moiety comprising a DLL3 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-matriglycan scFv1; and ii) a second polypeptide comprising from N’
  • Anti-matriglycan scFv1 and anti-matriglycan scFv2 can be the same or different.
  • anti- matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 237 (hereinafter denoted as “DLL3wt-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan scFv (anti-matriglycan scFv1); a second anti-matriglycan scFv (anti-matriglycan scFv2); a first unit of second binding moiety comprising a Jag1 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-matriglycan scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit
  • Anti-matriglycan scFv1 and anti-matriglycan scFv2 can be the same or different.
  • anti- matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the 132 sf-6122278 Attorney Docket No.: 340742000140 variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 238 (hereinafter denoted as “Jag1wt-Fc-ADG41scFv”).
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 239 (hereinafter denoted as “Jag1v-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first anti-matriglycan scFv (anti-matriglycan scFv1); a second anti-matriglycan scFv (anti-matriglycan scFv2); a first unit of second binding moiety comprising a Jag2 ECD or a variant thereof; a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – anti-matriglycan scFv1; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit
  • Anti-matriglycan scFv1 and anti-matriglycan scFv2 can be the same or different.
  • anti- matriglycan scFv1 and/or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 240 (hereinafter denoted as “Jag2wt-Fc-ADG41scFv”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising LAMA2 LG4-5, a second non-antibody moiety comprising LAMA2 LG4-5, a first unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non- antibody moiety.
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 241 (hereinafter denoted as “DLL4wt-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127.
  • a bifunctional protein construct comprising a 134 sf-6122278 Attorney Docket No.: 340742000140 first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 242 (hereinafter denoted as “DLL4v-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 243 (hereinafter denoted as “DLL4max-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the first and second units of the second binding moiety each comprises a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 244 (hereinafter denoted as “DLL4di-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising LAMA2 LG4-5, a second non-antibody moiety comprising LAMA2 LG4-5, a first unit of a second binding moiety comprising a DLL1 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119.
  • the first unit 135 sf-6122278 Attorney Docket No.: 340742000140 of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 245 (hereinafter denoted as “DLL1wt-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising LAMA2 LG4-5, a second non-antibody moiety comprising LAMA2 LG4-5, a first unit of a second binding moiety comprising a DLL3 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL3 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non- antibody moiety.
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 246 (hereinafter denoted as “DLL3wt-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising LAMA2 LG4-5, a second non-antibody moiety comprising LAMA2 LG4-5, a first unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the F
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • the first and second units of the second binding moiety each comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133.
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 249 (hereinafter denoted as “Jag2wt-Fc-LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non-antibody moiety; and ii) a
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non- antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL4 ECD or a variant thereof, and wherein the DLL4 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 339.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same 138 sf-6122278 Attorney Docket No.: 340742000140 or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 321 (hereinafter denoted as “DLL4(EGF1-6)-hIgG1-Fc- laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 345.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a DLL1 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL1 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non-anti
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non- antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD or a variant thereof, and wherein the DLL1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 340.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 324 (hereinafter denoted as “DLL1(EGF1-6)-hIgG1-Fc- laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 345.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, and an Fc domain comprising a first subunit and 139 sf-6122278 Attorney Docket No.: 340742000140 a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag1 ECD or a variant thereof, and wherein the Jag1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 341.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 327 (hereinafter denoted as “Jagged1(EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 345.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a Jag2 ECD or a variant thereof, a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non- antibody moiety
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD or a variant thereof, and wherein the Jag2 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 342.
  • the first and second units of the second binding 140 sf-6122278 Attorney Docket No.: 340742000140 moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 330 (hereinafter denoted as “Jagged-2(EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 345.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, a second unit of a second binding moiety comprising a DLL4 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non-antibody moiety; and ii) a
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non- antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL4 ECD or a variant thereof, and wherein the DLL4 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 339.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 331 (hereinafter denoted as “DLL4(EGF1-6)-hIgG4-Fc- laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 346.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a DLL1 ECD or a variant thereof, a second unit of a second binding moiety 141 sf-6122278 Attorney Docket No.: 340742000140 comprising a DLL1 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker –
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non- antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD or a variant thereof, and wherein the DLL1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 340.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 332 (hereinafter denoted as “DLL1(EGF1-6)-hIgG4-Fc- laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 346.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, a second unit of a second binding moiety comprising a Jag1 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non- antibody moiety
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag1 ECD or a variant thereof, and wherein the Jag1 ECD or a variant thereof comprises the 142 sf-6122278 Attorney Docket No.: 340742000140 amino acid sequence of SEQ ID NO: 341.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 333 (hereinafter denoted as “Jagged1(EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 346.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: a first non-antibody moiety comprising laminin-a2 LG4-5, a second non- antibody moiety comprising laminin-a2 LG4-5, a first unit of a second binding moiety comprising a Jag2 ECD or a variant thereof, a second unit of a second binding moiety comprising a Jag2 ECD or a variant thereof, and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety– optional linker – the first subunit of the Fc domain – optional linker – the first non-antibody moiety; and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – the second non- antibody moiety
  • the first and second non-antibody moieties can be the same or different.
  • the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343.
  • the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD or a variant thereof, and wherein the Jag2 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 342.
  • the first and second units of the second binding moiety can be the same or different; and/or can bind to the same or different Notch receptor.
  • a bifunctional protein construct comprising a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 334 (hereinafter denoted as “Jagged2(EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5”).
  • each subunit of Fc domain comprises the amino acid sequence of SEQ ID NO: 346.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the bifunctional construct comprises the amino acid sequence of any one of SEQ ID NOs: 250-252.
  • the bifunctional constructs provided herein can comprise a first unit of a first binding moiety comprising two or more LAMA2 LG4-5 connected in tandem, and a second unit of a first binding moiety comprising two or more LAMA2 LG4-5 connected in tandem.
  • the first and second units of the first binding moiety can be the same or different.
  • Heterodimeric fusions [0361]
  • the bifunctional protein constructs described herein e.g., comprising an Fc domain comprising a first subunit and a second subunit) may comprise two or more different polypeptide chains.
  • the bifunctional protein construct is heterodimeric.
  • a bifunctional protein construct comprising a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a muscle-specific molecule, a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein: i) the first binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional second linker; or ii) the first binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N-terminus of the first subunit
  • the Fc domain comprises knob-into-hole mutations and/or charged-pair mutations. In some embodiments, the Fc domain comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering), and the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional 144 sf-6122278 Attorney Docket No.: 340742000140 linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the bifunctional protein construct comprises an Fc domain comprising a first subunit and a second subunit, wherein one subunit of the Fc domain is fused to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a muscle-specific molecule (at N-terminus, C-terminus, or both), and the other subunit of the Fc domain is fused to a second binding moiety (e.g., scFv, Fab, sdAb, or non- antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor (at N-terminus, C-terminus, or both).
  • a first binding moiety e.g., scFv, Fab, s
  • the bifunctional protein construct can comprise two or more units of the first binding moiety, and/or two or more units of the second binding moiety.
  • the two or more units of the first binding moiety can be the same or different.
  • the two or more units of the second binding moiety can be the same or different.
  • a bifunctional protein constructs comprising: a first binding moiety comprising a Fab specifically binding to a muscle-specific molecule, a second binding moiety (e.g., comprising a Notch ligand ECD or variant thereof) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the Fab comprises a first polypeptide comprising VH-CH1 and a second polypeptide comprising VL-CL, wherein the C-terminus of the CH1 or CL of the Fab is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and wherein the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional second linker.
  • a first binding moiety comprising a Fab specifically binding to a muscle-specific molecule
  • a second binding moiety e.g., comprising a Notch ligand ECD
  • the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the first binding moiety comprises an anti-matriglycan Fab, such as an anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177.
  • the first binding moiety comprises an anti- LAMA2 Fab, such as an anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into- 145 sf-6122278 Attorney Docket No.: 340742000140 hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: i) a first unit of a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a first Notch receptor and activating the first Notch receptor, ii) a second unit of a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a second Notch receptor and activating the second Notch receptor; iii) a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a muscle-specific molecule; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the
  • the bifunctional protein construct comprises: i) a first unit of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a second muscle- specific molecule; wherein the first unit of the first binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the C-terminus of the second subunit of the Fc domain via an optional fourth linker.
  • a first binding moiety e.g., scFv, Fab, sdAb, or non-antibody moiety
  • a second unit of a first binding moiety e.g., scFv, Fab,
  • the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct.
  • 146 sf-6122278 Attorney Docket No.: 340742000140 the muscle-specific molecule is independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non- antibody moiety) specifically binding to one or more muscle-specific molecules, a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional linker; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the C-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain
  • the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct.
  • the one or more muscle- specific molecules is independently selected from the group consisting of LAMA2, CDH15, 147 sf-6122278 Attorney Docket No.: 340742000140 ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into- hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non- antibody moiety) specifically binding to one or more muscle-specific molecules, a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional linker; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of the Fc domain
  • the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct.
  • the one or more muscle- specific molecules is independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting 148 sf-6122278 Attorney Docket No.: 340742000140 of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into- hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: i) a first unit of a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a first Notch receptor and activating the first Notch receptor, ii) a second unit of a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a second Notch receptor and activating the second Notch receptor; iii) a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a muscle-specific molecule; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the
  • the bifunctional protein construct comprises: i) a first unit of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a second muscle- specific molecule; wherein the first unit of the first binding moiety is fused to the N-terminus 149 sf-6122278 Attorney Docket No.: 340742000140 of the first subunit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional fourth linker.
  • a first binding moiety e.g., scFv, Fab, sdAb, or non-antibody moiety
  • the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct.
  • the muscle-specific molecule is independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non- antibody moiety) specifically binding to one or more muscle-specific molecules, a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the second binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional linker; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of the Fc domain
  • the one or more muscle-specific molecules is independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • a bifunctional protein construct comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non- antibody moiety) specifically binding to one or more muscle-specific molecules, a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) specifically binding to a Notch receptor and activating the Notch receptor, and an Fc domain comprising a first subunit and a second subunit; wherein the second binding moiety is fused to the C-terminus of the second subunit of the Fc domain via an optional linker; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the N-terminus of the second subunit
  • the one or more muscle-specific molecules is independently selected from the group consisting of LAMA2, CDH15, ⁇ -DG, and matriglycan on ⁇ -DG.
  • the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.
  • the Fc domain comprises knob-into-hole mutations, wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.
  • the knob mutation is T366W (EU numbering)
  • the hole mutation is T366S/L368A/Y407V (EU numbering).
  • each subunit of the Fc domain further comprises an L234A/L235A/H435A mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.
  • each subunit of the Fc domain comprises an L234A/L235A/P329G “LALALPG” mutation (EU numbering).
  • the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.
  • the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the bifunctional protein constructs provided herein comprises i) one or more units of a second binding moiety each independently comprising an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267); and ii) one or more units of a first binding moiety each independently comprising an antibody moiety specifically binding to a muscle- specific molecule, optionally wherein the antibody moiety is independently selected from the group consisting of an scFv, a Fab, and an sdAb.
  • the two or more units of the second binding moiety are the same; and/or ii) the two or more units of the first binding moiety are the same. In some embodiments, i) the two or more units of the second binding moiety are different; and/or ii) at least one of the two or more units of the first binding moiety is different from the other(s).
  • Fc-containing bifunctional protein constructs comprising two or more different polypeptide chains (e.g., heterodimeric bifunctional protein constructs)
  • the Fc domain comprising knob-into-hole mutations and/or charged-pair mutations may be used.
  • the Fc domain comprises charged-pair mutations, wherein an amino acid residue in the first subunit of the Fc domain is replaced with a positively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced with a negatively charged residue.
  • the Fc domain comprises charged-pair mutations, wherein an amino acid residue in the first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced with a positively charged residue.
  • the Fc domain comprises charged-pair mutations, wherein an amino acid residue at D399 (EU numbering) in the first subunit of the Fc domain is replaced with a positively charged residue, and an amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue.
  • the Fc domain comprises charged-pair mutations, wherein an amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue at D399 (EU numbering) in the second subunit of the Fc domain is replaced with a positively charged residue.
  • the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255. In some embodiments, the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348. In some embodiments, the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257.
  • the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256. In some embodiments, the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 259. In some embodiments, the bifunctional protein construct comprises the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 259, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258.
  • the muscle-specific molecules allow the bifunctional protein construct to be preferentially delivered and anchored to muscle tissues.
  • the first binding moiety of the bifunctional protein constructs described herein specifically bind to a muscle-specific molecule.
  • the muscle-specific molecule is expressed only in muscle cells or tissue.
  • the muscle-specific molecule is a molecule primarily expressed in muscle cells or tissue, compared to cells or tissues of organs that are not muscle.
  • the muscle-specific molecule is also expressed in a cells or tissues of organs that are not muscle.
  • the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina.
  • the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M-cadherin (CDH15).
  • the target antigen is a component of the dystrophin- associated glycoprotein complex (DGC).
  • the target antigen is selected from the group consisting of ⁇ -dystroglycan ( ⁇ -DG), ⁇ -DG, laminin, perlecan, collagen (e.g., Type IV collagen), ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, biglycan, sarcospan, and matriglycan on ⁇ -DG.
  • the target antigen is a laminin subunit alpha-2 (LAMA2), ADG41, CDH15, ⁇ - DG, or matriglycan on ⁇ -DG.
  • the first binding moiety can comprise an antibody moiety or a non-antibody moiety that specifically binds to the muscle-specific molecule.
  • the antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a F(ab’) 2 , an scFv, and an sdAb.
  • Table 8 A list of antibody moieties that specifically binding to muscle-specific molecules is presented in Table 8.
  • the first binding moiety comprises an anti-LAMA2 antibody moiety which specifically binds to LAMA2.
  • Any of the anti-LAMA2 antibody moieties described herein can be used in the bifunctional protein constructs described herein.
  • Means for binding to LAMA2 described herein can be any of the anti-LAMA2 antibody moieties described herein and functional equivalents thereof.
  • the present application also provides novel anti-LAMA2 antibody moieties which can be used for purposes beyond being a component of the bifunctional protein constructs described herein.
  • an anti- LAMA2 antibody construct comprising (or consisting essentially of, or consisting of) any of the anti- LAMA2 antibody moieties described herein.
  • the antibody is monospecific.
  • the antibody is multispecific (such as bispecific). Multispecific antibodies have 155 sf-6122278 Attorney Docket No.: 340742000140 binding specificities for at least two different antigens or epitopes.
  • the antibody is monoclonal.
  • the antibody is polyclonal.
  • the antibody is ChIP-grade, or at least IP grade or western blot grade.
  • the antibody can react with protein of interest from any organism, such as human, mouse, rabbit, rat, monkey, fruit fly, zebrafish, chicken, worms, bacteria, etc.
  • the antibody is chimeric, mouse, rabbit, rat, sheep, human, partially humanized, fully humanized, semi-synthetic, or fully-synthetic antibody.
  • Laminins are glycoproteins of the mammalian extracellular matrix. Laminins are heterotrimeric proteins made up of alpha, beta, and gamma chains. They are major constituents of the basal lamina and function in cell differentiation, migration, and adhesion. LAMA2 is the alpha 2 chain of laminin-2.
  • the anti-LAMA2 antibody moiety described herein can specifically bind to LAMA2 derived from any source, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the LAMA2 is human LAMA2.
  • the anti-LAMA2 antibody moiety binds to human LAMA2 only and does not bind to LAMA2 derived from another species.
  • the anti-LAMA2 antibody moiety has cross-species reactivity to LAMA2 other than human LAMA2, such as mouse or cynomolgus monkey LAMA2.
  • Antibody moieties with cross- species reactivity can help extrapolate animal data to human clinical trials.
  • the anti-LAMA2 antibody moiety can be of any antibody format.
  • the anti-LAMA2 antibody moiety comprises a VH and a VL.
  • an anti-LAMA2 antibody moiety comprising: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)) within a reference VH having the amino acid sequence set forth in SEQ ID NO: 49, and
  • an anti-LAMA2 antibody moiety comprising a VH and a VL, wherein: (i) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and (ii) the VH comprises an H-CDR1 comprising the
  • an anti-LAMA2 antibody moiety comprising a VH comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 49; and a VL comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence homology to SEQ ID NO: 50.
  • the one or more amino acid variations are in one or more of the CDRs.
  • the one or more amino acid variations are in one or more 157 sf-6122278 Attorney Docket No.: 340742000140 of the framework regions (FRs).
  • the two or more amino acid variations are in both CDRs and FRs.
  • an anti-LAMA2 antibody moiety comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 49, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 50.
  • the anti-LAMA2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49, and a VL comprising the amino acid sequence of SEQ ID NO: 50.
  • an anti-LAMA2 antibody moiety comprising: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)) within a reference VH having the amino acid sequence set forth in any one of SEQ ID NOs:
  • an anti-LAMA2 antibody moiety comprising a VH and a VL, wherein: (i) the VH comprises an H-CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 7, 13, 16, 19, 22, 25, 28, 31, and 34, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); an H-CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 8, 14, 17, 20, 23, 26, 29, 32, and 35, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and an H-CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 9, 15, 18, 21, 24, 27,
  • an anti-LAMA2 antibody moiety comprising a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to any one of SEQ ID NOs: 54-64; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence homology to any one of SEQ ID NOs: 65-73.
  • the one or more amino acid variations are in one or more of the CDRs. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the framework regions (FRs). In some embodiments, the two or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in both CDRs and FRs.
  • an anti-LAMA2 antibody moiety comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 7, 13, 16, 19, 22, 25, 28, 31, and 34, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 8, 14, 17, 20, 23, 26, 29, 32, and 35, an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 9, 15, 18, 21, 24, 27, 30, 33, and 36, an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 10, 37, 40, 43, and 46, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 11, 38, 41, 44, and 47, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 12, 39, 42, 45, and 48.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID 160 sf-6122278 Attorney Docket No.: 340742000140 NO: 72.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, 161 sf-6122278 Attorney Docket No.: 340742000140 and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-LAMA2 antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti- LAMA2 antibody moiety comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, 162 sf-6122278 Attorney Docket No.: 340742000140 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, 163 sf-6122278 Attorney Docket No.: 340742000140 HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1 (SEQ ID NO: 7), HC- CDR2 (SEQ ID NO: 26), and HC-CDR3 (SEQ ID NO: 9) of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1 (SEQ ID NO: 10), LC-CDR2 (SEQ ID NO: 11), and LC-CDR3 (SEQ ID NO: 12) of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL 164 sf-6122278 Attorney Docket No.: 340742000140 comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC- 165 sf-6122278 Attorney Docket No.: 340742000140 CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC- 166 sf-6122278 Attorney Docket No.: 340742000140 CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, 167 sf-6122278 Attorney Docket No.: 340742000140 and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-LAMA2 antibody moiety comprises HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid 168 sf-6122278 Attorney Docket No.: 340742000140 sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti- LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC- CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-LAMA2 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73.
  • the anti-LAMA2 antibody moiety comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73.
  • the anti-LAMA2 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12.
  • the anti-LAMA2 antibody moiety comprises a VH selected from any of the exemplary VHs in Table 2 and a VL selected from any of the exemplary VLs in Table 3.
  • the anti-LAMA2 antibody moiety comprises a VH paired with a VL set forth in Table 4.
  • the anti-LAMA2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising the amino acid sequence of SEQ ID NO: 65.
  • the anti-LAMA2 antibody moiety is an anti-LAMA2 scFv.
  • the anti-LAMA2 scFv comprises from N’ to C’: VH-optional linker- VL.
  • the anti-LAMA2 scFv comprises from N’ to C’: VL-optional 173 sf-6122278 Attorney Docket No.: 340742000140 linker-VH. Any suitable linker (e.g., see “Linker” subsection below) can be used herein, including but are not limited to SEQ ID NO: 212.
  • the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 51.
  • the anti- LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145 or 268.
  • the anti-LAMA2 antibody moiety is an anti-LAMA2 Fab.
  • the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 52, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 55-64, and a second polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 65-73.
  • Anti-Matriglycan Antibody Moiety [0392] In some embodiments, the first binding moiety comprises an anti-matriglycan antibody moiety which specifically binds to matriglycan on ⁇ -dystroglycan ( ⁇ -DG).
  • any of the anti- ⁇ -DG antibody moieties described herein can be used in the bifunctional protein constructs described herein.
  • Means for binding to matriglycan described herein can be any of the anti-matriglycan antibody moieties described herein and functional equivalents thereof.
  • the present application also provides novel anti-matriglycan antibody moieties which can be used for purposes beyond being a component of the bifunctional protein constructs described herein.
  • an anti-matriglycan antibody construct comprising (or consisting essentially of, or consisting of) any of the anti-matriglycan antibody moieties described herein.
  • the antibody is monospecific.
  • the antibody is multispecific (such as bispecific).
  • Multispecific antibodies have binding specificities for at least two different antigens or epitopes.
  • the antibody is monoclonal.
  • the antibody is polyclonal.
  • the antibody is ChIP-grade, or at least IP grade or western blot grade.
  • the antibody can react with protein of interest from any organism, such as human, mouse, rabbit, rat, monkey, fruit fly, zebrafish, chicken, worms, bacteria, etc.
  • the antibody is chimeric, mouse, rabbit, rat, sheep, human, partially humanized, fully humanized, semi-synthetic, or fully-synthetic antibody.
  • ⁇ -dystroglycan a laminin-binding protein and a component of the dystrophin associated glycoprotein complex (DGC), is uniquely modified on O-mannose sites by alternating xylose and glucuronic acid subunits, called matriglycan.
  • Matriglycan is a 174 sf-6122278 Attorney Docket No.: 340742000140 receptor for laminin-G domain-containing proteins. Matriglycan links ⁇ -DG to the basement membrane.
  • the anti-matriglycan antibody moiety described herein can specifically bind to matriglycan derived from any source, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the matriglycan is human matriglycan.
  • the anti-matriglycan antibody moiety binds to human matriglycan only and does not bind to matriglycan derived from another species.
  • the anti-matriglycan antibody moiety has cross-species reactivity to matriglycan other than human matriglycan, such as mouse or cynomolgus monkey matriglycan. Antibody moieties with cross-species reactivity can help extrapolate animal data to human clinical trials.
  • the anti-matriglycan antibody moiety can be of any antibody format. In some embodiments, the anti-matriglycan antibody moiety comprises a VH and a VL.
  • the anti-matriglycan antibody moiety comprises: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)) within a reference VH having the amino acid sequence set forth in any one of SEQ ID NOs: 89-
  • the anti-matriglycan antibody moiety comprising a VH and a VL
  • the VH comprises an H-CDR1 comprising the amino acid sequence of 175 sf-6122278 Attorney Docket No.: 340742000140 SEQ ID NO: 74 or 80, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 75 or 81, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 76 or 82, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (
  • the anti-matriglycan antibody moiety comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to any one of SEQ ID NOs: 89-93; and a VL comprising the amino acid sequence of SEQ ID NO: 94-98, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence homology to any one of SEQ ID NOs: 94-98.
  • the one or more amino acid variations are in one or more of the CDRs. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the framework regions (FRs). In some embodiments, the two or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in both CDRs and FRs.
  • the anti-matriglycan antibody moiety comprises an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 74 or 80, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75 or 81, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76 or 82, an LC-CDR1 comprising the amino acid 176 sf-6122278 Attorney Docket No.: 340742000140 sequence of any one of SEQ ID NO: 79, 83, and 86, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 78, 84, and 87, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 79, 85, and 88.
  • the anti- matriglycan antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, and LC-CDR1, LC- CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98.
  • the anti-matriglycan antibody moiety comprises an HC- CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 89, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 94.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC- 177 sf-6122278 Attorney Docket No.: 340742000140 CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC- CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-matriglycan antibody moiety comprising an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98.
  • an anti-matriglycan antibody moiety comprising a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98.
  • the anti-matriglycan antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising 178 sf-6122278 Attorney Docket No.: 340742000140 the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79.
  • an anti-matriglycan antibody moiety comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 83, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 85.
  • the anti-matriglycan antibody moiety comprises a VH selected from any of the exemplary VHs in Table 5 and a VL selected from any of the exemplary VLs in Table 6.
  • Table 5 Exemplary anti-ADG41 VH
  • Table 6 Exemplary anti-ADG4 VL
  • an anti-matriglycan antibody moiety comprising a VH paired with a VL set forth in Table 7.
  • an anti-matriglycan antibody moiety comprising a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 95.
  • the anti-matriglycan antibody moiety is an anti- matriglycan scFv.
  • the anti-matriglycan scFv comprises from N’ to C’: VH-optional linker-VL.
  • the anti-matriglycan scFv comprises from N’ to C’: VL-optional linker-VH.
  • Any suitable linker e.g., see “Linker” subsection below can be used herein, including but are not limited to SEQ ID NO: 212.
  • the anti-matriglycan antibody moiety comprises the amino acid sequence of SEQ ID NO: 99.
  • the anti-matriglycan antibody moiety is an anti- matriglycan Fab.
  • the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.
  • Anti-CDH15 Antibody Moiety [0406]
  • the first binding moiety comprises an anti-CDH15 antibody moiety which specifically binds to CDH15. Any of the anti-CDH15 antibody moieties described herein can be used in the bifunctional protein constructs described herein.
  • Means for binding to CDH15 described herein can be any of the anti-CDH15 antibody moieties described herein and functional equivalents thereof.
  • the present application also 180 sf-6122278 Attorney Docket No.: 340742000140 provides novel anti-CDH15 antibody moieties which can be used for purposes beyond being a component of the bifunctional protein constructs described herein.
  • Cadherin-15 (CDH15) is a member of the cadherin superfamily. Cadherins consist of an extracellular domain containing five cadherin domains, a transmembrane region, and a conserved cytoplasmic domain. CDH15 may be essential for myogenesis and may serve as a trigger for terminal muscle cell differentiation.
  • the anti-CDH15 antibody moiety described herein can specifically bind to CDH15 derived from any source, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the CDH15 is human CDH15.
  • the anti-CDH15 antibody moiety binds to human CDH15 only and does not bind to CDH15 derived from another species.
  • the anti-CDH15 antibody moiety has cross-species reactivity to CDH15 other than human CDH15, such as mouse or cynomolgus monkey CDH15.
  • Antibody moieties with cross- species reactivity can help extrapolate animal data to human clinical trials.
  • the anti-CDH15 antibody moiety can be of any antibody format.
  • the anti-CDH15 antibody moiety comprises a VH and a VL.
  • the anti-CDH15 antibody moiety comprises: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)), and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)) within a reference VH having the amino acid sequence set forth in SEQ ID NO: 108, and ii)
  • the anti-CDH15 antibody moiety comprising a VH and a VL
  • the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions); and (ii)
  • the anti-CDH15 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 108; and a VL comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence homology to SEQ ID NO: 109.
  • the one or more amino acid variations are in one or more of the CDRs. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the framework regions (FRs). In some embodiments, the two or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in both CDRs and FRs.
  • the anti-CDH15 antibody moiety comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, an HC-CDR2 comprising the 182 sf-6122278 Attorney Docket No.: 340742000140 amino acid sequence of SEQ ID NO: 103, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
  • the anti- CDH15 antibody moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 108, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 109.
  • the anti-CDH15 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 108, and a VL comprising the amino acid sequence of SEQ ID NO: 109.
  • the anti-CDH15 antibody moiety is an anti-CDH15 scFv.
  • the anti-CDH15 scFv comprises from N’ to C’: VH-optional linker- VL. In some embodiments, the anti-CDH15 scFv comprises from N’ to C’: VL-optional linker-VH. Any suitable linker (e.g., see “Linker” subsection below) can be used herein, including but are not limited to SEQ ID NO: 212. In some embodiments, the anti-CDH15 scFv comprises the amino acid sequence of SEQ ID NO: 110. [0416] In some embodiments, the anti-CDH15 antibody moiety is an anti-CDH15 Fab.
  • the anti-CDH15 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 111, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 112.
  • the first binding moiety is a non-antibody binding moiety specifically binding to the muscle-specific molecule, for example a protein domain (e.g., a laminin G-like domain (LG domain)) of an extracellular matrix (ECM) protein that can bind to matriglycan.
  • ECM extracellular matrix
  • Means for binding to a muscle-tissue specific molecule described herein can be any of the non-antibody binding moieties described herein and functional equivalents thereof.
  • the non-antibody moiety comprises a protein domain selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, an LG domain of perlecan, the laminin coiled-coil binding domain of agrin, and the laminin ⁇ binding domain of nidogen.
  • the non-antibody moiety provided herein comprises an LG domain of laminin.
  • the non-antibody moiety comprises an LG domain of any one of laminin subunit ⁇ -1 (LAMA1), LAMA2, laminin subunit ⁇ -3 (LAMA3), laminin subunit ⁇ -4 (LAMA4), or laminin subunit ⁇ -5 (LAMA5).
  • the non-antibody moiety comprises the amino acid sequence of any of SEQ ID NOs: 118-124.
  • the non-antibody moiety is a variant comprising a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NOs: 118-124.
  • the non- antibody moiety comprises an LG domain of LAMA2. In some embodiments, the non- antibody moiety comprises LG4-5 domains of LAMA2 (LAMA2 LG4-5). In some embodiments, the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119. [0420] In some embodiments, the non-antibody moiety comprises an LG domain of perlecan. In some embodiments, the non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 120. [0421] In some embodiments, the non-antibody moiety comprises an LG domain of agrin. In some embodiments, the non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 121.
  • the non-antibody moiety comprises a laminin coiled-coil binding domain of agrin.
  • the laminin coiled-coil binding domain of agrin comprises the amino acid sequence of SEQ ID NO: 122.
  • the non-antibody moiety comprises a laminin ⁇ , ⁇ , and/or ⁇ binding domain of agrin.
  • the non-antibody moiety comprises amino acids 1-271 of human agrin.
  • the non-antibody moiety comprises a laminin ⁇ binding domain of nidogen.
  • the laminin ⁇ binding domain of nidogen comprises the amino acid sequence of SEQ ID NO: 123 or 124.
  • the non-antibody moiety comprises a C-terminal domain of human nidogen-1.
  • the C-terminal domain of human nidogen-1 comprises amino acids 390-1247 of human nidogen-1.
  • the non-antibody moiety comprises a C-terminal domain of human nidogen-2.
  • the C-terminal domain of human nidogen-2 comprises amino acids 515-1372 of human nidogen-2. 184 sf-6122278 Attorney Docket No.: 340742000140 B.
  • Notch signaling pathways play critical roles in cell-fate decision and differentiation in many tissues during embryonic and postnatal development. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of Duchenne muscular dystrophy (DMD) is the progressive depletion of satellite cells, leading to the failure of muscle repair. Satellite cells from mdx mice, a DMD mouse model, were found to have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal (see, e.g., Bjornson, et al., Stem Cells.
  • DMD Duchenne muscular dystrophy
  • Notch intracellular C-terminal domain inhibits myoblast differentiation and muscle injury repair (see, e.g., Wen, et al., Mol Cell Biol. 2012, 32(12):2300-11).
  • NBD Notch intracellular C-terminal domain
  • Notch receptors are activated by single-pass transmembrane ligands of the Delta, Serrated, Lag-2 (DSL) family, including Delta-like 1 (DLL1; e.g., GenBank Accession No. NP005609), Delta-like 3 (DLL3; e.g., GenBank Accession Nos. NP_982353.1 or NP_058637.1), Delta-like 4 (DLL4; e.g., GenBank Accession No. NP_061947.1), Jagged-1 (Jag1; e.g., GenBank Accession No. AAC51731), and Jagged-2 (Jag2; e.g., GenBank Accession No. AAD15562).
  • DLL1 Delta-like 1
  • DLL3 Delta-like 3
  • DLL4 Delta-like 4
  • Jagged-1 Jagged-1
  • Jag2 e.g., GenBank Accession No. AAD15562
  • Jagged-2 Jagged-2
  • the ligands are characterized by an N-terminus of Notch ligand (MNNL) region, a Delta/Serrate/LAG-2 (DSL) domain, and tandem epidermal growth factor (EGF)-like repeats within the extracellular domain.
  • MNNL N-terminus of Notch ligand
  • DSL Delta/Serrate/LAG-2
  • EGF tandem epidermal growth factor
  • the extracellular domains of the Notch receptor and ligands interact leading to cleavage of Notch by furin-like convertase, ADAM (a disintegrin and metalloproteinase) metalloprotease, and/or a gamma secretase.
  • the gamma- secretase-mediated cleavage generates and releases the Notch intracellular domain (NICD), 185 sf-6122278 Attorney Docket No.: 340742000140 which translocates into the nucleus, where NICD binds to the transcription factor CSL (CBF1, Suppressor of Hairless, Lag-1) to induce expression of downstream target genes.
  • CBF1 transcription factor CSL
  • DLL1, DLL3, and DLL4 are members of the Delta family of Notch ligands.
  • DLL1 is a type I cell surface protein composed of an MNNL domain, DSL domain, and eight EGF-like repeats within the extracellular domain.
  • the first two EGF-like domains of DLL1 are unusual in that they have very short loop sequences that resemble a motif seen in the OSM-11 protein in C. elegans.
  • DLL1 contains a PDZ-ligand domain on the cytosolic side of the ligand.
  • the MNNL domain is amino acids 18-176
  • the DSL domain is amino acids 177- 221
  • EGF-like repeat 1 is amino acids 226-254
  • EGF-like repeat 2 is amino acids 257-285
  • EGF-like repeat 3 is amino acids 292-325
  • EGF-like repeat 4 is amino acids 332-363
  • EGF-like repeat 5 is amino acids 370-402
  • EGF-like repeat 6 is amino acids 409-440
  • EGF-like repeat 7 is amino acids 447-478
  • EGF-like repeat 8 is amino acids 485-516 of human DLL1.
  • DLL3 is the shortest of the mammalian Delta-like ligands, containing an MNNL domain, DSL domain, and six EGF-like repeats within the extracellular domain.
  • the MNNL domain is amino acids 27-175, the DSL domain is amino acids 176-215, EGF-like repeat 1 is amino acids 216-249, EGF-like repeat 2 is amino acids 274-310, EGF-like repeat 3 is amino acids 312-351, EGF-like repeat 4 is amino acids 353-389, EGF-like repeat 5 is amino acids 391-427, and EGF-like repeat 6 is amino acids 429-465 of human DLL3. See, e.g., Ladi et al., J Cell Biol., 2005, 170(6): 983–992.
  • DLL4 is a type I cell surface protein composed of an MNNL domain, DSL domain, and eight EGF-like repeats within the extracellular domain.
  • DLL4 contains a PDZ- ligand domain on the cytosolic side of the ligand.
  • the MNNL domain is amino acids 27-172
  • the DSL domain is amino acids 173-217
  • EGF-like repeat 1 is amino acids 218-251
  • EGF-like repeat 2 is amino acids 252-282
  • EGF-like repeat 3 is amino acids 284-322
  • EGF-like repeat 4 is amino acids 324-360
  • EGF-like repeat 5 is amino acids 362-400
  • EGF-like repeat 6 is amino acids 402-438
  • EGF-like repeat 7 is amino acids 440-476
  • EGF-like repeat 8 is amino acids 480-518 of human DLL4.
  • the Serrate family differs from the Delta family in that the Serrate family contain a cysteine-rich domain 186 sf-6122278 Attorney Docket No.: 340742000140 (CRD) following the EGF-like repeats.
  • Both Jag1 and Jag2 are composed of an MNNL domain, DSL domain, 16 EGF-like repeats, and a CRD domain within the extracellular domain.
  • the first two EGF-like domains of Jag1 and Jag2 are unusual in that they have very short loop sequences that resemble a motif seen in the OSM-11 protein in C. elegans.
  • Jag1 contains a PDZ-ligand domain on the cytosolic side of the ligand.
  • the MNNL domain is amino acids 34-184
  • the DSL domain is amino acids 185-229
  • EGF-like repeat 1 is amino acids 230-263
  • EGF-like repeat 2 is amino acids 264-294
  • EGF-like repeat 3 is amino acids 296-334
  • EGF-like repeat 4 is amino acids 336-372
  • EGF-like repeat 5 is amino acids 374-410
  • EGF-like repeat 6 is amino acids 412- 448
  • EGF-like repeat 7 is amino acids 450-485
  • EGF-like repeat 8 is amino acids 487-523
  • EGF-like repeat 9 is amino acids 525-561
  • EGF-like repeat 10 is amino acids 586-627
  • EGF- like repeat 11 is amino acids 629-665
  • EGF-like repeat 12 is amino acids 667-703
  • EGF-like repeat 16 is amino acids 820-856, and the CRD domain is amino acids 857-1067.
  • the MNNL domain is amino acids 27-195
  • the DSL domain is amino acids 196-240
  • EGF-like repeat 1 is amino acids 241-274
  • EGF-like repeat 2 is amino acids 275-305
  • EGF-like repeat 3 is amino acids 307-345
  • EGF-like repeat 4 is amino acids 347-383
  • EGF-like repeat 5 is amino acids 385-421
  • EGF-like repeat 6 is amino acids 423- 459
  • EGF-like repeat 7 is amino acids 461-496
  • EGF-like repeat 8 is amino acids 498-534
  • EGF-like repeat 9 is amino acids 536-572
  • EGF-like repeat 10 is amino acids 574-634
  • EGF-like repeat 11 is amino acids 636-672
  • EGF-like repeat 12 is amino acids 674-710
  • EGF-like repeat 13 is amino acids 712-748
  • the second binding moiety of any of the bifunctional protein construct provided herein comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).
  • Means for binding to a Notch receptor described herein can be any of the Notch ligand ECD or variant described herein and functional equivalents thereof.
  • the 187 sf-6122278 Attorney Docket No.: 340742000140 ECD or a variant thereof of a Notch ligand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 EGF-like domains.
  • the DLL1 ECD comprises EGF1-8 domains.
  • the DLL3 ECD comprises EGF1-6 domains.
  • the DLL4 ECD comprises EGF1-8 domains.
  • the Jag1 ECD comprises EGF1-16 domains.
  • the Jag2 ECD comprises EGF1-16 domains.
  • the ECD or variant thereof of a Notch ligand comprises a C2(MNNL) domain.
  • the ECD or variant thereof of a Notch ligand comprises a DSL domain. In some embodiments, the ECD or variant thereof of a Notch ligand comprises EGF1-3 domains, EGF1-4 domains, EGF1-4 domains, EGF1-5 domains, or EGF- 1-6 domains. In some embodiments, the ECD or a variant thereof of a Notch ligand comprises EGF1-5 domains.
  • the ECD or variant thereof of a Notch ligand comprises a C2(MNNL)-DSL-EGF1-3, C2(MNNL)-DSL-EGF1-4, C2(MNNL)-DSL- EGF1-5, or C2(MNNL)-DSL-EGF1-6 domain.
  • the non-antibody moiety binds to a human Notch receptor described herein can be any of the Notch ligand ECD or variant described herein and functional equivalents thereof.
  • the non-antibody moiety has cross- species reactivity to a Notch receptor described herein can be any of the Notch ligand ECD or variant described herein and functional equivalents thereof, such as a mouse, rat, or cynomolgus monkey non-antibody moiety.
  • Antibody moieties with cross-species reactivity can help extrapolate animal data to human clinical trials.
  • the present application provides engineered DLL ECDs, which can be incorporated into the bifunctional proteins described here.
  • an engineered DLL4 ECD wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N26.
  • the further mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D.
  • the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (i) G2S, T52N, F81L, T135N, and H168Y; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y
  • the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264. [0435] In some embodiments, there is provided an engineered DLL4 ECD, wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of G2, E14, T52, R66, P80, F81, T135, H168, Q220, N231, and N260, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO.
  • the mutation is selected from the group consisting of G2S, E14H, T52N, R66S, R66T, P80L, F81L, T135N, H168Y, Q220H, N231D, and N260D, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126.
  • the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) G2S, E14H, T52N, F81L, R66T, P80L, T135N, H168Y, and N231D; and (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N231D;
  • the second binding moiety comprises a DLL4 ECD or a variant thereof.
  • the second binding moiety comprises a DLL4 ECD, and optionally wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125.
  • the second binding moiety comprises a variant DLL4 ECD, and optionally wherein the variant DLL4 ECD comprises an MNNL domain, a DSL domain, and/or an EGF1-5 domains of DLL4.
  • the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267, or a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NOs: 126-129 and 260-267.
  • the variant DLL4 ECD comprises no more any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in reference to SEQ ID NO: 126-129 and 260-267.
  • the present invention also provides a protein construct comprising any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267).
  • the protein construct is a bifunctional protein construct, such as any of the bifunctional protein constructs described herein comprising an engineered DLL4 ECD.
  • the protein construct further comprises a 189 sf-6122278 Attorney Docket No.: 340742000140 binding moiety that specifically binds to a muscle-specific molecule. In some embodiments, the binding moiety that specifically binds to a muscle-specific molecule.
  • the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina.
  • the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M- cadherin (CDH15).
  • the second binding moiety comprises a DLL1 ECD or a variant thereof.
  • the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD of DLL1 comprises the amino acid sequence of SEQ ID NO: 130.
  • the second binding moiety comprises a variant DLL1 ECD, and optionally wherein the variant DLL1 ECD comprises a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NOs: 130.
  • the variant DLL1 ECD comprises no more any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in reference to SEQ ID NO: 130.
  • the second binding moiety comprises a DLL3 ECD or a variant thereof.
  • the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.
  • the second binding moiety comprises a variant DLL3 ECD, and optionally wherein the variant DLL3 ECD comprises a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 131.
  • the variant DLL3 ECD comprises no more any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in reference to SEQ ID NO: 131.
  • the second binding moiety comprises a Jag1 ECD or a variant thereof.
  • the second binding moiety comprises a Jag1 ECD, and wherein the Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 132.
  • the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises MNNL domain, DSL domain, and EGF1-6 domains of Jag1, or a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the sequence of MNNL domain, DSL domain, and EGF1-6 domains of Jag1.
  • the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.
  • the second binding moiety comprises a Jag2 ECD or a variant thereof.
  • the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.
  • the second binding moiety comprises a variant of Jag2 ECD, and optionally wherein the Jag2 ECD variant comprises a sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 135.
  • the Jag2 ECD variant comprises no more any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in reference to SEQ ID NO: 135.
  • the non-antibody moiety further comprises an aptamer, a SOMAmer, a DARpin, or other engineered binding molecules or scaffolds.
  • the Notch activating moiety is conjugated to the 5’ or 3’ end of the aptamer or SOMAmer by chemical crosslinking.
  • Anti-Notch Antibody Moiety [0443]
  • the second binding moiety is an anti-Notch antibody moiety that activates the Notch receptor.
  • Anti-Notch antibodies capable of activating the Notch receptor are known in the art, including, for example Asano et al., J Immunol., 180(5):2796-804 (2008); Hoare et al., Nat Cell Biol., 18(9):979-92 (2016); Falk et al., Methods., 58(1):69-78 (2012); Thermo Fisher Scientific Cat.
  • the anti-Notch antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a F(ab’)2, an scFv, and an sdAb.
  • the first binding moiety, the second binding moiety, and other components of bifunctional protein constructs described herein can be connected via one or more optional linkers.
  • the domains within the first binding moiety can be connected via one or more optional linkers.
  • the domains within the second binding moiety can be connected via one or more optional linkers.
  • the linker is a peptide linker.
  • the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a 191 sf-6122278 Attorney Docket No.: 340742000140 sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/034103.
  • the peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more, amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer, amino acids long.
  • the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.
  • the peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’Acqua et al.
  • the peptide linker does not promote the formation of any secondary structures.
  • the linkage of the domains to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well-known in the art (e.g. WO1999/054440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y.
  • the peptide linker can be a stable linker, which is not cleavable by proteases, especially by Matrix metalloproteinases (MMPs).
  • MMPs Matrix metalloproteinases
  • the peptide linker is a flexible linker.
  • Exemplary flexible linkers include glycine polymers (G)n, where n is an integer of at least one, and glycine- serine polymers (including, for example, (GS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • the linker is a GS linker.
  • the peptide linker comprises the amino acid sequence of LE (SEQ ID NO: 222) or the amino acid sequence of any one of SEQ ID NOs: 207-221, 337 192 sf-6122278 Attorney Docket No.: 340742000140 and 338.
  • the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.
  • the bifunctional protein constructs and binding moieties e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • binding moieties e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • Methods of making proteins constructs and constructing nucleic acids or vectors encoding thereof, as well as purifying protein constructs are known in the art. Also see US20220204602 and Examples 1 and 2 as described herein for exemplary methods.
  • Proteins comprising antibody moieties can be obtained using methods known in the art, such as by immunizing a non-human mammal and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and subsequence selection or by using phage display. Nucleic acid constructs encoding any one of the first binding moieties, second binding moieties, or bifunctional protein constructs described herein, vectors, and host cells for preparation are also provided.
  • polypeptide portion of the bifunctional protein constructs or binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein may be prepared by any of the known protein expression and purification methods in the art.
  • DNA sequence encoding the polypeptide portion of the bifunctional protein constructs or binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) can be fully synthesized. After obtaining such sequence, it is cloned into a suitable expression vector, then transfected into a suitable host cell.
  • the transfected host cells are cultured, and the supernatant is harvested and purified to obtain the polypeptide portion of the bifunctional protein constructs or binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein.
  • binding moieties e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • the antibody moieties described herein can also be obtained by conventional immunization methods, such as by immunizing a mammal (e.g., mouse, llama), and obtaining the antibody 193 sf-6122278 Attorney Docket No.: 340742000140 moieties from the serum; or from a hybridoma, such as by fusing B cells from lymph nodes and/or spleen of immunized animal with myeloma cells. Purification can be followed.
  • the expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector.
  • Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter.
  • Enhancer sequences may be selected to enhance the transcription of the nucleic acids.
  • Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.
  • the two or more polypeptides of a bifunctional protein construct or a binding moiety are encoded by a single vector.
  • the two or more polypeptides of a bifunctional protein construct or a binding moiety are encoded by two or more vectors.
  • the two or more polypeptides of a functional protein construct or a binding moiety are Host cells containing the vector may be useful in expression or cloning of the isolated nucleic acids.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • binding moieties e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • prokaryotic cells such as E. coli is well established in the art.
  • Suitable higher eukaryotic cells include, without 194 sf-6122278 Attorney Docket No.: 340742000140 limitation, invertebrate cells and insect cells, and vertebrate cells.
  • the host cell is E. coli.
  • the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.
  • the vector can be introduced to the host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.
  • the host cells comprise two or more vectors each encoding a polypeptide of any of the protein constructs or targeting moieties described herein.
  • the host cells comprise a single vector comprising isolated nucleic acids encoding two or more polypeptides of any of the bifunctional protein constructs or binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein.
  • the two or more vectors are introduced to the host cell at the same time. In some embodiments, the two or more vectors are introduced to the host cell sequentially.
  • the present application provides methods of making the polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein, comprising i) culturing an isolated host cell comprising any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the isolated host cells described herein, under a condition suitable for the expression of the polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein, and ii) obtaining the expressed polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein from said
  • the method further comprises introducing one or more isolated nucleic acids or one or more vectors into the host cell.
  • the isolated host cells are cultured under conditions that allow expression of the isolated nucleic acids inserted in the vectors.
  • Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of 195 sf-6122278 Attorney Docket No.: 340742000140 supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants.
  • suitable conditions as appropriate for the purpose of the expression.
  • the methods of making further comprises purifying any of the obtained polypeptide portion of any of the binding (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) moieties or bifunctional protein constructs described herein.
  • the polypeptides expressed by the host cell can assemble together (e.g., form a polypeptide complex such as a dimer) and produce any of the polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein.
  • the polypeptide complex may be formed inside the host cell.
  • the polypeptide complex may be formed inside the host cell with the aid of relevant enzymes and/or cofactors.
  • the polypeptide complex may be secreted out of the cell.
  • individual polypeptides may be secreted out of the host cell then form a polypeptide complex outside of the host cell.
  • the two or more polypeptides of any of the binding moieties may be separately expressed and allowed to form (e.g., dimerize) a protein complex under suitable conditions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer and allow the first protein monomer and the second protein monomer to dimerize through appropriate interactions such as hydrophobic interactions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide and the second polypeptide.
  • the first polypeptide and the second polypeptide may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.
  • the expressed polypeptide(s) and/or the polypeptide complex can be collected using any suitable methods.
  • the polypeptide(s) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium.
  • the host cells containing the polypeptide and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the 196 sf-6122278 Attorney Docket No.: 340742000140 unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or the polypeptide complex is secreted into periplasmic space of E.
  • the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)).
  • agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)).
  • the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor and/or an antibiotic may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated microorganisms.
  • the expressed polypeptide(s) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor & Francis.
  • a suitable method such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC,
  • polypeptides and/or polypeptide complexes can be purified by affinity chromatography.
  • protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)); Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010).
  • protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgG ⁇ 3 heavy chain (Guss et al., EMBO J. 5:15671575 (1986)).
  • protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising ⁇ light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. et al., J. Biol. Chem., 267, 2234-2239 (1992)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • the method further comprises analyzing the purified protein product, such as by SDS-PAGE and/or SEC-HPLC.
  • nucleic Acids and Vectors [0464] In one aspect, provided herein is one or more nucleic acids (e.g., isolated) encoding one or more polypeptides of the bifunctional protein constructs, or various binding moieties described herein.
  • the one or more nucleic acids can be RNA (e.g., mRNA) or DNA.
  • the one or more nucleic acids are mRNA(s) and the mRNA(s) is formulated in liposomes or lipid nanoparticles.
  • vectors comprising any of the nucleic acids (e.g., isolated nucleic acids) described herein, such as viral vectors (e.g., AAV vector or lentiviral vector).
  • isolated host cell comprising or expressing (e.g., one or more polypeptide components of) any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein.
  • isolated host cells comprising one or more nucleic acids encoding (e.g., one or more polypeptide components of) any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein.
  • isolated host cells comprising one or more vectors comprising one or more nucleic acids encoding (e.g., one or more polypeptide components of) any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs described herein.
  • one vector comprises one or more isolated nucleic acids encoding the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct.
  • one vector comprises two or more isolated nucleic acids encoding two or more polypeptide components of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct.
  • two or more vectors comprise two or more isolated nucleic acids encoding two or more polypeptide components of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and 198 sf-6122278 Attorney Docket No.: 340742000140 protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct.
  • the two or more isolated nucleic acids encoding two or more polypeptide components of the binding moieties are under the control of the same promoter on the same vector (e.g., linked via IRES, or nucleic acid encoding self-cleaving 2A peptide such as P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A in between), the same promoter on different vectors (e.g., both TetOn), different promoters on the same vector, or different promoters on different vectors (e.g., one is inducible, one is constitutive).
  • the same promoter on the same vector e.g., linked via IRES, or nucleic acid encoding self-cleaving 2A peptide such as P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A in between
  • TetOn TetOn
  • different promoters on the same vector e.g., one is in
  • the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.
  • the present application also includes variants to these nucleic acid sequences.
  • the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the bifunctional protein constructs or various binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein under at least moderately stringent hybridization conditions.
  • the variants to nucleic acid sequences comprises degenerate nucleotide codes. Those skilled in the art can determine the different nucleotides comprising degenerate codons.
  • the present application also provides vectors in which a nucleic acid of the present application is inserted.
  • the nucleic acid(s) can be cloned into a number of types of vectors.
  • the nucleic acid(s) can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; 199 sf-6122278 Attorney Docket No.: 340742000140 WO 01/29058; and U.S. Pat. No. 6,326,193).
  • the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.
  • AAV adeno-associated virus
  • the nucleic acid encoding the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct comprises sequences or features in addition to the coding sequence encoding the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct.
  • the nucleic acid can be an mRNA (or a DNA sequence encoding the mRNA) comprising optional features that improve targeting of the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct encoded by the mRNA and to increase stability of the mRNA.
  • the nucleic acid(s) encoding polypeptide chain(s) of the bifunctional protein construct comprises a nucleic acid sequence encoding a signal peptide.
  • Signal peptides are short peptides that may be present at the N-terminus or the C-terminus of a newly synthesized protein, that may function to properly translocate the protein.
  • the signal peptide assists with translocating the polypeptide chain(s) of the bifunctional protein construct encoded by the nucleic acid(s).
  • the signal peptide translocates the polypeptide chain(s) of the bifunctional protein construct to the cellular membrane.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 253 or 336. IV.
  • compositions comprising i) any of the bifunctional protein constructs described herein, any of the engineered DLL4 ECD and protein constructs comprising the DLL4 ECD described herein, any of the isolated nucleic acids (DNA or RNA) encoding the bifunctional protein constructs, any of the isolated nucleic acids (DNA or RNA) encoding the engineered DLL4 ECD and protein constructs comprising the DLL4 ECD described herein, or any of the vectors (e.g., viral vector) comprising such isolated nucleic acids; and ii) an optional pharmaceutically acceptable carrier.
  • the vectors e.g., viral vector
  • Suitable formulations (e.g., pharmaceutical composition) of the bifunctional protein construct described herein, a nucleic acid encoding the bifunctional protein construct described herein, a vector (e.g., viral vector) comprising a nucleic acid encoding the 200 sf-6122278 Attorney Docket No.: 340742000140 bifunctional protein construct, the engineered DLL4 ECD and protein constructs comprising the DLL4 ECD described herein, a nucleic acid encoding the engineered DLL4 ECD and protein constructs comprising the DLL4 ECD described herein, a vector (e.g., viral vector) comprising a nucleic acid encoding the engineered DLL4 ECD and protein constructs comprising the DLL4 ECD described herein can be obtained by mixing the bifunctional protein construct (or the nucleic acid encoding the bifunctional protein construct, or the vector (e.g., viral vector) comprising a nucleic acid encoding the bifunctional protein construct), the engineered
  • the pharmaceutical composition further comprises additional ingredients.
  • Additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall (e.g., surfactant, such as polysorbate (e.g., polysorbate 80) or poloxamer).
  • surfactant such as polysorbate (e.g., polysorbate 80) or poloxamer.
  • the excipient is different depending on whether a pharmaceutical composition comprises a protein, nucleic acid, or vector (e.g., viral vector).
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or
  • compositions to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
  • the pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology.
  • Preparations include but are not limited to, bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3- butane diol, for example.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for parenteral, intraperitoneal, intramuscular, intradermal, subcutaneous, or intravenous routes of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a unit dose is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • kits comprising any one of the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein constructs (or the nucleic acid encoding the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct described herein, or the vector comprising a nucleic acid encoding the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) or bifunctional protein construct) described herein.
  • the binding moiety e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • bifunctional protein constructs or the nucleic acid encoding the binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein construct
  • the container holds a composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a sterile access port for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • 203 sf-6122278 Attorney Docket No.: 340742000140 V.
  • Methods of Treatment [0483] Also provided herein are methods of treating a muscle-related disease in an individual (e.g., human), comprising administering to the individual an effective amount of any of the bifunctional protein constructs described herein, or nucleic acid(s) or vector(s) (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof).
  • the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy.
  • a list of muscular dystrophies with associated genetic defects that may be treated by the claimed bifunctional protein constructs are presented in Table 9.
  • Table 9 Muscular Dystrophies with Associated Genetic Defects 204 sf-6122278 Attorney Docket No.: 340742000140 205 sf-6122278 Attorney Docket No.: 340742000140 [0484]
  • the muscle disease is muscular dystrophy.
  • the muscular dystrophy comprises one or more of: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disorder, and Emery-Dreifuss muscular dystrophy (EDMD).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • LGMD limb-girdle muscular dystrophy
  • CMD congenital muscular dystrophy
  • FSHD facioscapulohumeral muscular dystrophy
  • DM myotonic dystrophy
  • OPMD oculopharyngeal muscular dystrophy
  • DD Charcot-Marie-Tooth
  • the limb-girdle muscular dystrophy type is selected from the group consisting of LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2G, LGMD2H, LGMD2I, LGMD2J, LGMD2K, LGMD2L, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2Q, LGMD2R, LGMD2S, LGMD2T, LGMD2U, LGMD2V, LGMD2W, LGMD2X, and LGMD2Y.
  • the myotonic dystrophy type is selected from the group consisting of DM1 and DM2.
  • the Charcot-Marie-Tooth disorder type is selected from the group consisting of Type 1a, Type 1b, and Type X.
  • the muscle disease is sarcopenia.
  • a method of renewing satellite cells in a muscle tissue comprising contacting the muscle tissue with an effective amount of any of the bifunctional protein constructs or binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein, or the vector (e.g., viral vector) comprising a nucleic acid encoding the bifunctional protein constructs or binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein.
  • the bifunctional protein constructs or binding moiety e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • the method allows myoblast differentiation in the muscle tissue.
  • a method of repairing and/or regenerating a muscle tissue comprising contacting the muscle tissue with an effective amount of any of the bifunctional 206 sf-6122278 Attorney Docket No.: 340742000140 protein constructs or binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein, or the vector (e.g., viral vector) comprising a nucleic acid encoding the bifunctional protein constructs or binding moiety (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) described herein.
  • the vector e.g., viral vector
  • the muscle tissue is a damaged muscle tissue.
  • the muscle tissue is from an individual (e.g., human) with a muscle-related disease, such as Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy.
  • a muscle-related disease such as Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy.
  • the treatment methods for muscle-related diseases described herein can achieve one or more following effects: (i) increase or maintain muscle strength compared with a preexisting state or compared with a state which would occur in the absence of treatment, (ii) delay progression of muscular dystrophy, (iii) reduce loss of muscle, (iv) improve or maintain motor skills compared with a preexisting state or compared with a state which would occur in the absence of treatment, (v) repair or maintain muscle cell or tissue, (vi) increase regeneration of muscle cell or tissue compared with a preexisting state or compared with a state which would occur in the absence of treatment, (vii) enhance satellite cell renewal, and/or (viii) increase myoblast differentiation.
  • muscle strength is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared with a preexisting state or compared with a state which would occur in the absence of treatment.
  • motor skills is improved by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared with a preexisting state or compared with a state which would occur in the absence of treatment.
  • regeneration of muscle cell or tissue is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared with a preexisting state or compared with a state which would occur in the absence of treatment.
  • satellite cell renewal is enhanced by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, 207 sf-6122278 Attorney Docket No.: 340742000140 about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared with a preexisting state or compared with a state which would occur in the absence of treatment.
  • myoblast differentiation is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared with a preexisting state or compared with a state which would occur in the absence of treatment.
  • the bifunctional protein construct or binding moiety e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein
  • a nucleic acid or vector e.g., viral vector
  • the bifunctional protein construct, or a nucleic acid or vector (e.g., viral vector) encoding thereof, or pharmaceutical composition thereof is administered to the individual two or more times, for example every week, every two weeks, every three weeks, every month, every two months, every three months, every six months, and the like.
  • the bifunctional protein construct, or a nucleic acid or vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, subcutaneous, transdermal, mucosal, intraperitoneal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Example 1 Generation of an exemplary bifunctional fusion protein comprising mouse DLL4 variant containing C2-DSL and EGF1-5, mIgG1 Fc, and mouse Laminin-211 alpha2 LG4-5 domains (Construct muDLL4v-Fc-muLAMA2 LG4-5)
  • Mouse DLL4 (Accession: AAF76428.1) containing C2-DSL-EGF1-5 (aa 28-401) with rat DLL4EL12 variant mutations (Luca et al., 2015, Science 47(6224):847-53) was used to generate the DLL4v-Fc-LAMA2 LG4-5 construct (SEQ ID NO: 242).
  • the mouse DLL4 variant containing C2-DSL and EGF1-5 was fused to the N-terminus of mouse IgG1 Fc engineered to remove effector function.
  • the C-terminus of the Fc was fused to a (GGGGS) 3 linker (SEQ ID NO: 211) and mouse Laminin-2alpha (Accession: NP_032507.2) LG domains 4-5 (aa 2870-3118) (SEQ ID NO: 250).
  • the whole DNA sequence was assembled with codon optimization for expression in CHO or HEK293 cells.
  • the DNA was cloned into the pcDNA3.x vector (Thermo Fisher Scientific). pcDNA3.x with the cloned construct was transformed into E.
  • coli bacteria DH5 ⁇ competent cells
  • DNA sequence was validated
  • the colony was propagated by an overnight culture
  • 209 sf-6122278 Attorney Docket No.: 340742000140 plasmid DNA was obtained using a commercial plasmid purification kit.
  • pcDNA3.x plasmid DNA was transfected into CHO or HEK293 cells for expression, either transiently or stably after establishing a stable cell pool.
  • Recombinant protein was purified from the cell culture supernatants with a 25 ml MabSelectTM SuReTM column (Cytiva).
  • Proteins were eluted from the column with 0.1 M glycine pH 3.0, and immediately neutralized using 20% volume of 1M Tris pH 8.0. The eluted samples were then buffer exchanged to 1X PBS for monodispersity analysis using analytical size exclusion chromatography (SEC). A second step of purification with preparative SEC in 1X PBS was implemented when the monodispersity of the recombinant protein was below 93%. Purity and integrity of purified proteins were analyzed with analytical HPLC-SEC (FIG. 3B) and SDS-PAGE (FIG. 3A). HPLC-SEC (FIG. 3B) and SDS-PAGE analysis (FIG.
  • Example 2 Generation of an exemplary bifunctional fusion protein comprising mouse DLL4 variant containing C2-DSL and (EGF1-5), mIgG1 Fc, and anti-laminin-2alpha (Construct muDLL4v-Fc-LAMA2scFv)
  • Mouse DLL4 (Accession: AAF76428.1) containing C2-DSL-EGF1-5 (aa 28-401) with rat DLL4EL12 variant mutations (Luca et al., 2015, Science. 47(6224):847-53) was used to generate the DLL4v-Fc-LAMA2scFv construct.
  • the mouse DLL4 variant containing C2- DSL and EGF1-5 was fused to the N-terminus of mouse IgG1 Fc engineered to remove effector function.
  • the C-terminus of the Fc was fuse to a (GGGGS)3 linker and anti-mouse Laminin-2alpha (SEQ ID NO: 251).
  • the whole DNA sequence was assembled with codon optimization for expression in CHO or HEK293 cells, which was cloned into the pcDNA3.x vector (Thermo Fisher Scientific).
  • pcDNA3.x with the cloned construct was transformed into E.
  • coli bacteria DH5 ⁇ competent cells
  • DNA sequence was validated
  • the colony propagated by overnight culture and plasmid DNA was obtained using a commercial plasmid purification kit.
  • pcDNA3.x plasmid DNA was transfected into CHO or HEK293 cells for expression, either transiently or stably after establishing a stable cell pool.
  • Recombinant protein was purified from the cell culture supernatants with a 25 ml 210 sf-6122278 Attorney Docket No.: 340742000140 MabSelectTM SuReTM column (Cytiva).
  • Proteins were eluted from the column with 0.1 M glycine pH 3.0, and immediately neutralized using 20% volume of 1M Tris pH 8.0. The eluted samples were then buffer exchanged to 1X PBS for monodispersity analysis using analytical size exclusion chromatography (SEC). A second step of purification with preparative SEC in 1X PBS was implemented when the monodispersity of the recombinant protein was below 93%. Purity and integrity of purified proteins were analyzed with analytical HPLC-SEC (FIG. 5B) and SDS-PAGE (FIG. 5A). SDS-PAGE (FIG. 5A) and HPLC-SEC (FIG.
  • the recombinant protein was expressed in HEK293 cells as described above and was purified from cell culture supernatant on a Ni-NTA nickel-chelating resin column (ThermoFisher Scientific) using manufacturer’s protocol.
  • the eluent containing the recombinant protein was buffer exchanged into PBS in Slide-A-Lyzer dialysis cassettes (ThermoFisher Scientific) with molecular weight cutoffs (MWCOs) of 10 kDa.
  • Example 4 Confirmation of binding of murine bifunctional constructs to targets [0498] Binding to Notch of the muDLL4v-Fc-LAMA2scFv, muDLL4wt-Fc-LAMA2 LG4-5, and muDLL4v-Fc-LAMA2 LG4-5 constructs was confirmed using a sandwich ELISA with a mouse recombinant Notch1 protein (Sino Biologicals). A 96-well ELISA plate was coated with 2 ug/ml of recombinant Notch 1 protein at 100 ⁇ L/well at 4°C.
  • DLL4v-Fc-LAMA2 LG4-5 and DLL4v-Fc-LAMA2scFv have higher binding affinity to Notch 1, compared to the DLL4wt-Fc-LAMA2 LG4-5.
  • Example 5 Binding of recombinant bifunctional proteins containing murine laminin-2 LG4-5 to matriglycan
  • Matriglycan on alpha-dystroglycan was purified with WGA agarose beads from rabbit muscle homogenate following published methods (Michele et al., 2002, Nature. 418(6896):417-22). Binding between the DLL4v-Fc-LG4-5 construct and matriglycan was analyzed using Biacore T200 (Cytiva). The DLL4v-Fc-LG4-5 construct was captured on anti- Fc chips at a concentration of 10 ug/ml, and TBST buffer was used as a reference for signal subtraction on the same anti-Fc chip.
  • Example 6 Confirmation of binding of murine DLL4v-Fc-LAMA2scFv to recombinant human LG4-5 by Biacore [0500] Binding of the muLL4v-Fc-LAMA2scFv construct was tested as described above. The DLL4v-Fc-LAMA2scFv construct was captured on anti-Fc chips at a concentration of 10 ug/ml, and TBST buffer was used as a reference for signal subtraction on the same anti-Fc chip. Using single cycle kinetics, recombinant LG5 at different concentrations of 0.16 nM, 0.8 nM, 4 nM, 20 nM, and 100 nM were injected to confirm binding.
  • mice were housed 3–5 mice per cage, provided access to food (NIH-31 Open formulation diet; Envigo #7917) and water ad libitum, and enrichment. Mice were maintained on a 12 h light/dark cycle. All animal study protocols were reviewed and approved by the Tufts University’s 212 sf-6122278 Attorney Docket No.: 340742000140 Institutional Animal Care and Use Committee (IACUC) and were conducted accordingly. Male mice of the DBA/2J and D2.mdx backgrounds at 5-6 weeks of age were purchased from the Jackson Laboratory. After about one week of acclimation, mice were trained for forelimb grip strength testing. D2.mdx mice were randomly assigned into control and treatment groups based on forelimb grip strength.
  • D2.mdx male mice at about 7-8 weeks of age were injected with 5 mg/kg of muDLL4v-Fc-LAMA2scFv i.p. (FIG. 9A).
  • D2.mdx male mice of similar age in the control group were injected with 20 mg/kg of an isotype control antibody against trinitrophenol (TNP).
  • TNP trinitrophenol
  • the D2.mdx mice in the control and treatment groups were given injections every three weeks. From the second dose onwards, mice were pretreated with 5 mg/kg of diphenhydramine (i.p.) 10 minutes prior to dosing with the isotype control or DLL4v-Fc-LAMA2scFv to avoid potential anaphylactic reaction.
  • Forelimb grip strength test Grip strength of the forelimbs was assessed using a grid attached to an isometric force transducer (Columbus Instruments, Columbus, OH, USA) according to a previously published protocols (Aartsma-Rus and van Putten, 2014, J Vis Exp. (85):51303) and TREAT-NMD Standard Operation Protocol DMD_M.2.2.001). Briefly, mice were picked up by their tails and their forelimbs were allowed to grab the wire attached to the force transducer on the grip strength meter. Mice were pulled backwards by the tail until they released the wire. The maximum force required to break the mice’s grip from the mesh surface was recorded by the transducer.
  • mice administered DLL4v-Fc-LAMA2scFv was 65% greater than that of mice administered the isotype control (FIG. 9B).
  • the body weights of both control and treatment groups plateaued and were overall less than that of the DBA/2J wildtype mice (FIG. 9C), suggesting that despite low body weight, mice provided the DLL4v-Fc-LAMA2scFv construct were able to maintain muscle function.
  • Example 8 Generation of a bifunctional fusion protein comprising human DLL4 variant containing C2-DSL and EGF1-5, hIgG1 Fc, and anti-LAMA2 scFv [0505] Human DLL4 containing C2-DSL-EGF1-5 with G2S, F81L, and H168Y mutations, relative to wild-type DLL4 comprising the N-terminus to EGF5 (SEQ ID NO: 126), was used to generate the parental human DLL4v (SEQ ID NO: 260).
  • the parental human DLL4v was fused to the N-terminus of human IgG1 Fc engineered to remove effector function.
  • the C-terminus of the Fc was fuse to a (GGGGS)3 linker and anti-human LAMA2 scFv (SEQ ID NO: 268). Since rat DLL4EL12 variant mutations (US2019/0248894; Luca et al., 2015, Science 47(6224):847-53; WO2020/223610; and WO2021/007573) led to low yields and high aggregation in the context of human DLL4, mutations were introduced to overcome the poor chemistry, manufacturing and controls (CMC) developability.
  • CMC chemistry, manufacturing and controls
  • N-glycosylation sites were reintroduced to positions T52 and T135 of human DLL4v (SEQ ID NO: 260) and new mutations identified from yeast display screening were incorporated to determine DLL4 mutations that would confer higher affinity to Notch receptors.
  • the following seven variant human DLL4 were generated based off of the parental human DLL4v (SEQ ID NO: 260): DLL4v11 (SEQ ID NO: 261), DLL4v12 (SEQ ID NO: 262), DLL4v13 (SEQ ID NO: 263), DLL4v14 (SEQ ID NO: 264), DLL4v22 (SEQ ID NO: 265), DLL4v23 (SEQ ID NO: 266), and DLL4v24 (SEQ ID NO: 267).
  • the seven variant human DLL4 were further fused to an anti-LAMA2 scFv (SEQ ID NO: 268) to make bifunctional constructs (e.g., SEQ ID NOs: 270-276).
  • the N-glycosylation site and yeast display mutations are outlined in Table 10.
  • Bold amino acids indicate an amino acid mutations and non-bold amino acids are the original amino acids, relative to wild-type DLL4 (SEQ ID NO: 126).
  • Table 10 Human DLL4 mutations 214 sf-6122278 Attorney Docket No.: 340742000140 [0506] The whole DNA sequence was assembled with codon optimization for expression in CHO or HEK293 cells. The DNA was cloned into the pcDNA3.x vector (Thermo Fisher Scientific). pcDNA3.x with the cloned construct was transformed into E.
  • coli bacteria DH5 ⁇ competent cells
  • DNA sequence was validated
  • the colony was propagated by an overnight culture
  • plasmid DNA was obtained using a commercial plasmid purification kit.
  • pcDNA3.x plasmid DNA was transfected into CHO or HEK293 cells for expression, either transiently or stably after establishing a stable cell pool.
  • Recombinant protein was purified from the cell culture supernatants with a 25 ml MabSelectTM SuReTM column (Cytiva). Proteins were eluted from the column with 0.1 M glycine pH 3.0, and immediately neutralized using 20% volume of 1M Tris pH 8.0.
  • the constructs with N- glycosylation sites demonstrated excellent expression, stability, and high recovery of protein, compared to the v22, v23 and v24 constructs. As shown in FIGs.

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Abstract

L'invention concerne des compositions et des méthodes de traitement de dystrophies musculaires par administration d'une quantité efficace d'une composition contenant une protéine bifonctionnelle comprenant une première fraction de liaison qui se lie à des molécules spécifiques aux muscles et un second domaine fonctionnel qui active la signalisation Notch, activant ainsi de façon spatio-temporelle la signalisation Notch dans des tissus musculaires pour amplifier le renouvellement des cellules satellites in situ, et améliorant ainsi la réparation/régénération musculaire pour améliorer la fonction musculaire en tant que traitement de diverses dystrophies musculaires et de la sarcopénie. L'invention concerne également des domaines extracellulaires DLL4 modifiés et des constructions protéiques les comprenant.
PCT/US2024/045653 2023-09-08 2024-09-06 Protéines bifonctionnelles et leurs utilisations Pending WO2025054500A2 (fr)

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