WO2025222100A2 - Protéines de liaison à cd3, acides nucléiques codant de telles protéines, et leurs procédés de préparation et d'utilisation - Google Patents

Protéines de liaison à cd3, acides nucléiques codant de telles protéines, et leurs procédés de préparation et d'utilisation

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Publication number
WO2025222100A2
WO2025222100A2 PCT/US2025/025335 US2025025335W WO2025222100A2 WO 2025222100 A2 WO2025222100 A2 WO 2025222100A2 US 2025025335 W US2025025335 W US 2025025335W WO 2025222100 A2 WO2025222100 A2 WO 2025222100A2
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antibody
igg
binding
binding fragment
antigen binding
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WO2025222100A3 (fr
Inventor
Alexander John MARTINKO
David Geoffrey KUGLER
Burcu YIGIT
Mary MATHIEU
Prasuna PALURU
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Cartography Biosciences Inc
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Cartography Biosciences Inc
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Publication of WO2025222100A3 publication Critical patent/WO2025222100A3/fr
Pending legal-status Critical Current
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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
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • CD3 BINDING PROTEINS NUCLEIC ACIDS ENCODING SUCH PROTEINS,
  • the present invention relates to the preparation of immunoglobulin complementarity determining regions (“CDRs”) and immunoglobulin binding domains that bind human CD3s comprising those CDRs, and their use for the preparation of CD3- binding proteins in various formats.
  • CDRs immunoglobulin complementarity determining regions
  • CD3- binding proteins in various formats.
  • T-lymphocytes are involved in the cell-mediated arm of the immune response. T cells are activated by foreign antigens to proliferate and differentiate into effector cells when the antigen is displayed on the surface of antigen-presenting cells as fragments of protein antigens that have been partly degraded inside the antigen- presenting cell. The fragments are presented on the surface as a complex with MHC proteins. T cells recognize these antigen complexes via T-cell receptors (TCR). In humans, the majority of T cells express a TCR composed of an alpha chain and a beta chain, whereas a minor T-cell population is characterized by the TCR gamma/delta.
  • TCR T-cell receptors
  • TCR signaling within the T cell relies on its relationship to the CD3 complex.
  • the TCR-CD3 complex is composed of a TCR heterodimer in noncovalent association with invariant CD3 dimers: CD3ey, CD3e5, and CD3C .
  • Phosphorylation of immunoreceptor tyrosine-based activation motifs (“ITAMs”) follows ligand engagement with the TCR, leading to T cell activation, proliferation, cytokine signaling, and differentiation.
  • CD3 The crucial role played by CD3 in T cell activation makes CD3 an attractive drug target for modulation of cell-mediated immunity.
  • Anti-CD3 antibodies have been investigated for their ability to induce tolerance in autoimmune diseases such as diabetes, multiple sclerosis, and ulcerative colitis and tolerance to transplanted organs.
  • CD3 antibodies have also been used for ex vivo expansion of T cells for adoptive T cell therapy.
  • bispecific antibodies comprising a CD3-specific binding arm and a tumor-associated antigen binding arm fused together in a single bivalent antibody-like construct have garnered much attention in cancer immunotherapy.
  • the mechanism of action of CD3 bispecifics is based on redirecting and guiding T cell effector function in an antitumor manner by increasing the number of CD3 engagers in the tumor cell proximity.
  • CD3 bispecifics are able to direct T cell activation by tethering to tumor cells using their tumor-binding arm due to the monovalency of the TCR binding arm. Thus, these bispecifics are often referred to as a class by the name T cell engager, or “TCE.”
  • TCE T cell engager
  • the present invention relates to CD3 -binding proteins and related compositions and methods.
  • the present invention provides target binding proteins comprising an immunoglobulin variable region that binds T-cell surface glycoprotein CD3 epsilon chain (CD3s; Swiss-Prot P07766), wherein the immunoglobulin variable region comprises heavy chain CDRs Hl, H2, and H3 and light chain CDRs LI, L2, and L3 amino acid sequences as recited for one of the Identifiers in Table 1.
  • Table 1 SEQ ID NOs: 1-1542, with numbering across rows (so row 1 is SEQ ID NOs: 1-6, row 2 is SEQ ID NOs: 2-12, etc ): *US2015/016661Al, provided as a comparison to the sequences of the present invention
  • the present invention provides target binding proteins comprising an immunoglobulin variable region that binds CD3s, wherein the immunoglobulin variable region comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences as recited for one of the Identifiers in Table 2.
  • Table 2 (SEQ ID NOs: 1543-2056, with numbering across rows (so row 1 is SEQ ID NOs: 1543-1544, row 2 is SEQ ID NOs: 1545-1546, etc.):
  • the target binding protein is an antibody.
  • antibody as used herein is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies) and multispecific antibodies (e.g., bispecific antibodies) such as based on the Duobody ® technology (Genmab) or Hexabody ® technology (Genmab), antibody fragments, and artificial constructs such as single-chain variable fragments (scFvs) that comprise immunoglobulin variable regions.
  • scFvs single-chain variable fragments
  • Antibody fragment and “antibody binding fragment” mean antigen-binding fragments and analogues of an antibody, typically including at least a portion of the antigen binding or variable regions (e.g. one or more CDRs) of the parental antibody.
  • An antibody fragment retains at least some of the binding specificity of the parental antibody.
  • an antibody fragment retains at least 10% of the parental binding activity when that activity is expressed on a molar basis.
  • an antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parental antibody’s binding affinity for the target.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv, unibodies (technology from Genmab); nanobodies (technology from Ablynx); domain antibodies (technology from Domantis); and multispecific antibodies formed from antibody fragments.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.
  • an Antibody of the invention is a T cell engaging antibody (e.g., a bispecific T cell engaging antibody, or BiTE), a pro-Bispecific T Cell Engager (pro-BiTE) molecule, pro-Chimeric Antigen Receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cell, such as a CAR modified NK cell.
  • a T cell engaging antibody e.g., a bispecific T cell engaging antibody, or BiTE
  • pro-BiTE pro-Bispecific T Cell Engager
  • pro-CAR pro-Chimeric Antigen Receptor
  • an antibody of the invention is a BiTE® (Micromet) which acts through the simultaneous engagement of tumor-associated antigens and CD3, resulting in the activation of T-cells irrespective of MHC.
  • An “Fab fragment” is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • An “Fc” region contains two heavy chain fragments comprising the CHI and CH2 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • An “Fab 1 fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.
  • An “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • a “single-chain Fv antibody” refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • scFv see Pluckthun, 1994, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946, 778 and 5,260,203.
  • the present invention provides target binding proteins comprising an immunoglobulin variable region that binds CD3s, wherein the immunoglobulin variable region is an scFv comprising an amino acid sequence as recited for one of the Identifiers in Table 3.
  • a “diabody” is a small antibody fragment with two antigen-binding sites.
  • the fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL or VL-VH variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448.
  • a “Duobody®” (Genmab) are bispecific antibodies with normal IgG structures (Labrijn et al., 2013, Proc. Natl.
  • “Hexabodies” are antibodies that, while retaining regular structure and specificity, have an increased killing ability (Diebolder et al., 2014, Science 343(6176): 1260-3).
  • a “domain antibody fragment” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody fragment.
  • the two VH regions of a bivalent domain antibody fragment may target the same or different antigens.
  • the target binding protein is a single chain variable fragment (scFv), a BiTE®, a (SCFV)2, a NANOBODY®, a nanobody -HSA VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four-in-one), a DUTAMAB®, a DT- TgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)
  • the target binding protein is or comprises an IgG, IgM, IgA, IgE, or IgD antibody or fragment thereof.
  • the target binding protein is an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the target binding protein is humanized.
  • An antibody fragment of the invention may comprise a sufficient portion of the constant region to permit dimerization (or multimerization) of heavy chains that have reduced disulfide linkage capability, for example where at least one of the hinge cysteines normally involved in inter-heavy chain disulfide linkage is altered as described herein.
  • an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC (antibody dependent cellular cytotoxicity) function, and/or complement binding (for example, where the antibody has a glycosylation profile necessary for ADCC function or complement binding).
  • the target binding protein of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g. U.S. Pat. No. 5,624,821; W02003/086310; W02005/120571; W02006/0057702; Presta, 2006, Adv. Drug Delivery Rev . 58:640-656. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc.
  • the antibodies of the present invention also include antibodies with intact Fc regions that provide full effector functions, e.g. antibodies of isotype IgGl, which induce complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC) in a targeted cell.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • the target binding protein further comprises a masking moiety that inhibits binding of the target binding protein to CD3 in an inactive state.
  • the masking moiety is coupled to the target binding protein via a cleavable moiety (either directly or indirectly, e.g., via one or more linkers), and the cleavable moiety is a substrate for a protease.
  • the protease is ADAMS, AD AMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAMI 7/T ACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, Aspartate proteases, B ACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Cysteine proteinases, Cruzipain, Legumain, Otubain-2, KLKs,
  • the target binding protein further comprises a second immunoglobulin variable region that specifically binds to a second target antigen.
  • the target binding protein is an antibody-drug conjugate.
  • the antibody is conjugated to a toxin, radioisotope, small molecule, diagnostic agent, therapeutic macromolecule, targeting moiety, or detectable moiety, via a conjugating moiety.
  • the conjugating moiety is cleavable by a protease. In some embodiments, the conjugating moiety is non- cleavable by a protease.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the target binding protein of the invention and a pharmaceutically acceptable carrier.
  • the present disclosure provides a nucleic acid encoding target binding proteins comprising an immunoglobulin variable region that binds CD3, wherein the immunoglobulin variable region comprises heavy chain CDRs Hl, H2, and H3 and light chain CDRs LI, L2, and L3 as recited for one of the Identifiers in Table 1.
  • the invention also provides isolated nucleic acids encoding anyone of the anti-CD3 antibodies or antigen binding fragments of the invention.
  • the invention also provides expression vectors comprising one or more nucleic acids of the present invention.
  • An expression vector is a DNA molecule comprising the regulatory elements necessary for transcription of a target nucleic acid in a host cell.
  • the target nucleic acid is placed under the control of certain regulatory elements including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancer elements.
  • Such a target nucleic acid is said to be “operably linked to” the regulatory elements when the regulating element controls the expression of the gene.
  • These isolated nucleic acids and the expression vectors comprising them may be used to express the antibodies of the invention or antigen binding fragments thereof in recombinant host cells.
  • the invention also provides host cells comprising an expression vector of the present invention.
  • the invention also provides a vessel or injection device comprising anyone of the anti-CD3 antibodies or antigen binding fragments of the invention.
  • the invention also provides a method of producing an anti-CD3 antibody or antigen binding fragment of the invention comprising: culturing a host cell comprising a polynucleotide encoding a heavy chain and/or light chain of an antibody of the invention (or an antigen binding fragment thereof) under conditions favorable to expression of the polynucleotide; and optionally, recovering the antibody or antigen binding fragment from the host cell and/or culture medium.
  • the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in a single vector.
  • the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in different vectors.
  • the present disclosure provides a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the target binding protein described herein or the pharmaceutical composition described herein.
  • the subject in need thereof has been identified or diagnosed as having a cancer.
  • the present disclosure provides a method of producing a target binding protein of the invention, comprising: culturing the target cell described herein in a culture medium under a condition sufficient to produce the target binding protein; and recovering the target binding protein from the cell or the culture medium.
  • the method further comprises isolating the target binding protein recovered from the cell or the culture medium.
  • the method further comprises formulating the target binding protein into a pharmaceutical composition.
  • Figure 1 Design summary for Round 1 of CD3 binder engineering.
  • Figure 2 Antibody schematics for IgGs produced from Round 1 CD3 binder engineering.
  • Figure 3 Antibody schematics for T-cell engagers produced from Round 1 CD3 binder engineering.
  • Figure 4 Supernatant expression titers for Round 1 CD3 binder engineering of IgGs and T-cell engagers determined by Biolayer Interferometry with Protein A quantitation probes.
  • Figure 5 Analytical size exclusion chromatography by HPLC demonstrated monomeric purity with minimal aggregation for CD3-10, CD- 12, and CD3-15.
  • Figure 6 Representative thermostability analysis determined by Sypro Organge based differential scanning fluorimetry.
  • Figure 7 Representative BLI sensograms for selected clones from round 1 engineering. Antibodies were captured on anti-human Fc probes followed by association with a concentration series of soluble CD3 antigen.
  • FIG. 8 Top CD3 binders coupled with a tumor associated antigen (TAA1 ) target into TCEs are functional against cell lines with low, medium and high tumor antigen expression.
  • TAA1 tumor associated antigen
  • Figure 9 Antibody schematics for T-cell engagers produced from Round 2 CD3 binder engineering.
  • Figure 10 Supernatant expression titers determined by Biolayer Interferometry with Protein A quantitation probes showed improved expression in Round 2 of CD3 binder engineering compared to Round 1.
  • Figure 11 Representative analytical size exclusion chromatography by HPLC from round 2 CD3 binder engineering demonstrated monomeric purity with minimal aggregation for CD3-32, CD-42, and CD3-46.
  • Figure 12 Representative thermostability analysis for selected clones CD3-32, CD-42, and CD3-46 from round 2 CD3 binder engineering determined by Sypro Organge based differential scanning fluorimetry.
  • Figure 13 Representative BLI sensograms for selected clones from round 4 engineering. Antibodies were captured on anti-human Fc probes followed by association with a concentration series of soluble CD3 antigen.
  • Figure 14 Top CD3 binders from round 2 are functional as TCEs against a cell line expressing the target tumor antigen.
  • Figure 15 Efficacy of four CD3 binders as TCEs selected to test in vivo. Potent anti -tumor activity observed upon administration of TCEs.
  • Figure 16 Pharmacokinetics of TAAxCD3-42 evaluated at 5 mg/kg.
  • Figure 17 Pharmacokinetics of TAAxCD3-15 evaluated at 5 mg/kg.
  • target binding proteins that specifically bind to cluster of differentiation 3 (CD3).
  • the target binding protein may include a heavy chain variable domain and a light chain variable domain, which form an immunoglobulin variable region that specifically binds to CD3.
  • the target binding proteins preferably comprise one or more mutations in their amino acid sequences that makes them more humanized, e.g., increases their similarities to a CD3- binding molecule produced naturally in humans.
  • the target binding proteins of the invention may be single chain proteins, such as single chain antibodies.
  • the target binding proteins may be single chain fragment variable (scFv) antibodies.
  • the target binding proteins may be multichain proteins (e.g., intact IgG antibodies).
  • the target binding proteins may be activatable molecules, such as activatable CD3 -binding molecules that include a masking moiety coupled to the CD3 -binding domain via a cleavable moiety (either directly or indirectly, e.g., via one or more linkers).
  • the cleavable moiety may be cleaved under certain conditions (e.g., when exposed to a protease in a tumor microenvironment) to thereby release the masking moiety from the CD3 -binding domain.
  • the target binding proteins may be multispecific (e.g., bispecific) binding proteins that bind to one or more additional targets other than CD3.
  • the multispecific proteins may specifically bind to CD3 and a tumor associated antigen, e g., HER2, CD 19, EpCAM, LY6G6D, KLK2, CD20, CD22, CD38, FLT3, STEAP1, PD-1, PRAME, GPRC5D, GP100, BCMA EGFRvIII, gpA33, 4-1BB, PSMA, MUC16, MUC17, DLL3, CEA, cMET, IMCgplOO, SSTR2, and the like.
  • a tumor associated antigen e.g., HER2, CD 19, EpCAM, LY6G6D, KLK2, CD20, CD22, CD38, FLT3, STEAP1, PD-1, PRAME, GPRC5D, GP100, BCMA EGFRvIII, gpA33, 4-1BB, PSMA, MUC16, M
  • the tumor associated antigen is a personalized neoantigen. See, e.g., Douglass et al., Sci Immunol 2021;6:eabd5515 (doi: 10.1126/sciimmunol.abd5515); Xie et al., Sig Transduct Target Ther 8, 9 (2023) (doi.org/10.1038/s41392-022-01270-x).
  • compositions, kits, nucleic acids, vectors, and recombinant cells as well as related methods, including methods of using and methods of producing any of the target binding proteins described herein.
  • a and “an” refers to one or more (i.e., at least one) of the grammatical object of the article.
  • a cell encompasses one or more cells.
  • the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives.
  • the term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • exemplary is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion.
  • a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to and does find direct support for embodiments related to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.
  • a “target binding protein” of the invention comprises a heavy chain variable domain and a light chain variable domain, which together form at least one immunoglobulin variable region that specifically binds to human CD3.
  • a target binding protein of the invention that comprises two or more binding domains may comprise only CD3 binding domains, or may comprise at least one CD3 binding domain and a binding domain that specifically binds to another target (e.g., a tumor associated antigen.
  • references to a target binding protein or an anti-CD3 antibody herein refer to both a monospecific (only CD3 binding) or a multispecific (e.g., bispecific) target binding protein or anti-CD3 antibody.
  • the heavy chain variable domain and the light chain variable domain are disposed within the same polypeptide.
  • the heavy chain variable domain and the light chain variable domain are coupled together by one or more linkers.
  • the linker may be a peptide linker described in the Linkers section below.
  • the immunoglobulin variable region in the single chain polypeptide may be an scFv.
  • the heavy chain variable domain and the light chain variable domain are disposed within two different polypeptides.
  • the target binding protein may be a protein complex that comprises multiple polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides). In some examples, some or all (e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides) of the multiple polypeptides may be identical. In some examples, each of the multiple polypeptides in the target binding complex is different from the other.
  • the immunoglobulin variable region may reside within any of a variety of different constructs, including an antibody or a fragment thereof, a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), BiTE® or a component thereof, a (scFv)2, a NANOBOD Y®, a nanobody-HSA, VHH-scAb, a VHH- Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four-in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogon
  • a target binding protein specifically binds to a polypeptide sequence can be determined using any assay known in the art. Examples of assays known in the art to determining binding affinity include surface plasmon resonance (e.g., BIACORE® (Cytiva Sweden AB) or a similar technique (e.g. KinExa® (Sapidyne Instruments Inc.) or OCTET® (Sartorius)).
  • a target binding protein of the invention is an antibody.
  • the term “antibody” refers to any form of antibody that exhibits the desired biological activity.
  • antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • antigen binding sites e.g., fragments, sub
  • Single chain antibodies are also included by reference in the term “antibody.”
  • Preferred therapeutic antibodies are intact IgG antibodies.
  • the term “intact IgG” as used herein is meant as a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises IgGl, IgG2, IgG3, and IgG4. In mice this class comprises IgGl, IgG2a, IgG2b, IgG3.
  • the known Ig domains in the IgG class of antibodies are VH, Cyl, Cy2, Cy3, VL, and CL.
  • the present invention includes anti-CD3 antigen-binding fragments and methods of use thereof.
  • a “full length antibody” is, in the case of an IgG, a bivalent molecule comprising two heavy chains and two light chains. Each heavy chain comprises a VH domain followed by a constant domain (CHI), a hinge region, and two more constant (CH2 and Cm) domains; while each light chain comprises one VL domain and one constant (CL) domain.
  • a full length antibody in the case of an IgM is a decavalent or dodecavalent molecule comprising 5 or 6 linked immunoglobulins in which each immunoglobulin monomer has two antigen binding sites formed of a heavy and light chain.
  • antibody fragment or “antigenbinding fragment” refers to anti gen -binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions.
  • antigenbinding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • the present invention includes anti-CD3 Fab fragments and methods of use thereof.
  • a “Fab fragment” is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab fragment” can be the product of papain cleavage of an antibody.
  • the present invention includes anti-CD3 antibodies and antigen-binding fragments thereof which comprise an Fc region and methods of use thereof.
  • An “Fc” region contains two heavy chain fragments comprising the CH3 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • the present invention includes anti-CD3 Fab’ fragments and methods of use thereof.
  • a “Fab 1 fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CHI and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.
  • the present invention includes anti-CD3 F(ab’)2 fragments and methods of use thereof.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • An “F(ab')2 fragment” can be the product of pepsin cleavage of an antibody.
  • the present invention includes anti-CD3 Fv fragments and methods of use thereof.
  • the “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • the present invention includes anti-CD3 scFv fragments and methods of use thereof.
  • the term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv 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 “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody may target the same or different antigens.
  • the present invention includes anti-CD3 bivalent antibodies and methods of use thereof.
  • a “bivalent antibody” comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
  • the present invention includes anti-CD3 diabodies and methods of use thereof.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL or VL-VH a linker that is too short to allow pairing between the two domains on the same chain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl.
  • an antibody or antigen-binding fragment of the invention which is modified in some way retains at least 10% of its binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis.
  • an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the CD3 binding affinity as the parental antibody.
  • an antibody or antigen-binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
  • the present invention includes isolated anti-CD3 antibodies and antigenbinding fragments thereof and methods of use thereof.
  • isolated is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.
  • An “isolated” antibody, antigen-binding fragment, nucleic acid, etc., is one which has been identified and separated and/or recovered from one or more components of its natural environment.
  • the antibody, antigenbinding fragment, nucleic acid, etc. is purified to 75% by weight or more, more preferably to 90% by weight or more, still more preferably to 95% by weight or more, and still more preferably to 98% by weight or more.
  • isolated biological molecules are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium.
  • An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.
  • the present invention includes anti-CD3 chimeric antibodies (e.g., human constant domain/mouse variable domain) and methods of use thereof.
  • a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species.
  • variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.
  • parental antibody an antibody from an experimental animal
  • the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.
  • the present invention includes anti-CD3 humanized antibodies and antigenbinding fragments thereof (e.g., rat or mouse antibodies that have been humanized) and methods of use thereof.
  • humanized antibody refers to forms of antibodies that contain sequences from both human and non-human (e.g., mouse or rat) antibodies.
  • the humanized antibody will comprise substantially 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 framework (FR) regions are those of a human immunoglobulin sequence.
  • the humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy -terminal portion of the heavy chain may define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W ., ed., 2nd ed. Raven Press, N.Y. (1989).
  • variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the two binding sites are, in general, the same.
  • variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al:, National Institutes of Health, Bethesda, MD; 5 th ed.; NIH Publ. No.
  • hypervariable region refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain).
  • CDR complementarity determining region
  • isolated nucleic acid molecule or “isolated polynucleotide” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
  • a nucleic acid molecule comprising a particular nucleotide sequence does not encompass intact chromosomes.
  • Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.
  • a nucleic acid or polynucleotide is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a “pre” sequence 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 phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • germline sequence refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used.
  • Human germline sequences may be obtained, for example, from JOINSOLVER germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health.
  • Mouse germline sequences may be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.
  • the antibodies and antigen-binding fragments disclosed herein may bind human CD3 bivalently with a KD value of 10 x 10' 9 M or lower as determined by surface plasmon resonance (e.g., BIACORE®) or a similar technique (e.g. KinExa® or bio-layer interferometry (OCTET®)).
  • the antibodies and antigen-binding fragments disclosed herein may bind human CD3 bivalently with a KD value of about 5-10 x 10' 9 M as determined by surface plasmon resonance (e.g., BIACORE®) or a similar technique (e.g. KinExa® or OCTET®).
  • r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot.
  • Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
  • the present invention includes methods for making an anti-CD3 antibody or antigen-binding fragment thereof of the present invention.
  • the anti-CD3 antibodies disclosed herein may be produced recombinantly (e.g., in an A. colU l expression system, a mammalian cell expression system or a lower eukaryote expression system).
  • nucleic acids encoding the antibody immunoglobulin molecules of the invention e.g., VH or VL
  • VH or VL may be inserted into a pET- based plasmid and expressed in the A. colU l system.
  • the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter.
  • a host cell e.g., bacterial host cell such as E.coli such as BL21 or BL21DE3
  • T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter.
  • a bacterial host cell such as a A.
  • coli includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • Transformation can be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.
  • the present invention includes recombinant methods for making an anti- CD3 antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g., heavy and/or light immunoglobulin chain); culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under condition favorable to such expression and, optionally, isolating the antibody or fragment or chain from the host cell and/or medium in which the host cell is grown.
  • Anti-CD3 antibodies can also be synthesized by any of the methods set forth in U.S. Patent No. 6,331,415.
  • Eukaryotic and prokaryotic host cells including mammalian cells as hosts for expression of the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels.
  • Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia fmlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyverom
  • Pichia sp. any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium luckno ense, any Fusarium sp., Yarrow ia lipolytica, and Neurospora crassa.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody or fragment or chain in the host cells or secretion into the culture medium in which the host cells are grown.
  • Antibodies and antigen-binding fragments thereof and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies and antigen-binding fragments thereof and immunoglobulin chains of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques.
  • the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
  • the GS system is discussed in whole or part in connection with European Patent Nos. 0216846, 0256055, and 0323997 and 0338841.
  • the mammalian host cells e.g., CHO
  • the present invention includes methods for purifying an anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprising introducing a sample comprising the antibody or fragment to a purification medium (e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., protein-A, protein-G, protein-A/G, protein-L)) and either collecting purified antibody or fragment from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound antibody or fragment from the medium and collecting the eluate.
  • a purification medium e.g., cation exchange medium, anion exchange medium, hydrophobic exchange medium, affinity purification medium (e.g., protein-A, protein-G, protein-A/G, protein-L)
  • the medium is in a column to which the sample is applied.
  • the purification method is conducted following recombinant expression of the antibody or fragment in a host cell, e.g., wherein the host cell is first lysed and, optionally, the lysate is purified of insoluble materials prior to purification on a medium.
  • glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody.
  • all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein comprise the instant invention, independent of the glycosylation pattern the antibodies may have.
  • antibodies with a glycosylation pattern comprising only non-fucosylated N- glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These antibodies with non-fucosylated -gl yeans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.
  • the present invention further includes anti-CD3 antigen-binding fragments of the anti-CD3 antibodies disclosed herein.
  • the antibody fragments include F(ab)2 fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin.
  • Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine.
  • Immunoglobulins may be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. In some embodiments, different constant domains may be appended to humanized VL and VH regions derived from the CDRs provided herein. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3 and IgG4; IgAl and IgA2.
  • the invention comprises antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.
  • the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g. a human constant region, such as yl, y2, y3, or y4 human heavy chain constant region or a variant thereof.
  • the antibody or antigen-binding fragment comprises a light chain constant region, e.g. a human light chain constant region, such as lambda or kappa human light chain region or variant thereof.
  • the human heavy chain constant region can be y4 and the human light chain constant region can be kappa.
  • the Fc region of the antibody is y4 with a Ser228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).
  • the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgGl subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG2 subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG4 subtype.
  • the anti-CD3 antibodies and antigen-binding fragments thereof are engineered antibodies to include modifications to framework residues within the variable domains the antibody, e.g. to improve the properties of the antibody or fragment.
  • framework modifications are made to decrease the immunogenicity of the antibody or fragment. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e. framework residues) in a parental (e.g. rodent) antibody or fragment with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g. human residues in the case of human therapeutics.
  • a parental antibody or fragment e.g. rodent antibody or fragment with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g. human residues in the case of human therapeutics.
  • Such an antibody or fragment is referred to as a “humanized” antibody or fragment.
  • an engineered (e.g. humanized) antibody it is desirable to increase the affinity, or alter the specificity of an engineered (e.g. humanized) antibody.
  • One approach is to mutate one or more framework residues to the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or fragment framework sequences to the germline sequences from which the antibody or fragment is derived.
  • Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Patent No. 5,693,762, U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101).
  • the anti-CD3 antibodies and antigen-binding fragments thereof are engineered (e.g. humanized) to include modifications in the framework and/or CDRs to improve their properties.
  • engineered changes can be based on molecular modelling.
  • a molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen.
  • Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions.
  • CDRs regions classified as “CDRs” and “hypervariable loops”.
  • Later studies (Raghunathan et al, (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody -antigen crystal complexes and observed that the antigen binding regions in antibodies do not necessarily conform strictly to the “CDR” residues or “hypervariable” loops.
  • the molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice the potential antigen binding regions based on the model differ from the conventional “CDR”s or “hypervariable” loops.
  • Commercial scientific software such as Discovery Studio (BIO VIA, Dassault Systems)) can be used for molecular modeling.
  • Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs.
  • FR4 framework 4
  • VJ regions for the human germlines are compared with the corresponding non-human region.
  • FR4 (framework 4) in VL J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non-human region.
  • the CDRs are grafted into the selected human frameworks.
  • certain residues in the VL-VH interface can be retained as in the non-human (parental) sequence.
  • Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen.
  • these residues are retained as in the non-human (parental) sequence.
  • Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation.
  • Developability filters can be introduced early on in the design stage to eliminate/minimize these potential problems.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent No. 7,125,689.
  • the antibodies of the present disclosure do not contain deamidation or asparagine isomerism sites.
  • an asparagine (Asn) residue may be changed to Gin or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR.
  • a similar problem may occur at a Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60: 1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734.
  • the asparagine is changed to glutamine (Gin).
  • any methionine residues may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id.
  • Another type of framework modification involves mutating one or more residues within the framework regions to prevent aggregation.
  • the risk of an antibody to aggregate can be assessed using the spatial aggregation propensity. See, Chennamsetty, et al. (2010) ./. Phys. Chem. 114, 6614-6624.
  • the method requires the calculation of the Solvent Accessible Area (SAA) for each atom.
  • SAA Solvent Accessible Area
  • the molecular aggregation score is then calculated as the sum of all atomic scores. For a given radius and size of molecule, this is an approximate indication of its overall tendency to aggregate. Residues with a high aggregation score are replaced by residues with a lower score (e.g. more hydrophilic amino acids).
  • the antibodies (e.g., humanized antibodies) and antigen-binding fragments thereof disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigendependent cellular cytotoxicity).
  • the antibodies and antigen-binding fragments thereof disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more properties of the antibody or fragment.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the antibodies and antigen-binding fragments thereof disclosed herein also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; W02003/086310;
  • Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc regions. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.
  • the antibody or antigen-binding fragment of the invention is an IgG4 isotype antibody or fragment comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region.
  • S228P Serine to Proline mutation at a position corresponding to position 228
  • This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al (1993). Mol. Immunol. 30: 105-108; position 241 is based on the Kabat numbering system).
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent No. 5,677,425.
  • the number of cysteine residues in the hinge region of CHI is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc- hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the antibody or antigen-binding fragment of the invention is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement.
  • the proteins of the invention which are preferably antibodies and most preferably IgG antibodies or fragments thereof, may have altered (e.g., relative to an unmodified antibody) FcyR binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (KD), dissociation and association rates (k o ff and k on respectively), binding affinity and/or avidity) and that certain alterations are more or less desirable.
  • KD equilibrium dissociation constant
  • K a is the reciprocal of KD.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-FcyR interactions, i.e., specific binding of an Fc region to an FcyR including but not limited to, equilibrium methods (e.g., enzyme- linked immuno absorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g.
  • ELISA enzyme- linked immuno absorbent assay
  • RIA radioimmunoassay
  • BIACORE® Octet®, or KinExa® analysis
  • other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • FRET fluorescence resonance energy transfer
  • chromatography e.g., gel filtration
  • the proteins of the present invention bind to one or more human FcyRs selected from the group consisting of FcyRI, FcyRIIB, FcyRIIC, FcyRIIIA-F158, and FcyRIIIA-V158 with an affinity at least 10-fold, preferably at least 30-fold, and more preferably at least 100-fold, less than equivalent protein having a wildtype human IgGl heavy chain constant domain Fc region or a wild-type human IgG4 heavy chain constant domain Fc region.
  • the proteins of the invention comprise an immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region and an immunoglobulin hinge region.
  • the immunoglobulin Fc region may be an IgG Fc region, an IgE Fc region, or an IgA Fc region.
  • the protein comprises two immunoglobulin Fc regions, each immunoglobulin Fc region comprising an immunoglobulin C2 region and an immunoglobulin C3 region and an immunoglobulin hinge region, wherein the hinge region of one of the immunoglobulin Fc regions is bound to the hinge region of the other immunoglobulin Fc region to form a dimeric Fc structure.
  • a protein is a human or humanized IgG protein.
  • the proteins of the invention comprise a mutated IgG4 Fc region, and preferably the protein is an IgG comprising two mutated IgG4 Fc regions to form a dimeric Fc structure.
  • a mutated IgG4 Fc region may comprise one of the mutations, or mutational combinations, recited in Table 3.
  • the numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
  • the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.
  • each mutation in the combination is separated by a “/”. Deletions are indicated by “A
  • the proteins of the invention comprise a mutated IgGl Fc region, and preferably the protein is an IgG comprising two mutated IgGl Fc regions to form a dimeric Fc structure.
  • a mutated IgGl Fc region may comprise one of the mutations recited in Table 4.
  • the numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
  • the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.
  • a mutated IgGl Fc region may comprise one of the mutational combinations recited in Table 5.
  • the numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
  • the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.
  • each mutation in the combination is separated by a “/” and deletions are indicated by a “A”.
  • the proteins of the invention comprise a wild type or mutated IgG2 Fc region, and preferably the protein is an IgG comprising two wild type or mutated IgG2 Fc regions to form a dimeric Fc structure.
  • a mutated IgG2 Fc region may comprise one of the mutations, or mutational combinations, recited in Table 6.
  • the numbering system of the constant region referred to in this table is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
  • the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position. For those entries that include combinations of more than one mutation, each mutation in the combination is separated by a “/”.
  • the antibodies or antigen-binding fragments of the invention comprise a particular glycosylation pattern.
  • an afucosylated or an aglycosylated antibody or fragment can be made (z.e., the antibody lacks fucose or glycosylation, respectively).
  • the glycosylation pattern of an antibody or fragment may be altered to, for example, increase the affinity or avidity of the antibody or fragment for a CD3 antigen.
  • modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody or fragment sequence.
  • one or more amino acid substitutions can be made that result in removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Antibodies and antigen-binding fragments disclosed herein may further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns (See for example, Choi et al, (2003) Proc. Natl. Acad. Set. 100: 5022-5027; Hamilton et al., (2003) Science.
  • a particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular A-glycan structure predominates (see, e.g., U.S. Patent No. 7,029,872 and U.S. Patent No. 7,449,308).
  • These genetically modified host cells have been used to produce antibodies that have predominantly particular N-gl yean structures (See for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).
  • the antibodies and antigen-binding fragments thereof disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and non-fucosylated hybrid and complex A-glycans, including bisected and multi antennary species, including but not limited to A-glycans such as GlcNAc(i-4)Man3GlcNAc2; Gal(i-4)GlcNAc(i-4)Man3GlcNAc2; NANA(i-4)Gal(i- 4)GlcNAc(i-4)Man3GlcNAc2.
  • A-glycans such as GlcNAc(i-4)Man3GlcNAc2; Gal(i-4)GlcNAc(i-4)Man3GlcNAc2; NANA(i-4)Gal(i- 4)GlcNAc(i-4)Man3GlcNAc2.
  • the antibodies and antigen-binding fragments thereof provided herein may comprise antibodies or fragments having at least one hybrid A-glycan selected from the group consisting of GlcNAcMansGlcNAc2;
  • the hybrid A-glycan is the predominant A-gly can species in the composition.
  • the antibodies and antigen-binding fragments thereof provided herein comprise antibodies and fragments having at least one complex A-glycan selected from the group consisting of GlcNAcMan3GlcNAc2;
  • GalGlcNAcMan3GlcNAc2 NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2;
  • the complex A-glycan are the predominant A-glycan species in the composition.
  • the complex A-glycan is a particular A-gly can species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex -gl yeans in the composition.
  • the antibody and antigen binding fragments thereof provided herein comprise complex 7V-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N- glycans comprise the structure NANA2Gal2GlcNAc2Man3GlcNAc2, wherein such structure is afucosylated.
  • complex N- glycans comprise the structure NANA2Gal2GlcNAc2Man3GlcNAc2, wherein such structure is afucosylated.
  • Such structures can be produced, e.g., in engineered Pichia pastoris host cells.
  • the A-glycan is fucosylated.
  • the fucose is in an al,3-linkage with the GlcNAc at the reducing end of the A-glycan, an al, 6- linkage with the GlcNAc at the reducing end of the A-glycan, an al,2-linkage with the Gal at the non-reducing end of the A-glycan, an al,3-linkage with the GlcNac at the nonreducing end of the A-glycan, or an al,4-linkage with a GlcNAc at the non-reducing end of the A-glycan.
  • the glycoform is in an al,3-linkage or al,6-linkage fucose to produce a glycoform selected from the group consisting of MansGlcNAc2(Fuc), GlcNAcMansGlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc); in an al,3-linkage or al,4-linkage fucose to produce a glycoform selected from the group
  • the antibodies e.g., humanized antibodies
  • antigenbinding fragments thereof comprise high mannose A-glycans, including but not limited to, Ma GlcN AC2, Man?GlcNAc2, ManeGlcNAc2, MamGlcN Ac2, Man4GlcNAc2, or N- glycans that consist of the Man3GlcNAc2 A-glycan structure.
  • the complex A-glycans further include fucosylated and non-fucosylated bisected and multi antennary species.
  • A-glycan and “glycoform” are used interchangeably and refer to an TV-linked oligosaccharide, for example, one that is attached by an asparagine-TV-acetylglucosamine linkage to an asparagine residue of a polypeptide.
  • TV-linked glycoproteins contain an TV-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • glycoproteins The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, TV-acetylgalactosamine (GalNAc), TV-acetylglucosamine (GlcNAc) and sialic acid (e.g., TV-acetyl -neuraminic acid (NANA)).
  • GalNAc TV-acetylgalactosamine
  • GlcNAc TV-acetylglucosamine
  • sialic acid e.g., TV-acetyl -neuraminic acid (NANA)
  • TV-glycans have a common pentasaccharide core of MamGlcNAc? (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to TV-acetyl; GlcNAc refers to TV-acetylglucosamine).
  • Man refers to mannose; “Glc” refers to glucose; and “NAc” refers to TV-acetyl; GlcNAc refers to TV-acetylglucosamine.
  • TV-glycan structures are presented with the nonreducing end to the left and the reducing end to the right.
  • the reducing end of the N- glycan is the end that is attached to the Asn residue comprising the glycosylation site on the protein.
  • TV-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the MamGlcNAc? (“Man3”) core structure which is also referred to as the “trimannose core”, the “pentasaccharide core” or the “paucimannose core”.
  • branches comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the MamGlcNAc? (“Man3”) core structure which is also referred to as the “trimannose core”, the “pentasaccharide core” or the “paucimannose core”.
  • TV-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
  • a “high mannose” type TV-glycan has five or more mannose residues.
  • a “complex” type TV-gly can typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a “trimannose” core.
  • Complex TV-glycans may also have galactose (“Gal”) or TV-acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl).
  • Complex TV-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”).
  • Complex TV-glycans may also have multiple antennae on the “trimannose core,” often referred to as “multiple antennary glycans.”
  • a “hybrid” TV-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.
  • the various TV-glycans are also referred to as “glycoforms.”
  • G-2 refers to an TV-glycan structure that can be characterized as Man3GlcNAc2
  • G-l refers to an -glycan structure that can be characterized as GlcNAcMan3GlcNAc2
  • GO refers to an A-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2
  • Gl refers to an A-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2
  • G2 refers to an A-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2
  • Gal2 refers to an A-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2
  • Al refers to an A-glycan structure that can be characterized as NANAGal
  • the “F” indicates that the A-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N- glycan.
  • the A-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N- glycan.
  • GOF, GIF, G2F, A1F, and A2F all indicate that the A-glycan further includes a fucose residue attached to the GlcNAc residue at the reducing end of the A-glycan.
  • Lower eukaryotes such as yeast and filamentous fungi do not normally produce A-glycans that produce fucose.
  • multiantennary N- glycan refers to A-glycans that further comprise a GlcNAc residue on the mannose residue comprising the non-reducing end of the 1,6 arm or the 1,3 arm of the A-glycan or a GlcNAc residue on each of the mannose residues comprising the non-reducing end of the 1,6 arm and the 1,3 arm of the A-glycan.
  • multiantennary A-glycans can be characterized by the formulas GlcNAc(2-4)Man3GlcNAc2, Gal(
  • 1-4 refers to 1, 2, 3, or 4 residues.
  • bisected A-glycan refers to N- glycans in which a GlcNAc residue is linked to the mannose residue at the reducing end of the A-glycan.
  • a bisected A-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2 wherein each mannose residue is linked at its non-reducing end to a GlcNAc residue.
  • a multiantennary A-glycan is characterized as GlcNAc3Man3GlcNAc2
  • the formula indicates that two GlcNAc residues are linked to the mannose residue at the non-reducing end of one of the two arms of the A-glycans and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the A-glycan.
  • the proteins of the invention comprise an aglycosylated Fc region.
  • an IgGl Fc region may be aglycosylayed by deleting or substituting residue N297.
  • the antibodies and antigen-binding fragments thereof disclosed herein may further contain one or more glycosylation sites in either the light or heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or fragment or an alteration of the pK of the antibody due to altered antigen-binding (Marshall et al. (1972) Annu Rev Biochem 41 :673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence.
  • Each antibody or antigen-binding fragment will have a unique isoelectric point (pl), which generally falls in the pH range between 6 and 9.5.
  • the pl for an IgGl antibody typically falls within the pH range of 7-9.5 and the pl for an IgG4 antibody typically falls within the pH range of 6-8.
  • Each antibody or antigen-binding fragment will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71).
  • the TMI the temperature of initial unfolding
  • the melting point of an antibody or fragment can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20: 1952- 60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).
  • antibodies and antigen-binding fragments thereof are selected that do not degrade rapidly. Degradation of an antibody or fragment can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).
  • CE capillary electrophoresis
  • MALDI-MS Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).
  • antibodies and antigen-binding fragments thereof are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties.
  • antibodies and fragments are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.
  • the anti-CD3 antibodies and antigen-binding fragments thereof disclosed herein may also be conjugated to a chemical moiety.
  • the chemical moiety may be, inter alia, a polymer, a radionucleotide or a cytotoxic factor.
  • the chemical moiety is a polymer which increases the half-life of the antibody or fragment in the body of a subject.
  • Suitable polymers include, but are not limited to, hydrophilic polymers which include but are not limited to polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2kDa, 5 kDa, 10 kDa, 12kDa, 20 kDa, 30kDa or 40kDa), dextran and monomethoxypolyethylene glycol (mPEG).
  • PEG polyethylene glycol
  • mPEG monomethoxypolyethylene glycol
  • the antibodies and antigen-binding fragments thereof disclosed herein may also be conjugated with labels such as "Tc, " m Tc, 86 Y, 88 Y, 90 Y, n i In, 32 P, 14 C, 123 I, 124 I, 125 1, 3 H, 131 I, n C, 15 O, 13 N, 18 F, 19 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 Eu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 45 Ti, 89 Zr, 217 Ci, 211 At, 212 Pb, 177 Lu, 44 Sc, 47 Sc, 109 p d , 234 Th , and 40 K , 157 Gd 55 Mn , 52 ⁇ an( J 56 Fe
  • the antibodies and antigen-binding fragments disclosed herein may also be PEGylated, for example to increase its biological (e.g., serum) half-life.
  • PEG polyethylene glycol
  • the antibody or fragment typically is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody or fragment to be PEGylated is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401 384.
  • the antibodies and antigen-binding fragments disclosed herein may also be conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152 Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3 -dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.
  • fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate,
  • the antibodies and antigen-binding fragments thereof of the invention may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
  • a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and
  • a metal chelator(s) is a molecule having one or more polar groups that act as a ligand for, and complex with, a paramagnetic metal. Suitable chelators are known in the art and include acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups, carboxy ethylidene groups, or carboxymethylene groups.
  • chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTP A), l,4,7,10-tetraazacyclo-tetradecane-l,4,7,10-tetraacetic acid (DOTA), 1- substituted 1,4, 7, -tricarboxymethyl- 1,4, 7, 10-teraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and l,4,8,l l-tetra-azacyclotetradecane-l,4,8,l l- tetraacetic acid (TETA).
  • DTP A diethylenetriaminepentaacetic acid
  • DO3A 1- substituted 1,4, 7, -tricarboxymethyl- 1,4, 7, 10-teraazacyclododecane
  • EDTA ethylenediaminetetraacetic acid
  • TETA l,4,8,l l-tetra-azacyclotetradecane-l,4,8,l l-
  • Additional chelating ligands are ethylene bis-(2-hydroxy- phenylglycine) (EHPG), and derivatives thereof, including 5-C1-EHPG, 5Br-EHPG, 5- Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; benzodi ethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTP A; bis-2 (hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds, which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA
  • any method known in the art for conjugating the antibodies and antigenbinding fragments thereof of the invention to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13: 1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies and fragments are conventional and very well known in the art.
  • Chemical cross-linkers may be classified on the basis of the following:
  • Reactive groups on antibodies and labels that can be targeted using a crosslinker include primary amines, carbonyls, carbohydrates and carboxylic acids.
  • many reactive groups can be coupled nonselectively using a cross-linker such as photoreactive phenyl azides.
  • suitable reagents see Pierce 2003-2004 Applications Handbook and Catalog # 1600926, which is hereby incorporated by reference.
  • cross-linker-to-target molar ratios Many factors must be considered to determine optimum cross-linker-to-target molar ratios. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is normally desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal to ensure that the biological activity of the protein is retained. It is also important to consider the number of reactive groups on the surface of the protein. If there are numerous target groups, a lower cross-linker-to-protein ratio can be used. For a limited number of potential targets, a higher cross-linker-to-protein ratio may be required. This translates into more cross-linker per gram for a small molecular weight protein.
  • Conformational changes of proteins associated with a particular interaction may also be analyzed by performing cross-linking studies before and after the interaction. A comparison is made by using different arm-length cross-linkers and analyzing the success of conjugation.
  • the use of cross-linkers with different reactive groups and/or spacer arms may be desirable when the conformation of the protein changes such that hindered amino acids become available for cross-linking.
  • Cross-linkers are available with varying lengths of spacer arms or bridges connecting the reactive ends.
  • the most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites for cross-linking, different lengths of bridges may be considered for the interaction. Shorter spacer arms are often used in intramolecular cross-linking studies, while intermolecular cross-linking is favored with a cross-linker containing a longer spacer arm.
  • polymer portions e.g., polyethylene glycol (“PEG”) homopolymers, polypropylene glycol homopolymers, other alkyl-polyethylene oxides, bis-polyethylene oxides and co-polymers or block co-polymers of poly(alkylene oxides)
  • PEG polyethylene glycol
  • polypropylene glycol homopolymers other alkyl-polyethylene oxides, bis-polyethylene oxides and co-polymers or block co-polymers of poly(alkylene oxides)
  • cross-linkers can, under certain circumstances be advantageous. See, e.g., U.S. Patents 5,643,575, 5,672,662, 5,705,153, 5,730,990, 5,902,588, and 5,932,462; and Topchieva et al., Bioconjug. Chem. 6: 380-8, 1995).
  • U.S. Patent 5,672,662 discloses bifunctional cross-linkers comprising a PEG polymer portion and a single este
  • Designing a cross-linker involves selection of the functional moieties to be employed.
  • the choice of functional moieties is entirely dependent upon the target sites available on the species to be crosslinked.
  • Some species e.g., proteins
  • may present a number of available sites for targeting e.g., lysine 8-amino groups, cysteine sulfhydryl groups, glutamic acid carboxyl groups, etc.
  • selection of a particular functional moiety may be made empirically in order to best preserve a biological property of interest (e.g., binding affinity of an antibody, catalytic activity of an enzyme, etc.)
  • Coupling through Amine Groups e.g., binding affinity of an antibody, catalytic activity of an enzyme, etc.
  • Homobifunctional NHS-ester conjugations are commonly used to cross-link amine- containing proteins in either one-step or two-step reactions.
  • Primary amines are the principle targets for NHS -esters. Accessible a-amine groups present on the N-termini of proteins react with NHS-esters to form amides.
  • a-amines on a protein are not always available, the reaction with side chains of amino acids become important. While five amino acids have nitrogen in their side chains, only the s-amino group of lysine reacts significantly with NHS-esters.
  • a covalent amide bond is formed when the NHS-ester cross-linking agent reacts with primary amines, releasing N- hydroxysuccinimide.
  • maleimides, alkyl and aryl halides, a-haloacyls, and pyridyl disulfides are typically employed as sulfhydryl-specific functional moieties.
  • the maleimide group is specific for sulfhydryl groups when the pH of the reaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reaction of the maleimides with sulfhydryls is 1000-fold faster than with amines.
  • Maleimides do not react with tyrosines, histidines or methionines. When free sulfhydryls are not present in sufficient quantities, they can often be generated by reduction of available disulfide bonds.
  • Carbodiimides couple carboxyls to primary amines or hydrazides, resulting in formation of amide or hydrazone bonds.
  • Carbodiimides are unlike other conjugation reactions in that no cross-bridge is formed between the carbodiimide and the molecules being coupled; rather, a peptide bond is formed between an available carboxyl group and an available amine group.
  • Carboxy termini of proteins can be targeted, as well as glutamic and aspartic acid side chains. In the presence of excess cross-linker, polymerization may occur because proteins contain both carboxyls and amines. No crossbridge is formed, and the amide bond is the same as a peptide bond, so reversal of the cross-linking is impossible without destruction of the protein.
  • a photoaffinity reagent is a compound that is chemically inert but becomes reactive when exposed to ultraviolet or visible light.
  • Arylazides are photoaffinity reagents that are photolyzed at wavelengths between 250-460 nm, forming a reactive aryl nitrene. The aryl nitrene reacts nonselectively to form a covalent bond. Reducing agents must be used with caution because they can reduce the azido group.
  • Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH 5- 7.
  • the reaction with hydrazides is faster than with amines, making this useful for sitespecific cross-linking.
  • Carbonyls do not readily exist in proteins; however, mild oxidation of sugar moieties using sodium metaperiodate will convert vicinal hydroxyls to aldehydes or ketones.
  • the target binding proteins of the invention may comprise one or more immunoglobulin variable regions in addition to the CD3 -binding domain described herein.
  • the target binding protein may comprise an additional light chain variable domain and an additional heavy chain variable domain.
  • the additional light chain variable domain and the additional heavy chain variable domain may form an additional immunoglobulin variable region.
  • the CD3- binding domain and the additional immunoglobulin variable region may be identical.
  • the CD3 -binding domain and the additional immunoglobulin variable region may be different from each other (e.g., may specifically bind to the same or different antigens or epitopes).
  • the CD3 -binding domain and the additional immunoglobulin variable region may be both Fv fragments, or at least one may be a Fv fragment. In some embodiments, the CD3 -binding domain and the additional immunoglobulin variable region may be both Fab fragments, or at least one may be a Fab fragment. In some embodiment, the CD3 -binding domain and an additional immunoglobulin variable region may be a Fab' fragment, or at least one can be a Fab’ fragment.
  • the target binding protein may be multispecific (e.g., bispecific, trispecific, tetraspecific, and other multispecific target binding proteins), e.g., binding to CD3 and one or more additional targets.
  • the multispecific target binding protein may be multivalent, e g , comprising multiple target binding sites regardless of whether the binding sites recognize the same or different targets.
  • the target binding protein may be bispecific.
  • the term “bispecific” means that target binding protein is able to specifically bind to two distinct targets.
  • a bispecific target binding protein comprises two immunoglobulin variable regions, each of which is capable of specifically binding to a different target.
  • the bispecific target binding protein may be capable of simultaneously binding two targets, e.g., two target proteins expressed on two distinct cells.
  • the target binding protein may comprise the CD3- binding domain and an additional immunoglobulin variable region capable of binding to a molecule on the surface of a cell associated with a disease (e.g., a tumor cell).
  • a disease e.g., a tumor cell
  • target binding proteins may simultaneously bind to an immune cell (e.g., T cell) and a cell associated with a disease (e.g., a tumor cell), thus activating the immune cell and crosslinking the activated immune cell to the cell associated with the disease.
  • the target binding protein may be formulated as part of chimeric antigen receptor (CAR), a T cell engaging antibody (e.g., bispecific T cell engaging antibody or BiTE), a pro-Bispecific T Cell Engager (pro-BiTE) molecule, pro-Chimeric Antigen Receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cell, such as a CAR modified NK cell.
  • CAR chimeric antigen receptor
  • a T cell engaging antibody e.g., bispecific T cell engaging antibody or BiTE
  • pro-BiTE pro-Bispecific T Cell Engager
  • pro-CAR pro-Chimeric Antigen Receptor
  • the target binding protein may be a monovalent bispecific antibody comprising the CD3-binding immunoglobulin variable region and an additional immunoglobulin variable region described herein.
  • the term “monovalent bispecific antibody” refers to a bispecific antibody, in which only one antigen-binding domain is directed against a given target.
  • the inventors have surprisingly discovered that certain CD3 -binding domains described herein display improved stability, manufacturability, and/or CD3 binding affinity in the context of a monovalent CD3 binding protein, including monovalent bispecific antibodies that specifically bind CD3 (e.g., an anti-CD3 scFv).
  • the target of the additional immunoglobulin variable region may be a protein or other type of molecules, e.g., cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g. collagen, fibronectin) and the like.
  • cell surface receptors and secreted binding proteins e.g., growth factors
  • soluble enzymes e.g., soluble enzymes
  • structural proteins e.g. collagen, fibronectin
  • the additional immunoglobulin variable region may bind to a target that is a molecule on or inside a cell that is associated with a disease.
  • the additional immunoglobulin variable region may bind to a tumor cell.
  • the additional immunoglobulin variable region may bind to a tumor associated antigen.
  • tumor associated antigen refers to any antigen including a protein, glycoprotein, ganglioside, carbohydrate, lipid that is associated with cancer. Such antigen may be expressed on tumor cells (e.g., malignant cells) or in the tumor microenvironment such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infdtrates.
  • the tumor associated antigen may be human epidermal growth factor receptor 2 (HER2).
  • the additional target binding domain may be trastuzumab or a fragment thereof.
  • the additional immunoglobulin variable region may be pertuzumab or a fragment thereof.
  • a bispecific antibody comprises heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation.
  • the bispecific antibody is generated using a knobs-into-holes (KIH) strategy.
  • the bispecific antibody comprises variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation).
  • the bispecific antibody comprises heavy chains with changes in amino acids that result in altered electrostatic interactions.
  • the bispecific antibodies comprise heavy chains with changes in amino acids that result in altered hydrophobic/hydrophilic interactions.
  • the target binding proteins herein include activatable target binding proteins.
  • an activatable target binding protein may comprise a prodomain, which refers to a polypeptide that, when linked to a target binding protein, functions to inhibit target binding by the target binding protein and includes an amino acid sequence that form a protease cleavable substrate.
  • the portion of the prodomain that inhibits target binding is referred to as a masking moiety (MM) and the amino acid sequence that is a protease cleavable substrate is referred to as a cleavable moiety (CM).
  • MM masking moiety
  • CM cleavable moiety
  • the prodomain may include a linker (L) between the MM and the CM and/or at the prodomain’s terminus (e.g., carboxyl and/or amino terminus to facilitate the linkage of the prodomain to the target binding protein).
  • a prodomain may comprise one of the following formulae (representing an amino acid sequence in an N- to C-terminal direction): MM-CM, MM-L-CM, MM-CM-L, MM-L-CM-L, CM-MM, CM- L-MM, L-CM-MM, or L-CM-L-MM, wherein each represents a direct or indirect (e.g., via a linker) linkage.
  • activatable target binding protein refers to a target binding protein in its inactive (uncleaved or native) state. It will be apparent to the ordinarily skilled artisan that in some embodiments a cleaved activatable target binding protein may be connected to a MM that is not reducing, inhibiting, or interfering with binding between the immunoglobulin variable region and its target. In some embodiments, a cleaved activatable target binding protein may lack a MM due to cleavage of the CM (e.g., by a protease), resulting in release of the MM.
  • cleaved state or “active state” refers to the condition of the activatable target binding proteins following cleavage of the CM by at least one protease.
  • uncleaved state or “inactive state” refers to the condition of the activatable target binding proteins in the absence of cleavage of the CM by a protease.
  • activatable is meant that the activatable target binding protein exhibits a first level of binding to a target when the activatable target binding protein is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target in the uninhibited, unmasked and/or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding.
  • the access of target to the immunoglobulin variable region of the activatable target binding protein is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent.
  • a cleaving agent capable of cleaving the CM i.e., a protease
  • the access of target to the immunoglobulin variable region of the activatable target binding protein is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease
  • the access of target to the immunoglobulin variable region of the activatable target binding protein is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent.
  • an activatable target binding protein may be designed by selecting an immunoglobulin variable region of interest and constructing the remainder of the activatable target binding protein so that, when conformationally constrained, the MM provides for masking of the immunoglobulin variable region or reduction of binding of the immunoglobulin variable region to its target. Structural design criteria can be to be taken into account to provide for this functional feature.
  • Activatable target binding proteins herein may exhibit an activatable phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited conformation.
  • Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions.
  • the dynamic range refers to the ratio of (a) a maximum detected level of target protein binding to an activatable target binding protein in the presence of a protease capable of cleaving a CM in the activatable target binding proteins to (b) a minimum detected level of target protein binding to an activatable target binding protein in the absence of the protease.
  • the dynamic range of an activatable target binding protein can be calculated as the ratio of the dissociation constant of an activatable target binding protein cleaving agent (e.g., enzyme) treatment to the dissociation constant of the activatable target binding proteins cleaving agent treatment.
  • Activatable target binding proteins having relatively higher dynamic range values exhibit more desirable activatable phenotypes such that target protein binding by the activatable target binding proteins occurs to a greater extent (e.g., predominantly occurs) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the activatable target binding proteins than in the absence of a cleaving agent.
  • a cleaving agent e.g., enzyme
  • the activatable target binding protein herein may comprise a immunoglobulin variable region (TB), one or more masking moieties (MMs) reducing, inhibiting, or interfering with the binding of the immunoglobulin variable region to its target(s), one or more cleavable moieties (CMs) that couple the one or more MMs to the TB, and optionally one or more half-life extending moieties (EMs).
  • TB immunoglobulin variable region
  • MMs masking moieties
  • CMs cleavable moieties
  • EMs half-life extending moieties
  • the activatable target binding protein may comprise the TB that specifically binds to CD3, and a masking moiety (MM) inhibiting the binding of the TB and CD3, wherein the MM is coupled to the TB via a cleavable moiety (CM) (either directly or indirectly, e.g., via one or more linkers).
  • CM cleavable moiety
  • components of the activatable target binding protein that are “coupled” may be coupled either via a direct covalent linkage or indirect covalent linkage, e.g., via one or more linking peptides (also referred to as “linkers”), cleavable moieties, or other components of the activatable protein.
  • the activatable target binding protein may comprise more than one immunoglobulin variable regions (TBs).
  • the activatable target binding protein may comprise the first TB that specifically binds to CD3, a first MM (MM1) inhibiting the binding of the TB1 and CD3, wherein the MM1 is coupled to the TB1 via a first cleavable moiety (CM1) (either directly or indirectly, e.g., via one or more linkers), a second immunoglobulin variable region (TB2) that specifically binds to a second target, a second masking moiety (MM2) inhibiting the binding of the TB2 and the second target, wherein the MM2 is coupled to the TB2 via a second cleavable moiety (CM2) (either directly or indirectly, e.g., via one or more linkers).
  • the activatable target binding protein may further comprise a half-life extending moiety (EM).
  • the activatable target binding protein comprise a sc
  • the activatable target binding proteins herein may comprise one or more masking moieties (MMs) capable of interfering with the binding of the TBs to the targets.
  • MMs masking moieties
  • a masking moiety in an activatable molecule (that is not yet activated) “masks” or reduces or otherwise inhibits the binding of the immunoglobulin variable region to its target.
  • the coupling or modifying of target binding protein with a MM may inhibit the ability of the protein to specifically bind its target by means of inhibition known in the art (e.g., structural change and competition for antigen-binding domain).
  • the coupling or modifying of a target binding protein with a MM may effect a structural change that reduces or inhibits the ability of the protein to specifically bind its target. In some embodiments, the coupling or modifying of a target binding protein with a MM sterically blocks, reduces or inhibits the ability of the antigenbinding domain to specifically bind its target.
  • a MM may be coupled to a TB by a CM and optionally one or more linkers described herein.
  • the MM prevents the TB from target binding; but when the activatable target binding protein is activated (when the CM is cleaved by a protease), the MMs does not substantially or significantly interfere with the TB’s binding to the target.
  • a MM interfering with the target binding of a TB may be coupled to the TB (either directly or indirectly, e.g., via one or more linkers).
  • a MM interfering with the target binding of a TB may be coupled, either directly or indirectly, to a component of the activatable target binding protein that is not the TB.
  • the MM may be coupled, either directly or indirectly, to a different TB.
  • the MM may be coupled, either directly or indirectly, with an EM.
  • the MM in the tertiary or quaternary structure of the activatable structure, may be in a position (e.g., proximal to the TB to be masked) that allows the MM to mask the TB.
  • a MM may interact with the TB, thus reducing or inhibiting the interaction between the TB and its binding partner.
  • the MM may comprise at least a partial or complete amino acid sequence of a naturally occurring binding partner of the TB.
  • the MM may be a fragment of a naturally occurring binding partner. The fragment may retain no more than 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, or 20% nucleic acid or amino acid sequence homology to the naturally occurring binding partner.
  • the MM may be a cognate peptide of the TB.
  • the MM may comprise a sequence of the TB’s epitope or a fragment thereof.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.
  • the MM may comprise an amino acid sequence that is not naturally occurring or does not contain the amino acid sequence of a naturally occurring binding partner or target protein.
  • the MM is not a natural binding partner of the TB.
  • the MM may be a modified binding partner for the TB which contains amino acid changes that decrease affinity and/or avidity of binding to the TB.
  • the MM may contain no or substantially no nucleic acid or amino acid homology to the TB’s natural binding partner.
  • the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to the natural binding partner of the TB.
  • the MM may not specifically bind to the TB, but still interfere with TB’s binding to its binding partner through non-specific interactions such as steric hindrance.
  • the MM may be positioned in the activatable target binding protein such that the tertiary or quaternary structure of the activatable target binding protein allows the MM to mask the TB through charge-based interaction, thereby holding the MM in place to interfere with binding partner access to the TB.
  • the MM may have a dissociation constant for binding to the TB that is no more than the dissociation constant of the TB to the target.
  • the MM may not interfere or compete with the TB for binding to the target in a cleaved state.
  • the structural properties of the MMs may be selected according to factors such as the minimum amino acid sequence required for interference with protein binding to target, the target protein-protein binding pair of interest, the size of the TB, the presence or absence of linkers, and the like.
  • the MM may be unique for the coupled TB.
  • MMs include MMs that were specifically screened to bind a binding domain of the TB or fragment thereof (e.g., affinity masks).
  • Methods for screening MMs to obtain MMs unique for the TB and those that specifically and/or selectively bind a binding domain of a binding partner/target are provided herein and can include protein display methods.
  • the term “masking efficiency” refers to the activity (e.g., EC50) of the activatable target binding protein in the inactivated state divided by the activity of a control antibody, wherein the control antibody may be either cleavage product of the activatable target binding protein or the antibody or fragment thereof used as the TB of the activatable target binding protein.
  • An activatable target binding protein having a reduced level of a TB activity may have a masking efficiency that is greater than 10.
  • the activatable target binding proteins described herein may have a masking efficiency that is greater than 10, 100, 1000, or 5000.
  • the MM may be a polypeptide of about 2 to 50 amino acids in length.
  • the MM may be a polypeptide of from 2 to 40, from 2 to 30, from 2 to 20, from 2 to 10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25 to 35, from 30 to 40, from 35 to 45, from 40 to 50 amino acids in length.
  • the MM may be a polypeptide with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
  • the MM may be a polypeptide of more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.
  • the binding affinity of the TB towards the target or binding partner with an interfering MM may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times lower than the binding affinity of the TB towards its binding partner without an interfering MM, or between 5-10, 10-100, 10- 1,000, 10-10,000, 10-100,000, 10-1,000,000, 10- 10,000,000, 100-1,000, 100-10,000, 100- 100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000- 10,000,000, 10,000-100,000, 10,000-1 ,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the TB towards its binding partner when there is no interfering MM.
  • the dissociation constant of the MM towards the TB it masks may be greater than the dissociation constant of the TB towards the target.
  • the dissociation constant of the MM towards the masked TB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater than the dissociation constant of the TB towards the target.
  • the binding affinity of the MM towards the masked TB may be lower than the binding affinity of the TB towards the target.
  • the binding affinity of MM towards the TB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lower than the binding affinity of the TB towards the target.
  • the MMs may contain genetically encoded or genetically nonencoded amino acids.
  • genetically non-encoded amino acids include but are not limited to D-amino acids, P-amino acids, and y-amino acids.
  • the MMs contain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% of genetically non-encoded amino acids.
  • the MM may have a biological activity or a therapeutic effect, such as binding capability.
  • the free peptide may bind with the same or a different binding partner.
  • the free MM may exert a therapeutic effect, providing a secondary function to the compositions disclosed herein.
  • the MM once uncoupled from the activatable target binding protein and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments the MM in a free state does not elicit an immune response in the subject.
  • Suitable MMs may be identified and/or further optimized through a screening procedure from a library of candidate activatable target binding proteins having variable MMs.
  • a TB and a CM may be selected to provide for a desired enzyme/target combination, and the amino acid sequence of the MM can be identified by the screening procedure described below to identify a MM that provides for a switchable phenotype.
  • a random peptide library e.g., of peptides comprising 2 to 40 amino acids or more
  • MMs with specific binding affinity for a TB may be identified through a screening procedure that includes providing a library of peptide scaffolds comprising candidate MMs wherein each scaffold is made up of a transmembrane protein and the candidate MM.
  • the library may then be contacted with an entire or portion of a protein such as a full length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein (also capable of binding the binding partner of interest), and identifying one or more candidate MMs having detectably bound protein.
  • the screening may be performed by one more rounds of magnetic-activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as determination of the binding affinity of MM towards the TB and subsequent determination of the masking efficiency, e.g., as described in W02009025846 and US20200308243A1, which are incorporated herein by reference in their entireties.
  • MCS magnetic-activated sorting
  • FACS fluorescence-activated sorting
  • a MM may be selected for use with a specific antibody or antibody fragment.
  • suitable MM for use with a TB that binds to an epitope may comprise the sequence of the epitope.
  • suitable MM for masking the anti-CD3 binding proteins disclosed herein include MMs comprising the sequences of GYLWGCEWNCGGITT (SEQ ID NO: 2315), NAFRCWWDPPCQPMT (SEQ ID NO: 2316), ARGLCWWDPPCTHDL (SEQ ID NO: 2317), or NHSLCYWDPPCEPST (SEQ ID NO: 2318).
  • Additional masking moieties for anti-CD3 binding proteins include the sequences of MMYCGGNEVLCGPRV (SEQ ID NO: 2319), GYRWGCEWNCGGITT (SEQ ID NO: 2320), MMYCGGNEIFCEPRG (SEQ ID NO: 2321), GYGWGCEWNCGGSSP (SEQ ID NO: 2322), and MMYCGGNEIFCGPRG (SEQ ID NO: 2323).
  • MMs examples include WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, W02020118109, W02020092881, W02020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, W02019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, W02017011580, WO2016179335, WO2016179285, WO2016179257, W02016149201, and WO2016014974, which are incorporated herein by reference in their entireties.
  • the activatable target binding protein may comprise one or more cleavable moieties (CMs).
  • CMs cleavable moieties
  • cleavable moiety and “CM” are used interchangeably herein to refer to a peptide, the amino acid sequence of which comprises a substrate for a sequence-specific protease.
  • the CM may be positioned between a TB and a MM.
  • the CM and the TB of the activatable target binding proteins may be selected so that the TB represents a binding moiety for a given target, and the CM represents a substrate for one or more proteases, where the protease is co-localized with the target in a tissue (e.g., at a treatment site or diagnostic site in a subject).
  • the protease may cleave the CM in the activatable target binding protein when the activatable target binding protein is exposed to the protease.
  • the activatable target binding proteins may find particular use where, for example, one or more proteases capable of cleaving a site in the CM, is present at relatively higher levels in target-containing tissue of a treatment site or diagnostic site than in tissue of non-treatment sites (for example in healthy tissue).
  • the CMs herein may comprise substrates for proteases that have known substrates have been reported in a number of cancers. See, e.g., La Roca et al., British J. Cancer 90(7): 1414-1421, 2004. Substrates suitable for use in the CM components employed herein include those which are more prevalently found in cancerous cells and tissue. Thus, in certain embodiments, the CM may comprise a substrate for a protease that is more prevalently found in diseased tissue associated with a cancer.
  • the cancers include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, a HER2-positive cancer, Kaposi sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell cancer (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, ovarian cancer, bladder cancer, BCG-resistant nonmuscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck carcinoma, uterine cancer, cervical cancer, or testicular cancer, and the like.
  • the CM components comprise substrates for protease(s) that is/are more prevalent in tumor tissue.
  • the activatable target binding protein may comprise two CMs (e.g., for coupling MMs to multiple TBs).
  • the first and the second CMs may comprise the substrates of the same protease.
  • the first and the second CMs may comprise the substrates of different proteases.
  • the first and the second CMs may comprise or consist of the same sequence. Tn some examples, the first and the second CMs may comprise or consist of different sequences.
  • CMs for use in the activatable target binding protein herein include any of the protease substrates that are known the art.
  • the CM may comprise a substrate of a serine protease (e.g., u-type plasminogen activator (uPA, also referred to as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1).
  • uPA u-type plasminogen activator
  • MMP matrix metalloprotease
  • the CM may comprise a substrate of cysteine protease (CP) (e.g., legumain).
  • the CM may comprise a substrate for a disintegrin and metalloproteinase (ADAM) or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)(e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAMI 7/T ACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), aspartate protease (e.g.
  • ADAM disintegrin and metalloproteinase
  • ADAMTS disintegrin and metalloproteinase with thrombospondin motifs
  • cysteine cathepsin e.g., Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P
  • cysteine proteinase e.g., Cruzipain, Legumain, Otubain-2
  • Chymase DESCI, DPP-4, FAP, Elastase, FVlIa, F1XA, FXa, FXIa, FX
  • the protease substrate in the CM may comprise a peptide sequence that is not substantially identical (e.g., no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence that is naturally cleaved by the same protease.
  • CMs include those described in WO 2010/081173,
  • the CM may be or comprise a combination, a C- terminal truncation variant, or an N-terminal truncation variant of the example sequences discussed above.
  • Truncation variants of the aforementioned amino acid sequences that are suitable for use in a CM may be any that retain the recognition site for the corresponding protease. These include C-terminal and/or N-terminal truncation variants comprising at least 3 contiguous amino acids of the above-described amino acid sequences, or at least 4, 5, 6, 7, 8, 9, or 10 amino acids of the foregoing amino acid sequences that retain a recognition site for a protease.
  • the truncation variant of the above-described amino acid sequences may be an amino acid sequence corresponding to any of the above, but that is C- and/or N-terminally truncated by 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, or 1 to 3 amino acids, and which: (1) has at least three amino acid residues; and (2) retains a recognition site for a protease.
  • the truncated CM is an N-terminally truncated CM.
  • the truncated CM is a C-terminally truncated CM.
  • the truncated C is a C- and an N-terminally truncated CM.
  • the CM may comprise a total of 3 amino acids to 25 amino acids. In some embodiments, the CM may comprise a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.
  • the CM may be specifically cleaved by at least a protease at a rate of about 0.001-1500 x 10 4 M/S or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M/S.
  • the rate may be measured as substrate cleavage kinetics (kcat/K m ) as disclosed in WO2016118629. Conjugation agents
  • the target binding proteins may further comprise one or more additional agents, e.g., a targeting moiety to facilitate delivery to a cell or tissue of interest, a therapeutic agent (e.g., an antineoplastic agent such as chemotherapeutic or anti -neoplastic agent), a toxin, a radioisotope, a small molecule, a diagnostic agent, a targeting moiety, or a detectable moiety, or a fragment thereof.
  • the additional agents may be conjugated to the target binding proteins.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • the antibody or antigen-binding fragment thereof is admixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).
  • Formulations of therapeutic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al.
  • Toxicity and therapeutic efficacy of the target binding proteins of the invention, administered alone or in combination with another therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index (LD50/ ED50).
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration.
  • Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration and oral administration.
  • Administration of target binding proteins, used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection.
  • the target binding protein of the invention is administered intravenously.
  • the target binding proteins of the invention is administered subcutaneously.
  • a further therapeutic agent that is administered to a subject in association with the target binding proteins of the invention in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).
  • Methods for co-administration or treatment with a second therapeutic agent are well known in the art, see, e.g., Hardman, et al.
  • the other agents include, but are not limited to, a cytotoxic, chemotherapeutic, cytostatic, anti-angiogenic or antimetabolite agents, a tumor targeted agent, an immune stimulating or immune modulating agent or an antibody conjugated to a cytotoxic, cytostatic, or otherwise toxic agent.
  • the pharmaceutical composition can also be employed with other therapeutic modalities such as surgery, chemotherapy and radiation.
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention can be administered by an invasive route such as by injection.
  • an anti-CD3 antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery.
  • Administration by non-invasive routes e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.
  • the present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the target binding proteins of the invention or a pharmaceutical composition thereof.
  • a vessel e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder
  • the present invention also provides an injection device comprising any of the target binding proteins of the invention or a pharmaceutical composition thereof.
  • An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous or intravenous.
  • an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., antibody or fragment or a pharmaceutical composition thereof), a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore.
  • an injection device that comprises an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof is an intravenous (IV) injection device.
  • Such a device includes the antibody or fragment or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaCL and optionally including glucose) introduced into the body of the patient through the cannula or trocar/needle.
  • fluid e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KC1, CaCL and optionally including glucose
  • the antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula.
  • the IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line).
  • an injection device is an autoinjector; a jet injector or an external infusion pump.
  • a jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a patient’s body.
  • External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a patient’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically.
  • Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery.
  • a set of rollers pinches down on a length of flexible tubing, pushing fluid forward.
  • fluids can be delivered from multiple reservoirs at multiple rates.
  • compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413;
  • Such needleless devices comprising the pharmaceutical composition are also part of the present invention.
  • the pharmaceutical compositions disclosed herein may also be administered by infusion.
  • Examples of well- known implants and modules for administering the pharmaceutical compositions include those disclosed in: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No.
  • the liposomes will be targeted to and taken up selectively by the afflicted tissue. Such methods and liposomes are part of the present invention.
  • the administration regimen depends on several factors, including the serum or tissue turnover rate of the target binding proteins (anti-CD3 antibody or antigen-binding fragment), the level of symptoms, the immunogenicity of the target binding proteins, and the accessibility of the target cells in the biological matrix.
  • the administration regimen delivers sufficient therapeutic antibody or fragment to effect improvement in the target disease state, while simultaneously minimizing undesired side effects.
  • the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies or fragments is available (see, e.g, Wawrzynczak 1996) Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g, using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent.
  • humanized and fully human antibodies may be desirable.
  • the target binding proteins disclosed herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc.
  • Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation.
  • a total weekly dose is generally at least 0.05 pg/kg body weight, more generally at least 0.2 pg/kg, 0.5 pg/kg, 1 pg/kg, 10 pg/kg, 100 pg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346: 1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych.
  • a target binding protein of the present invention is administered, e.g., subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.
  • the target binding proteins can be administered over a period of at least about 1 week, 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, or as deemed appropriate by the treating physician.
  • a chronic condition can exist for, e.g., at least about 6 weeks, 2 months, a year, or longer.
  • the target binding proteins can be administered over a period of at least about 6 weeks, 2 months, 3 months or 6 months, a year, or even multiple years as required for medical care of an individual.
  • the target binding proteins can also be administered in an irregular manner. Early achievement of an effective target antibody concentration (a therapeutic dose level) with a loading dose followed by maintenance dosing with the antibody (frontloading) may be more effective than conventional therapy in terms of requiring a lower total antibody dose and faster time to maximum target engagement. As used herein, such an administration protocol is referred to as a “loading/maintenance administration protocol.” An effective target antibody concentration may be reached in 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, most preferably 1 week or less, including 1 day or less using a loading dose. The target serum concentration is then maintained by administration of an equal or smaller (or less frequent) maintenance dose during the remainder of the treatment regimen or until suppression of disease symptoms is achieved.
  • frontloading when referring to drug administration refers to the initial loading dose, followed by the maintenance dose.
  • the initial loading dose (single or multiple) is intended to more quickly increase the serum drug concentration of an animal or human patient to an effective target serum concentration.
  • frontloading is accomplished by initial dosing delivered over 3 weeks or less so that the antibody reaches the target serum concentration.
  • the loading dose or series of doses is administered for 2 weeks or less, more preferably 1 week or less, e.g. 1 day or less.
  • the loading dosing is a single dosing, with no maintenance dosing thereafter for at least one week, and the loading dosing is administered in 1 day or less.
  • it may be preferred to deliver the loading dose of antibody is administered by intravenous injection.
  • the present invention includes loading and maintenance doses of frontloading drug delivery by intravenous or subcutaneous administration.
  • Administration of the loading dose can be, for example, one or more dosings at a time interval of at least about 1, 2, 3, 4, 5, 6, 7 or 8 weeks apart.
  • the at least one loading dose is administered by one or more intravenous injections and then at least one maintenance dose by one or more intravenous or subcutaneous administrations.
  • the instructions can be for administering at least one loading dose by, for example, one or more intravenous or subcutaneous administrations and at least one maintenance dose by one or more intravenous or subcutaneous administrations.
  • both the at least one loading dose as well as the at least one maintenance dose is administered subcutaneously.
  • the at least one loading dose is administered by intravenous infusion followed by at least one maintenance dose administered subcutaneously.
  • the method of treatment can comprise administering a loading dose of 150-1350 mg of the anti-CD3 antibody by intravenous infusion or subcutaneous injection.
  • a maintenance dose of 600 mg or less of the anti-CD3 antibody can be administered every 4 weeks or less, preferably every 3 weeks or less, more preferably every 2 weeks or less, and in embodiments every 1 week or less, by subcutaneous injection.
  • the choice of loading and maintenance dosages and intervals can be made according to the ability of the animal or human patient to tolerate administration of the antibody to the body and according to a desired serum level of therapeutic to achieve.
  • a loading dose of a drug can be larger (e.g., about 1.5, 2, 3, 4 or 5 times larger) than a subsequent maintenance dose.
  • the one or more therapeutically effective maintenance doses can be any therapeutically effective amount described herein.
  • the loading dose can be about 2 or 3 times larger than the maintenance dose.
  • the anti-CD3 antibody can be administered in two (or more) loading doses prior to the maintenance dose.
  • a first loading dose of the antibody or fragment thereof can be administered on day 1, a second loading dose can be administered, e.g., about 1 or 2 weeks later, and a maintenance dose can be administered, e.g., once weekly or once every 2 weeks thereafter for the duration of treatment.
  • the first loading dose can be about 3 or 4 times larger than the maintenance dose
  • the second loading dose can be about 2, 3, 4, 5, or more times larger than the maintenance dose.
  • inhibitor or “treat” or “treatment” includes a postponement of development of the symptoms associated with disease and/or a reduction in the severity of such symptoms that will or are expected to develop with said disease.
  • the terms further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the terms denote that a beneficial result has been conferred on a vertebrate subject with a disease.
  • the term "effective amount” refers to an amount of a target binding proteins f of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of disease, for example cancer or the progression of cancer.
  • An effective dose further refers to that amount of the antibody or fragment sufficient to result in at least partial amelioration of symptoms, e.g., tumor shrinkage or elimination, lack of tumor growth, increased survival time.
  • an effective dose refers to that ingredient alone.
  • an effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.
  • An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.
  • kits comprising one or more components that include, but are not limited to, a target binding protein as described herein in association with one or more additional components including, but not limited to a pharmaceutically acceptable carrier and/or a therapeutic agent, as discussed herein.
  • the antibody or fragment and/or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.
  • the kit includes an anti-CD3 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and/or a therapeutic agent and a pharmaceutical composition thereof in another container (e.g., in a sterile glass or plastic vial).
  • the kit comprises a combination of the invention, including an anti-CD3 antibody or antigen-binding fragment thereof of the invention along with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.
  • the kit can include a device for performing such administration.
  • the kit can include one or more hypodermic needles or other injection devices as discussed above.
  • the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit.
  • information concerning the pharmaceutical compositions and dosage forms in the kit aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
  • the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/ distributor information and patent information.
  • Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane ( ⁇ iUL) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160: 1029; Tang et al. (1999) J. Biol. Chem.
  • An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1 :837-839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21 :371- 377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).
  • Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998) J. Immunol. 161 :3493; Kita et o/. (1999) J. Immunol. 162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276: 12999; Propst et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).
  • Animals can be immunized with cells bearing the antigen of interest.
  • Splenocytes can then be isolated from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).
  • Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g., colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).
  • PEG polyethylene glycol
  • Fluorescent reagents suitable for modifying nucleic acids including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).
  • the second phase of engineering focused on optimization of a subset of clones.
  • Results from phase one engineering provided insight as to which amino acid positions affected yield, binding or biophysical characteristics.
  • Functional TDCC activity was tested in TCE format in the context of an appropriate target. Criteria for selection of optimized candidates was based on several parameters: CD3 antigen binding, stability, developability, tuned affinity for in vitro cell killing, in vivo efficacy and unique sequence identity for proprietary use in internal programs.
  • Example 2 Construction of light and heavy chain plasmids for transient mammalian expression.
  • Fc- fusion proteins were purified by Protein A (Cytiva MabSelect PrismA) affinity chromatography and His-tagged proteins were purified by Ni-NTA (Roche cOmpleteTM His-Tag Resin) affinity chromatography.
  • Protein A affinity chromatography resin was washed 3-5X with at least 30-50 total bed volumes of lx HBS (50mM HEPES- NaOH pH 7.4 + 150 mM NaCl). Purified protein was eluted with 5 bed volumes of Pro A Elution Buffer (20 mM Acetate, pH 3.5 (Ricca Chemical).
  • DSF Differential Scanning Fluorimetry
  • Samples were diluted to a final concentration of 2pM with Thermo Scientific’s Protein Thermal Shift Dye, and QS’d with formulation buffer.
  • Mixtures were aliquoted into a LightCycler 480 384-well plate and analyzed from 25°C to 95°C using a gradient of (0.06°C/s). Acquisition data was continuously acquired at an excitation of 578nm & emission of 604nm.
  • Example 4 Determination of antibody binding kinetics to recombinant antigen
  • Selected humanized anti-CD3 clones were screened for binding to commercial antigen by bio-layer interferometry (BLI) using a Gator Bio Biosensor. Apparent affinities were measured against human CD3 epsilon & CD3 delta heterodimer protein (ACRO Biosystems His Tag: CDD-H52W1). Binding was analyzed using an antibody capture format with anti-human Fc II probes, followed by a 300s association of CD3 antigen in a concentration series from lOOnM diluted 3-fold down to 0.137nM. A dissociation step was performed in Gator Q buffer and kinetics were determined using a 1 : 1 Langmuir binding model processed using Gator analysis software.
  • Tumor cells (2,000 cells) are plated in a 384-well plate the day before the assay setup. Next day, healthy donor PBMCs are thawed and plated at a 10: 1 E:T ratio (20,000 cells) and incubated in the presence of indicated TCEs (12-point dilutions, starting at 2 pg/ml) for 48 hours. Cytotoxicity is measured at 48 hours by luminescence (BrightGlo® (Promega)).
  • mice 6-8 weeks old NSG mice (The Jackson Laboratory) were subcutaneously injected with 5xl0 6 LS1034 cells in right flank. When tumor volumes reached 70-100 mm 3 , 10xl0 6 donor PBMCs were injected intraperitoneally. When tumor volumes reach 100-150mm 3 , mice are randomized and indicated TCEs were injected at Img/kg dose, twice and 7 days apart. Tumor size (2x week) and body weight (lx week) were measured throughout the study.
  • FIG. 1 A set of designed CD3-binding antibodies is shown in Fig. 1.
  • the antibodies were generated by grafting CDRs on to combinations of human acceptor frameworks.
  • the resulting 29 light and heavy chain sequence pairs contained combinations of the following: human germline heavy chains: IGHV1-18, IGHV1-24, IGHV2-28, IGHV3-23, IGHV3-11, IGHV3-48, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV5-2, IGHV5-51; human germline light chains: IGLV1-46, IGLV7-46, IGLV2-11, IGKV3-15, IGKV1-39.
  • Vernier back mutations were made singly or in combination. Non-Vernier substitutions were also introduced at positions where canonical residues differ between human and mouse. Additionally, conservative rational mutations were made in frameworks and CDRs based on predictions from molecular modeling.
  • Fig. 2 and Fig. 3 depict designs, either as a bivalent scFv-Fc, or as a monovalent bispecific with an anti-CD3 scFv-Fc hole paired with a tumor antigen (CB01) binding VHH-Fc knob.
  • CB01 tumor antigen binding VHH-Fc knob.
  • the parental murine antibody demonstrated poor expression titers, which we observed at the 30 mL scale. Improving expression was a primary goal of the humanization process.
  • compositions CD3-1 through CD3-6 human lambda IGLV7-46 and IGLV2-11 frameworks were selected for CDR grafts.
  • the original SP34 mouse lambda IGLVl*01 did not align closely with any single human lambda framework.
  • Each light chain was paired with a heavy chain graft on human framework IGHV3-23 (Trastuzumab heavy chain germline) with conservative mutations. The following clones either failed expression or completely lost binding to recombinant antigen.
  • CD3-7, CD3-8, and CD3-12 are CDR grafts on to IGLV7-46 and IGHV3-72, the closest human heavy and light chain matches for the parental mouse SP34 germlines.
  • Published sequences of productive antibodies with the same human frameworks were aligned with our sequences.
  • residues conserved within germline families could contribute to overall antibody stability and function.
  • IGLV7-46, and IGLV2-11 were a distant match for the original mouse sequence, we looked for a consensus across other known functional human lambda antibodies.
  • CD3-9, CD3-10 and CD3-11 were generated by grafting mouse CDRs into frameworks that share lower sequence identity with mouse SP34.
  • a common kappa light chain IGKV1-39 from the human repertoire, and 2 heavy chain frameworks (IGHV2 and IGHV5) were chosen. The aim was to test CDR grafts on more divergent frameworks without considerable loss in yield and binding. Light chain switching from lambda to kappa was permissible and functionally equivalent in most cases.
  • Heavy chain IGHV5 and IGHV2 accommodated CDR grafts and some representative clones bound antigen with similar or improved affinities.
  • CD3-13, CD3-14, CD3-15, and CD3-21 were produced to validate and compare published results. The constructs were modelled after previously reported humanizations. Results were reproduced and we elected CD3-13 and CD3-15 to use as benchmark antibodies.
  • CD3-16 through CD3-20 were made by combining some different lambda and kappa light chains from above with a common heavy chain IGHV5. Conservative, previously productive mutations to the backbone were introduced. Although some specific light chain point mutations appeared to be detrimental, the use of lambda vs. kappa did not appear to make a significant difference. Overall, the yields were consistently lower, suggesting that IGHV5 was a poor choice for humanization.
  • IgG formats with our humanized SP34 scFvs were made and screened for expression. Although some versions failed to produce antibody from 30ml culture, we confirmed that high quality IgG can be produced at 30ml scale.
  • the constructs represented use a small GGGGS linker to connect our scFv to IgGl CH2/CH3 in the following format: Heavy chain variable - GGGGSGGGGSGGGGS - light chain variable - GGGGS-CH2-CH3.
  • TCE formats were designed placing each humanized CD3 binding arm into a hole Fc constant region and pairing it with a knob Fc version of a VHH binder against a target of interest.
  • thermostability analysis determined by SYPRO Orange staining based differential scanning fluorimetry is shown in Fig. 6. Aa T m cutoff value of 62.0°C was used to select acceptable clones.
  • FIG. 7 Representative BLI sensograms for selected clones from round 1 engineering are shown in Fig. 7. Antibodies were captured on anti-human Fc probes followed by association with a concentration series of soluble CD3 antigen. A range of affinities from low single digit nM to 400nM KD was observed. Apparent KDs for clones CD3-10, CD3- 12 and CD3-15 were 39.7, 9.27 and 16.9nM, respectively. These mid-range affinities correlate with potent TDCC activity. [0325] Fig. 8 shows in vitro killing of cell lines with low(Hut-78), medium(HPB- ALL) and high(Nomol-OX) expression of TAA1 antigen.
  • TAA1 TCEs tumor associated antigen
  • CD3-10 Three selected heavy and light chain combinations, CD3-10, CD3- 12, and CD3-15 from round 1 engineering were shuffled in a matrix to test all combinations of the 6 individual chains.
  • CD3-30 - CD3-36 was comprised of grafts into the following humanized germline pairs in all combinations:
  • CD3-37 through CD3-41 were designed from consensus versions with added mutations to introduce sequence diversity. Each mutation was previously associated with improved T m , yield, or aggregation in another context. Specific mutations that were varied are as follows:
  • CD3-42 light chain consensus from 3 selected round 1 kappa designs.
  • CD3-43 light chain consensus from 3 selected human IGLV7 designs.
  • CD3-44 kappa light chain consensus with FW1 extracted from CD3-10.
  • CD3-45 kappa light chain consensus with FW1 extracted from CD3-15.
  • CD3-44 and CD3-45 were considered hybrid kappa clones, part consensus combined with a unique FW 1.
  • CD3-46 and CD3-47 were made with additional CDR mutations yet untested. No improvements were observed for either clone.
  • Round 1 results provided valuable and predictive information to use as guide for the second set of designs.
  • Round 2 titers were consistently higher overall.
  • Antibody schematics for T-cell engagers produced from round 2 CD3 binder engineering is shown in Fig. 9.
  • Supernatant expression titers determined by Biolayer Interferometry with Protein A quantitation probes is shown in Fig. 10.
  • Representative analytical size exclusion chromatography by HPLC from round 2 CD3 binder engineering (Fig. 11) demonstrated monomeric purity with minimal aggregation for CD3-32, CD-42, and CD3- 46. Values for each were 94.4, 96.3 and 96.7% monomeric species, respectively.
  • thermostability analysis for selected clones CD3-32, CD-42, and CD3-46 from round 2 CD3 binder engineering determined by SYPRO Orange based differential scanning fluorimetry is shown in Fig. 12.
  • T m values for TCE versions of the 3 binders were 64.7°C, 64.5°C, and 64.3°C, respectively, falling above a cutoff value of 62.0°C.
  • Representative BLI sensograms for selected clones from round 2 engineering are shown in Fig. 13. Antibodies were captured on anti-human Fc probes followed by association with a concentration series of soluble CD3 antigen.
  • Round 2 engineering produced clones with a range of affinities between 3 and 300nM. Clones with different CD3 arm affinities were produced to test and optimize binding as TCEs in the context of different tumor targets. Three clones displayed potent cytotoxic activity against 2 tumor antigens in TDCC assays. BLI binding affinities were predictive of downstream assays, the soluble antigen binding correlated well with cellbased functional data. These three selected clones from round 2 engineering were assayed and apparent KDs were recorded as follows:
  • CD3-32 1.89 xlO’ 8 M
  • CD3-42 2.82xlO’ 8 M
  • Fig. 14 is a graph showing in vitro killing of LS1034 cells by TAA2 TCE coupled with varying CD3 arms of different affinities.
  • PBMCs were added to each well to measure T cell mediated killing. Killing is quantified as % of cytotoxicity in a BrightGlo® assay.
  • TCEs were provided at 12, 5-fold dilutions starting at 2mg/ml concentration.
  • Fig. 15 is a graph showing tumor volume (mm 3 ) of xenograft LSI 034 tumors in NSG mice following treatment with bispecific T cell engagers coupling TAA2 with CD3 of varying affinities at Img/kg dose.
  • Mice were humanized with healthy donor peripheral blood mononuclear cells (PBMCs). Mice that received PBMCs but not TCE were used as control group. Potent in vivo anti -tumor activity observed upon administration of TCEs.
  • PBMCs peripheral blood mononuclear cells
  • TAA tumor associated antigen
  • Tg32 mice express the human FCGRT transgene to have the highest and most human-like protection of humanized IgG enabling human PK predictions with allometric scaling (Betts et al. 2018).
  • the pharmacokinetics of TAAxCD3-42 and TAAxCD3-15 were evaluated at 5 mg/kg.
  • PK in FcRn mice showed antibody like half-life with allometric scaling to 10-12 days half-life in humans.
  • the PK displayed biphasic kinetics characterized by low clearance and low volume of distribution common to other antibody-based therapeutics as shown in Fig. 16 (CD3-42) and Fig. 17 (CD3-15) and in the following table.
  • “about X” includes a range of values that are ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1% of X, where X is a numerical value.
  • the term “about” refers to a range of values which are 10% more or less than the specified value.
  • the term “about” refers to a range of values which are 5% more or less than the specified value.
  • the term “about” refers to a range of values which are 1% more or less than the specified value.
  • ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
  • any range including any of the two individual values as the two end points is also conceived in this disclosure.
  • the expression “a dose of about 100 mg, 200 mg, or 400 mg” can also mean “a dose ranging from 100 to 200 mg”, “a dose ranging from 200 to 400 mg”, or “a dose ranging from 100 to 400 mg”.

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Abstract

L'invention concerne des protéines de liaison à CD3, des séquences CDR, des séquences à domaines variables, et des compositions associées, et des procédés pour leur utilisation et leur fabrication.
PCT/US2025/025335 2024-04-19 2025-04-18 Protéines de liaison à cd3, acides nucléiques codant de telles protéines, et leurs procédés de préparation et d'utilisation Pending WO2025222100A2 (fr)

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