WO2025201242A1 - Anticorps ciblant fap et lrrc15 et protéines de fusion ciblant l'hyaluronane et leurs utilisations - Google Patents

Anticorps ciblant fap et lrrc15 et protéines de fusion ciblant l'hyaluronane et leurs utilisations

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Publication number
WO2025201242A1
WO2025201242A1 PCT/CN2025/084379 CN2025084379W WO2025201242A1 WO 2025201242 A1 WO2025201242 A1 WO 2025201242A1 CN 2025084379 W CN2025084379 W CN 2025084379W WO 2025201242 A1 WO2025201242 A1 WO 2025201242A1
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seq
nos
antigen
binding
antibody
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Inventor
Fulai ZHOU
Brian Whitaker
Mark Anthony TORNETTA
Honglei BI
Ying Jin
Mark Lawrence CHIU
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Tavotek Biotherapeutics Hong Kong Ltd
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Tavotek Biotherapeutics Hong Kong Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype

Definitions

  • the present disclosure relates to antibodies targeting FAP and LRRC15 and antigen binding fragments thereof, and Fc-fusions of human hyaluronidase (HYAL) enzyme and fragments thereof for targeting hyaluronan (HA) , and their applications as therapeutic approaches for treating various solid tumors.
  • HYAL human hyaluronidase
  • CAFs are a key component of the tumor stroma and play a crucial role in promoting tumor growth, invasion, and metastasis.
  • the acquisition of a CAF phenotype is associated with the expression of a variety of CAF-related markers (Han et al., 2020) .
  • Fibroblast activation protein (FAP) is one of the most viable CAF ⁇ markers and plays a role in tumor progression.
  • FAP + CAFs contribute to an immunosuppressive tumor microenvironment and are associated with poor patient outcomes in ovarian cancer (Hussain et al., 2020) , making FAP a promising therapeutic target.
  • FAP chimeric antigen receptor T cells
  • small molecule inhibitors including small molecule inhibitors, nanomaterials, monoclonal antibodies (mAbs) , antibody-drug conjugates (ADCs) , chimeric antigen receptor (CAR) T cells, and oncolytic adenovirus.
  • mAbs monoclonal antibodies
  • ADCs antibody-drug conjugates
  • CAR chimeric antigen receptor
  • FAP is one of the promising therapeutic targets for CAFs (Adams et al., 2004; Fabre et al., 2020; Freedman et al., 2018; Kakarla et al., 2013; Santos et al., 2009; Wang et al., 2014; Xin et al., 2021) .
  • Leucine-rich repeat-containing protein 15 is identified as another important marker for CAF that is highly expressed on stromal fibroblasts of multiple solid tumors, as well as cancer cells of mesenchymal origin. While its expression in normal tissues is low, making it another ideal target for tumor stroma, high LRRC15 expression has prognostic value to be a biomarker predictive of higher tumor grade and adverse outcome in both osteosarcoma and soft-tissue sarcomas (Ben-Ami et al., 2020; Cui et al., 2020) . Moreover, LRRC15 + CAFs promote tumor growth and suppress CD8 + T cell function.
  • Hyaluronan is a prominent component of the extracellular matrix in tumors, and its aberrant metabolism contributes to tumor progression (Donelan et al., 2022) .
  • the degradation of HA is catalyzed by hyaluronidase enzymes.
  • hyaluronidases include HYAL1, HYAL2, HYAL3, and PH20 (SPAM1) .
  • HYAL1, HYAL2, HYAL3, and PH20 hyaluronidases
  • These enzymes play important roles in various physiological and pathological processes, such as tissue remodeling, embryogenesis, wound healing, and cancer progression (Kaul et al., 2021) .
  • PH20 the only enzyme with neutral-active properties, is widely used to hydrolyze hyaluronic acid in the tumor or used to be co-administered with a subcutaneously injected mAbs to increase its permeability (Locke et al., 2019) .
  • a pegylated recombinant human PH20 (PEGPH20) variant has been shown to be able to remodel the stroma of HA-rich tumors, improving the intra-tumor distribution of anticancer drugs and enhancing their therapeutic efficacy without increasing toxicity (Morosi et al., 2021) .
  • phase III study of Pegvorhyaluronidase alfa (PEGPH20) in combination with chemotherapy for pancreatic cancer failed to meet its primary endpoint, indicating that HA depletion alone may not be sufficient for effective treatment (Hakim et al., 2019; Van Cutsem et al., 2020) .
  • CD3 antigens are cell surface proteins found on T cells, a type of white blood cell that plays a crucial role in the immune system's ability to recognize and destroy abnormal cells, including cancer cells.
  • T cell engagers are a type of immunotherapy that harnesses the power of T cells to target and kill cancer cells (Peri et al., 2023) .
  • These therapies work by using bispecific antibodies that bind to both the CD3 antigen on T cells and a tumor-associated antigen (TAA) on cancer cells, bringing the two cell types into close proximity and activating the T cells to attack the cancer cells (Vafa &Trinklein, 2020) .
  • New therapies can be developed to target CD3 and cancer associated fibroblasts.
  • the present disclosure provides novel multispecific (e.g., bispecific or trispecific) antibodies that combine one or more of anti-FAP antibody, anti-LRRC15 antibody and anti-CD3 antibody or their antigen binding fragments, and Fc-fusions of hyaluronidases.
  • These antibodies or enzymes integrate domains with a variety of affinities and activities into a single molecule with multiple specificities.
  • a multifunctional antibody platform disclosed herein comprises architectures and compositions such as: 1) anti-CD3 x anti-FAP, 2) anti-CD3 x anti-LRRC15, and 3) anti-FAP x anti-LRRC15 x hyaluronidase.
  • These molecules are designed to have varying binding affinities, activities, and molecular formats to enable optimal T-cell redirection for tumor cytotoxicity.
  • the combination of a human hyaluronidase fusion protein with CAF homing and a direct tumor-targeting molecule can remodel the TME and produce synergistic effects against solid tumors, offering promising anti-tumor activity for patients with unmet medical needs in multiple solid tumors.
  • the heavy chain variable domain of the binding moiety that has binding specificity for FAP are present in a human VH framework; and the light chain variable domain of the first binding moiety are present in a human V kappa framework.
  • the disclosure provides an anti-FAP antibody or antigen binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence with at least 85%identity to any one of SEQ ID NOs: 1-3, 6-8, 12-14, and 17 or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence with at least 85%identity to any one of SEQ ID NOs: 4-5, 9-11, 15-16, and 18 or an antigen-binding portion thereof.
  • the disclosure provides a multispecific antibody comprising the anti-FAP antibody or antigen-binding fragment disclosed herein.
  • the disclosure provides an anti-LRRC15 antibody or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence comprising: SEQ ID NOs: 129 and 130; SEQ ID NOs: 131 and 132; or SEQ ID NOs: 153 and 154; respectively.
  • the present disclosure provides an anti-LRRC15 antibody or an antigen-binding portion thereof, comprising at least one variable-heavy-chain-only single domain (VHO) or an antigen-binding portion thereof, wherein the at least one variable-heavy-chain-only single-domain comprises an amino acid sequence with at least 85%identity to any one of SEQ ID NOs: 19-128 or an antigen-binding portion thereof.
  • VHO variable-heavy-chain-only single domain
  • the disclosure provides a multispecific antibody comprising the anti-LRRC15 antibody or antigen-binding fragment disclosed herein.
  • the disclosure provides an anti-CD3 antibody or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence comprising: SEQ ID NOs: 133 and 136; SEQ ID NOs: 134 and 136; SEQ ID NOs: 135 and 136; SEQ ID NOs: 140 and 142; SEQ ID NOs: 137 and 143; SEQ ID NOs: 155 and 156; or SEQ ID NOs: 157 and 158; respectively.
  • the disclosure provides an Fc-hyaluronidase fusion, comprising at least one hyaluronidase domain or portion thereof, wherein the hyaluronidase domain sequence is selected from SEQ ID NOs: 352-358 and 144-148. In some embodiments the Fc-hyaluronidase fusion is soluble.
  • the disclosure provides a multispecific antibody comprising at least one hyaluronidase domain or portion thereof disclosed herein.
  • the disclosure provides an anti-EGFR antibody, comprising at least one variable-heavy-chain-only single domain (VHO) or an antigen-binding portion thereof, wherein the VHO is selected from SEQ ID NOs: 165 and 166.
  • the disclosure provides an anti-EGFR antibody comprising tandem (e.g., two, three, or four) variable-heavy-chain-only single domain (VHO) selected from SEQ ID NOs: 165 and 166.
  • the disclosure provides an anti-cMet antibody or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence comprising: SEQ ID NOs: 161 and 162; respectively.
  • the disclosure provides an anti-VEGF antibody or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence selected from SEQ ID NOs: 163 and 164; respectively.
  • the disclosure provides an anti-5T4 antibody or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence selected from SEQ ID NOs: 159 and 160; respectively.
  • anti-FAP, anti-LRRC15, anti-CD3, anti-EGFR, anti cMet, anti-VEGF, or anti-5T4 antibodies or antigen binding fragments thereof disclosed herein may be human, humanized, or chimeric antibodies, or antigen binding fragments.
  • the hyaluronidase domain or portion thereof disclosed herein may be fused to Fc regions from full length IgG1, IgG2, IgG3, or IgG4 antibodies.
  • the antibody backbones may be modified to affect functionality, e.g., to eliminate residual effector functions.
  • aspects of the disclosure include multiple specific (e.g., bispecific or trispecific) antibodies comprising: a first heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations L234A, L235A, and F405L; and a second heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations L234A, L235A, K409R, and H435R.
  • the mutated human IgG1 constant region of the first heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 176
  • the mutated human IgG1 constant region of the second heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 177.
  • multispecific antibodies comprising: a first heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations L234A, L235A, and F405L; and a second heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations L234A, L235A, and K409R.
  • the mutated human IgG1 constant region of the first heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 176
  • the mutated human IgG1 constant region of the second heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 178.
  • aspects of the disclosure include isolated multispecific (e.g., bispecific or trispecific) antibodies comprising: a first heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations F243L, R292P, Y300L, V305I, P396 and S354C, T366W; and a second heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations F243L, R292P, Y300L, V305I, P396 and T366S, L368A, Y407V, Y349C.
  • a first heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations F243L, R292P, Y300L, V305I, P396 and S354C, T366W
  • a second heavy chain polypeptide subunit comprising a mutated human IgG1 constant region comprising mutations F243L, R29
  • the mutated human IgG1 constant region of the first heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 189
  • the mutated human IgG1 constant region of the second heavy chain polypeptide subunit comprises a sequence of SEQ ID NO: 190.
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the first binding arm can target a CD3 associated pathway and comprise an anti-CD3 or antigen binding fragment as described herein; and a second binding arm comprising: a second heavy chain protein comprising, from the N- to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a second light chain protein comprising, from the N-to the C
  • the present disclosure provides a multispecific antibody, comprising: 1) a first binding arm targeting CD3 comprising: a first heavy chain protein comprising an IgG heavy chain or an antigen-binding fragment thereof, and a first light chain protein comprising an IgG light chain or an antigen-binding fragment thereof, wherein: the IgG heavy chain or antigen-binding fragment of the first binding arm comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 133-135 and 137-140 or an antigen-binding fragment thereof, the IgG light chain or antigen-binding fragment of the first binding arm comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 136 and 141-143 or an antigen-binding fragment thereof; and 2) a second binding arm targeting FAP comprising: a second heavy chain protein comprising an IgG heavy chain or an antigen-binding fragment thereof, and a second light chain protein comprising an IgG light chain or an antigen-
  • the present disclosure provides a multispecific (bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the first binding arm can target a CD3 associated pathway and comprise an anti-CD3 or antigen binding fragment as described herein; and a second binding arm comprising: a second heavy chain protein comprising, from the N- to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a second light chain protein comprising, from the N-to the C-terminus,
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the first binding arm can target a CD3 associated pathway and comprise an anti-CD3 or antigen binding fragment as described herein; and a second binding arm comprising: a second heavy chain protein comprising, from the N- to the C-terminus, an optional signal sequence A -an IgG heavy chain comprising at least one variable-heavy-chain only (VHO) single domain or an antigen-binding portion thereof
  • the present disclosure a multispecific antibody, comprising: 1) a first binding arm targeting CD3 comprising: a first heavy chain protein comprising an IgG heavy chain or an antigen-binding fragment thereof, and a first light chain protein comprising an IgG light chain or an antigen-binding fragment thereof, wherein: the IgG heavy chain or antigen-binding fragment thereof of the first binding arm comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 133-135 and 137-140 or an antigen-binding fragment thereof, the IgG light chain or antigen-binding fragment thereof of the first binding arm comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 136 and 141-143 or an antigen-binding fragment thereof; and 2) a second binding arm targeting LRRC15 comprising: a second binding arm comprising: a second heavy chain protein comprising an IgG heavy chain comprising at least one variable-heavy-chain only (VHO) single domain or
  • VHO
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a heavy chain fusion protein comprising: from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof -an optional linker A -at least one variable-heavy-chain only (VHO) single domain or an antigen-binding portion thereof, and a light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the first binding arm can target a FAP-associated pathway and comprise an anti-FAP or antigen binding fragment as described herein; and the VHO or antigen-binding portion thereof can target an LRRC15-associated pathway and comprise an anti-LRRC15 antibody or antigen binding fragment as described herein;
  • the present disclosure a multispecific antibody, comprising: a heavy chain fusion protein targeting FAP and LRRC15 comprising: from the N-to the C-terminus, an IgG heavy chain or an antigen-binding fragment thereof - an optional linker -at least one variable-heavy-chain only (VHO) single domain or an antigen-binding fragment thereof, and a light chain protein targeting FAP comprising an IgG light chain or an antigen-binding fragment thereof.
  • VHO variable-heavy-chain only
  • the IgG heavy chain or antigen-binding fragment comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 1-3, 6-8, 12-14, and 17 or an antigen-binding fragment thereof
  • the variable-heavy-chain only (VHO) single domain or antigen-binding fragment comprises a heavy chain variable region comprising any set of the HCDR1, HCDR2, and HCDR3 set forth in Table 4 or any of the heavy chain variable region sequences set forth in Table 4 or 5
  • the IgG light chain or antigen-binding fragment comprises an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 4-5, 9-11, 15-16, and 18 or an antigen-binding fragment thereof.
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -a hyaluronidase domain or portion thereof -an optional linker A -an Fc chain or an antigen-binding portion thereof, wherein the hyaluronidase domain or portion thereof of the first binding arm can hydrolyze the hyaluronic acid (HA) and comprise a hyaluronidase domain or portion as described herein; and a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof -an optional linker B -at least one variable-heavy-chain only (VHO) single domain or an antigen-binding portion thereof, and a second light chain protein comprising, from
  • the present disclosure a multispecific antibody, comprising: 1) a first binding arm targeting HA comprising: a first heavy chain fusion protein comprising a hyaluronidase domain or fragment thereof -an optional linker A -an Fc region or a fragment thereof; and 2) a second binding arm targeting FAP and LRRC15 comprising: a second heavy chain fusion protein comprising an IgG heavy chain or an antigen-binding fragment thereof -an optional linker B -at least one variable-heavy-chain only (VHO) single domain or an antigen-binding fragment thereof, and a second light chain protein comprising an IgG light chain or an antigen-binding fragment thereof, wherein: the hyaluronidase domain or fragment of the first binding arm comprises any one of SEQ ID NOs: 352-358 and 144-146 or an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 352-358 and 144-146, and wherein: the IgG heavy
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -a hyaluronidase domain or portion thereof -an optional linker A -an Fc chain or an antigen-binding portion thereof, wherein the hyaluronidase domain or portion thereof of the first binding arm can hydrolyze the hyaluronic acid (HA) and comprise a hyaluronidase domain or portion as described herein; and a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -at least one variable-heavy-chain only (VHO) single domain -an optional linker B -an Fc region, wherein the variable-heavy-chain only single domain or an antigen-binding portion thereof of the second binding arm can target an EG
  • the present disclosure provides a multispecific antibody comprising: 1) a first binding arm targeting HA comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N-to the C-terminus, a hyaluronidase domain or fragment thereof -an optional linker A -an Fc region or a fragment thereof, 2) a second binding arm targeting EGFR comprising: a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, at least one variable-heavy-chain only (VHO) single domain that is capable of binding EGFR -an optional linker B -an Fc region or a fragment thereof; wherein: the hyaluronidase domain or fragment of the first binding arm comprises any one of SEQ ID NOs: 352-358 and 144-146 or an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 352-358 and 144-146.
  • VHO variable-heavy-chain
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -tandem VHOs -an optional linker A -an Fc region, wherein the tandem VHOs or an antigen-binding portions thereof of the first binding arm can target an EGFR-associated pathway and comprise an anti-EGFR or antigen binding fragment as described herein; and a second binding arm comprising: a second heavy chain protein comprising, from the N- to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an anti
  • the present disclosure provides a multispecific (e.g., bispecific or trispecific) antibody comprising: a first binding arm comprising: a first heavy chain protein comprising, from the N-to the C-terminus, an optional signal sequence A -tandem VHOs -an optional linker A -an Fc region -an optional linker B -an scFv or an antigen-binding portion thereof, wherein the tandem VHOs and scFv or an antigen-binding portions thereof of the first binding arm can target EGFR-and VEGF-associated pathways, respectively; and comprise an anti-EGFR and an anti-VEGF or antigen binding fragments as described herein; a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -
  • the IgG is human IgG1, IgG2, IgG3, or IgG4.
  • the first binding arm is monovalent, bivalent, or multivalent; and the second binding arm is monovalent, bivalent, or multivalent.
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 133-135, 137-140, 155, and 157, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 133-135 and 137-140, or an antigen-binding portion thereof
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 136, 141-143, 156, and 158, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 136 and 141-143, or antigen-binding portion thereof
  • the IgG heavy chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149 and 151, an amino acid sequence having at least 85%identity to any one of
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 133-135, 137-140, 155 and 157, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 133-135 and 137-140, or an antigen-binding portion thereof
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 136, 141-143, 156 and 158, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 136 and 141-143, or antigen-binding portion thereof
  • the IgG heavy chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 129, 131, and 153, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs:
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 133-135, 137-140, 155, and 157, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 133-135 and 137-140, or an antigen-binding portion thereof
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 136, 141-143, 156, and 158, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 136 and 141-143, or antigen-binding portion thereof
  • the variable-heavy-chain-only single domain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 19-128, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 19
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, and 151 an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 1-3, 6-8, 12-14, and 17, or an antigen-binding portion thereof
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 4-5, 9-11, 15-16, 18, 150, and 152, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 4-5, 9-11, 15-16, and 18, or antigen-binding portion thereof
  • the variable-heavy-chain-only single domain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 19-128, an amino acid sequence having at least 85%identity to any one of SEQ
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 352-358 and 144-148, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 352-358 and 144-148, or a portion thereof;
  • the IgG heavy chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, and 151, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 1-3, 6-8, 12-14, and 17, or an antigen-binding portion thereof
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 4-5, 9-11, 15-16, 18, 150, and 152, an amino acid sequence having at least 85%identity
  • the IgG heavy chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 352-358 and 144-148, an amino acid sequence having at least 85%identity to any one of SEQ ID NOs: 352-358 and 144-148, or a portion thereof;
  • the variable-heavy-chain-only single domain of the second binding arm comprises an amino acid sequence selected from SEQ ID NOs: 165 and 166.
  • tandem variable-heavy-chain-only single domains of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 165 and 166;
  • the IgG heavy chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NO: 161
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NO: 162.
  • the tandem variable-heavy-chain-only single domain or an antigen-binding portion of the first binding arm comprises an amino acid sequence selected from SEQ ID NOs: 165 and 166; and an scFv or an antigen-binding portion thereof of the first binding arm (VEGF-binding portion) comprises an amino acid sequence selected from SEQ ID NOs: 163 and 164;
  • the IgG heavy chain of the second binding arm comprises an amino acid sequence selected from SEQ ID NO: 161
  • the IgG light chain of the first binding arm comprises an amino acid sequence selected from SEQ ID NO: 162.
  • an antibody or multispecific (e.g., bispecific or trispecific) antibody disclosed herein comprises a modified Fc to extend the half-life of the multispecific (e.g., bispecific or trispecific) antibody, enhance resistance of the multispecific (e.g., bispecific or trispecific) antibody to proteolytic degradation, reduce effector functionality of the multispecific (e.g., bispecific or trispecific) antibody, facilitate generation of the multispecific (e.g., bispecific or trispecific) antibody by Fc heterodimerization, facilitate multimerization of the multispecific (e.g., bispecific or trispecific) antibody, and/or improve manufacturing and drug stability of the multispecific (e.g., bispecific or trispecific) antibody.
  • a modified Fc to extend the half-life of the multispecific (e.g., bispecific or trispecific) antibody, enhance resistance of the multispecific (e.g., bispecific or trispecific) antibody to proteolytic degradation, reduce effector functionality of the multispecific (e.g., bispecific or trispecific) antibody, facilitate generation
  • the present disclosure provides a composition comprising an antibody or multispecific (e.g., bispecific or trispecific) antibody disclosed herein.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or bispecific or trispecific antibody disclosed herein and a pharmaceutically acceptable carrier.
  • the present disclosure provides a nucleic acid encoding an anti-LRRC15 antibody or an antigen-binding portion thereof.
  • the present disclosure provides a nucleic acid encoding a human hyaluronidase enzyme domain or portion thereof or an Fc-fusion thereof.
  • the present disclosure provides a nucleic acid encoding an anti-EGFR or an antigen-binding portion thereof.
  • the present disclosure provides a nucleic acid encoding an anti-VEGF antibody or an antigen-binding portion thereof.
  • the present disclosure provides a nucleic acid encoding the multispecific (e.g., bispecific or trispecific) antibody, the first heavy chain protein or first heavy chain fusion protein, the first light chain protein, the second heavy chain protein or heavy chain fusion protein, and/or the second light chain protein disclosed herein.
  • the multispecific antibody e.g., bispecific or trispecific
  • the present disclosure provides a recombinant vector, such as an expression vector, comprising a nucleic acid disclosed herein.
  • the present disclosure provides a host cell comprising a recombinant vector such as an expression vector or a nucleic acid disclosed herein.
  • the present disclosure provides a method for preparing an antibody or multispecific (e.g., bispecific or trispecific) antibody disclosed herein, comprising culturing a host cell disclosed herein, growing the host cell in a host cell culture, providing host cell culture conditions wherein a nucleic acid disclosed herein is expressed, and recovering the antibody or multispecific (e.g., bispecific or trispecific) antibody from the host cell or from the host cell culture.
  • the multispecific (e.g., bispecific or trispecific) antibody is obtained using controlled Fab arm exchange.
  • aspects of the invention include methods for treating a disease or condition characterized by expression of FAP, comprising administering to an individual in need an effective dose of a multispecific (e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • the disease is a cancer.
  • the cancer is a glioblastoma.
  • the cancer is a human non-small cell lung cancer.
  • aspects of the invention include methods for treating a disease or condition characterized by expression of LRRC15, comprising administering to an individual in need an effective dose of a multispecific (e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • the disease is a cancer.
  • the cancer is a glioblastoma.
  • the cancer is an osteosarcoma.
  • aspects of the invention include methods for treating a disease or condition characterized by expression of EGFR, comprising administering to an individual in need an effective dose of a multispecific (e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • a multispecific (e.g., bispecific or trispecific) antibody e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • the disease is a cancer.
  • the cancer is a non-small cell lung cancer.
  • aspects of the invention include methods for treating a disease or condition characterized by expression of VEGF, comprising administering to an individual in need an effective dose of a multispecific (e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • a multispecific (e.g., bispecific or trispecific) antibody e.g., bispecific or trispecific) antibody, or a pharmaceutical composition, described herein.
  • the disease is a cancer.
  • the cancer is a non-small cell lung cancer.
  • the present disclosure provides a method for mediating FAP, LRRC15, HA, EGFR, cMet, or VEGF in a subject in need thereof, comprising administering to the subject an effective amount of an antibody or multispecific (e.g., bispecific or trispecific) antibody or a pharmaceutical composition disclosed herein.
  • an antibody or multispecific (e.g., bispecific or trispecific) antibody or a pharmaceutical composition disclosed herein comprising administering to the subject an effective amount of an antibody or multispecific (e.g., bispecific or trispecific) antibody or a pharmaceutical composition disclosed herein.
  • Figure 1 illustrates concentration-dependent ELISA binding of anti-FAP antibodies to recombinant human FAP, recombinant cynomolgus monkey FAP, and recombinant mouse FAP.
  • the Y-axis units represent OD 450nm, while the X-axis units represent the concentration of the respective test articles in nM (nanomoles per liter) .
  • the results show that all test molecules exhibited various levels of binding affinity to human, mouse, and cynomolgus FAP proteins, with the exception of a few that only showed binding to mouse FAP proteins.
  • Figure 2 displays concentration-dependent ELISA binding of FAP x CD3 bispecific antibodies to recombinant human FAP, recombinant cynomolgus monkey FAP, and recombinant mouse FAP.
  • the Y-axis units represent OD 450nm, while the X-axis units represent the concentration of the respective test articles in nM (nanomoles per liter) .
  • the bispecific antibodies contain a unique FAP targeting arm (with heavy chain variable sequences and light chain variable sequences provided as SEQ ID NOs: 1, 3, 7 and SEQ ID NOs: 5, 11 for FAP_MO36_v6, FAP_MO33_v6, FAP_MO33_v2, respectively) paired with an anti-CD3 arm (with heavy chain variable sequences and light chain variable sequences provided as SEQ ID NOs: 137, 140 and SEQ ID NOs: 142, 143 for Cris7_v3 and Cris7_v4, respectively) .
  • the results showed that the test molecules had varying binding capabilities to human, mouse, and cynomolgus FAP proteins.
  • Figure 3 depicts concentration-dependent flow cytometry binding of anti-FAP antibodies to the U-87 MG ( Figures 3A and 3B) and IMR-90 ( Figures 3C) cell lines bearing FAP.
  • the Y-axis units represent geometric Mean Fluorescence Intensity (gMFI)
  • the X-axis units represent the concentration of the respective test articles in nM (nanomoles per liter) .
  • the results show that all the test molecules exhibited varying levels of binding to FAP + U-87 MG and IMR-90 cells.
  • FAP_BIBH1_v1 and FAP_4B9 are provided as SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, 151 and SEQ ID NOs: 4-5, 9-11, 15-16, 18, 150, 152, respectively.
  • FIG. 4 illustrates concentration-dependent T cell activation by FAP x CD3 bispecific antibodies in NFAT reporter assay.
  • the bispecific antibodies contain a unique FAP targeting arm (with heavy chain variable sequences and light chain variable sequences for FAP_MO36_v6, FAP_MO33_v6 and FAP_4B9 provided as SEQ ID NOs: 3, 7, 149 and SEQ ID NOs: 5, 11, 150, respectively) paired with an anti-CD3 arm (with heavy chain variable sequences and light chain variable sequences for Cris7_v3, v4, 40G5_CD3 and 1979_CD3 provided as SEQ ID NOs: 137, 140, 155, 157 and SEQ ID NOs: 142, 143, 156, 158, respectively) .
  • T cell activation was measured using a reporter gene activation assay.
  • the Y-axis units represent the luminescence signal of reporter gene activation, while the X-axis units represent the concentration of the respective test articles in nM (nanomoles per liter) .
  • the results show that all the test molecules exhibited varying levels of T cell activation activity.
  • Figure 5 displays the ability of FAP x CD3 bispecific antibodies to induce PBMC (peripheral blood mononuclear cell) killing in the U-87 MG cell line.
  • PBMC peripheral blood mononuclear cell
  • the experiment involved mixing PBMCs with U-87MG cells in a 5: 1 E: T (Effector: Tumor) ratio along with the addition of the bispecific antibody. Varying levels of T cell killing were observed.
  • the x-axis represents the concentration of the antibody used in nM (nanomoles per liter)
  • the y-axis represents the percentage of cell lysis of tumor cells 48 hours after the addition of the antibody.
  • Figure 6 illustrates antitumor effectiveness of FAP x CD3 bispecific antibodies combined with EGFR x cMet x VEGF (TAVO412) trispecific antibodies in treating A549 models of non-small cell lung cancer using PBMC humanized solid tumor xenograft models.
  • FAP x CD3 (with heavy chain variable sequences and light chain variable sequences for FAP_4B9 provided as SEQ ID NOs: 149 and 150, respectively; and for 40G5_CD3 provided as SEQ ID NOs: 155 and 156, respectively) and TAVO412 (with heavy chain variable sequences and light chain variable sequences provided as SEQ ID NOs: 161, 163, 165-166 and 162, 164, respectively) were tested at 1 mg/kg and 2 mg/kg, respectively, in A549 xenograft models reconstituted with human PBMC.
  • the schematic structures of the tested antibodies are depicted in Figure 6B and 6C. The combination treatment showed better tumor growth inhibition effect compared to any monotherapy.
  • Figure 9 displays concentration-dependent ELISA binding of anti-LRRC15 antibodies to recombinant mouse LRRC15 ( Figure 9A-9L) .
  • the ELISA binding assays demonstrated the binding to recombinant mouse LRRC15 protein coated on a plate by a series of anti-LRRC15 antibodies.
  • the Y-axis units are OD 450nm, and the X-axis units are the concentration of the respective test articles in nM (nmol/L) units.
  • Figure 11 depicts concentration-dependent flow cytometry binding of anti-LRRC15 antibodies to the U-118 MG human glioma cell line bearing LRRC15 ( Figures 11A-11J) .
  • the cell binding assays demonstrated the binding to LRRC15 + U-118 MG cells by a serial dilution of anti-LRRC15 antibodies.
  • the Y-axis units are geometric Mean Fluorescence Intensity (gMFI)
  • the X-axis units are the concentration of the respective test articles in nM (nmol/L) units.
  • murines e.g., rats, mice
  • lagomorphs e.g., rabbits
  • non-human primates humans
  • canines felines
  • ungulates e.g., equines, bovines, ovines, porcines, caprines
  • the present disclosure provides anti-FAP antibodies and antigen-binding portions thereof.
  • the disclosure provides amino acid sequences for anti-FAP antibodies set forth as SEQ ID NOs: 1-18 in Table 2, and SEQ ID NOs: 149-152 in Table 10.
  • Molecules such as antibody-drug conjugates and multispecific antibodies, which comprise the anti-FAP antibodies or antigen-binding fragments disclosed herein, are also within the scope of the present disclosure.
  • the anti-FAP or antigen-binding portion thereof is selected from the group consisting of a whole antibody, an antibody fragment, a human antibody, humanized antibody, a single chain antibody, a conjugate, an antibody mimetic, and a defucosylated antibody.
  • the anti-FAP fragment is selected from the group consisting of a UniBody, a variable heavy only single domain antibody, and a Nanobody.
  • the anti-FAP fragment is selected from the group consisting of a single domain VHH, a single domain VHO, an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, and a Duocalin.
  • the disclosure provides an anti-LRRC15 or antigen binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NO: 129 and 131 or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 130 and 132, or an antigen-binding portion thereof.
  • a heavy chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 129 and 131 or an antigen-binding portion thereof
  • a light chain sequence comprising an amino acid sequence with at least 85%ident
  • the disclosure provides an anti-LRRC15 or antigen binding fragment thereof, comprising a heavy chain variable region sequence and a light chain variable region sequence comprising: SEQ ID NOs: 129 and 130; SEQ ID NOs: 131 and 132; SEQ ID NOs: 153 and 154; respectively.
  • the present disclosure provides an anti-LRRC15 or an antigen-binding portion thereof, comprising at least one variable-heavy-chain-only single domain or an antigen-binding portion thereof, wherein the at least one variable-heavy-chain-only (VHO) single-domain comprises an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 19-128 or an antigen-binding portion thereof.
  • VHO variable-heavy-chain-only
  • the disclosure provides an anti-CD3 or antigen binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 133-135 and 137-140 or an antigen-binding portion thereof, and a light chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 136 and 141-143 or an antigen-binding portion thereof.
  • a heavy chain sequence comprising an amino acid sequence with at least 85%identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of SEQ ID NOs: 136 and 141-143 or an antigen-binding portion thereof.
  • the anti-CD3 or antigen-binding portion thereof is selected from the group consisting of a whole antibody, an antibody fragment, a human antibody, humanized antibody, a single chain antibody, a conjugate, an antibody mimetic, and a defucosylated antibody.
  • the anti-CD3 fragment is selected from the group consisting of a UniBody, a variable heavy only single domain antibody, and a Nanobody.
  • the anti-CD3 fragment is selected from the group consisting of a single domain VHH, a single domain VHO, an Affibody, a DARPin, an Anticalin, an Avimer, a Versa body, and a Duocalin. Table 6. Variable Region Sequences of humanized anti-CD3 antibodies Table 7. Anti-CD3 antibodies Human hyaluronidase variants
  • the disclosure provides engineered hyaluronidases set forth as SEQ ID NOs: 352-358 and 144-146 for fragments thereof (Table 8) and hyaluronidases set forth as SEQ ID NOs: 147-148 (Table 9) .
  • the disclosure provides amino acid sequences of anti-cMet antibodies set forth as SEQ ID NOs: 161 and 162 in Table 10.
  • the disclosure provides an anti-cMet antibody or antigen binding fragment thereof, comprising a light chain sequence and a heavy chain sequence comprising: SEQ ID NOs: 161 and 162; respectively.
  • the disclosure provides an anti-cMet antibody or antigen binding fragment thereof, comprising a light chain sequence and a heavy chain sequence comprising: SEQ ID NOs: 163 and 164; respectively.
  • the disclosure provides an anti-EGFR antibody or antigen binding fragment thereof, comprising at least one variable-heavy-chain-only single domain or an antigen-binding portion thereof, comprising a variable-heavy-chain-only region sequence comprising: SEQ ID NOs: 165-166; respectively.
  • the anti-EGFR antibodies of the present disclosure may comprise, in tandem, two to four, such as two or three, variable-heavy-chain-only single domains or antigen-binding portion thereof, wherein the two or three variable heavy chain only single domains or an antigen-binding portion thereof are optionally connected via one or more linkers.
  • the present disclosure provides a bispecific antibody that targets and binds to human FAP and CD3 simultaneously and is capable of effectively blocking FAP proteins at the protein level.
  • the bispecific antibody binds both CD3 and FAP proteins and binds to one protein without affecting the binding of the other protein, that is, having the ability to bind CD3 and FAP simultaneously.
  • the bispecific antibody disclosed herein inhibits FAP-mediated disease or disorders, such as the glioblastoma or non-small cell lung cancer.
  • the binding arm that targets CD3 has a binding valency of one.
  • the bispecific antibody is preferably designed to have monovalent CD3-binding (i.e., one Fab arm binding to the epitope of CD3) .
  • Bivalent CD3 binding is linked to excess of activation-induced cell death in effector cells that would limit the efficacy of T cell redirection.
  • a bivalent CD3 redirection molecule can result in tumor antigen-independent immune effector cell activation that can increase systemic toxicity in the patient.
  • the use of a high affinity of anti-CD3 Fab arm can also increase toxicity.
  • High affinity variants of anti-CD3 Fab or scFv are typically poorly tolerated in cynomolgus monkeys because of resulting extensive cytokine release. High affinity for CD3 also shifted bispecific antibody biodistribution from tumors to CD3 rich tissue that led to increased risk of cytokine release syndrome. Thus, it is highly desirable for the binding affinity of the anti-CD3 arm to be lower than that of anti-FAP arm. Bivalent anti-CD3 agents can increase avidity in binding to CD3 and could lead to increase in cytokine release syndrome from off-tumor CD3 positive cells.
  • a bispecific antibody disclosed herein may comprise a first binding arms targeting CD3.
  • a bispecific antibody disclosed herein comprises a first CD3 binding arm comprising a human IgG1 heavy chain and a light chain sequence selected from SEQ ID NOs: 133-135, 137-140, 155, 157 and 136, 141-143, 156, 158, respectively.
  • a bispecific antibody disclosed herein may comprise a second binding arm targeting FAP.
  • a bispecific antibody disclosed herein comprises a second FAP binding arm comprising a human IgG1 heavy chain and a light chain sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, 151 and 4-5, 9-11, 15-16, 18, 150, 152, respectively.
  • Bispecific 5T4 x CD3 antibodies comprising a human IgG1 heavy chain and a light chain sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, 151 and 4-5, 9-11, 15-16, 18, 150, 152, respectively.
  • the present disclosure provides a 5T4 x CD3 bispecific antibody that targets simultaneously proteins linked to 5T4-associated pathways as well as proteins that can activate the CD3 T cell activity.
  • the present disclosure provides a bispecific antibody that targets and binds to human 5T4 and CD3 simultaneously and is capable of effectively blocking 5T4 proteins at the protein level.
  • the bispecific antibody binds both CD3 and 5T4 proteins and binds to one protein without affecting the binding of the other protein, that is, having the ability to bind CD3 and 5T4 simultaneously.
  • the bispecific antibody disclosed herein inhibits 5T4-mediated disease or disorders, such as the glioblastoma or non-small cell lung cancer.
  • a bispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 133-135, 137-140, 155, 157 and 159, and a light chain sequence selected from SEQ ID NOs: 136, 141-143, 156, 158 and 160, respectively.
  • the present disclosure provides an LRRC15 x CD3 bispecific antibody that targets simultaneously proteins linked to LRRC15-associated pathways as well as proteins that can activate the CD3 T cell activity.
  • the present disclosure provides a bispecific or trispecific antibody comprising: a first binding arm comprising: a first heavy chain protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof of the first binding arm can target a CD3 associated pathway and comprise an anti-CD3 or antigen binding fragment as described herein; and a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -at least one VHH or an antigen-binding portion thereof-an optional linker A -an IgG heavy chain or an antigen-binding portion thereof, where
  • the present disclosure provides a bispecific antibody that can be generated using well established point mutations in the CH1, CH2, and CH3 domains via controlled Fab arm exchange or via co-expression.
  • all constructs are symmetric so that there is no preference for the selection of point mutations of the respective parental antibodies.
  • the binding arm that targets CD3 has a binding valency of one.
  • the bispecific antibody is preferably designed to have monovalent CD3-binding (i.e., one Fab arm binding to the epitope of CD3) .
  • Bivalent CD3 binding is linked to excess of activation-induced cell death in effector cells that would limit the efficacy of T cell redirection.
  • a bivalent CD3 redirection molecule can result in tumor antigen-independent immune effector cell activation that can increase systemic toxicity in the patient.
  • the use of a high affinity of anti-CD3 Fab arm can also increase toxicity.
  • the second binding arm targeting an LRRC15 associated pathway is preferably designed to have monovalent LRRC15-binding (i.e., one Fab or VHO arm binding to the epitope of LRRC15) .
  • the second binding arm targeting an LRRC15 associated pathway can be monovalent, bivalent, trivalent, tetravalent, etc.
  • a bispecific antibody disclosed herein may comprise a second binding arms targeting LRRC15.
  • a bispecific antibody disclosed herein comprises a second LRRC15 binding arm comprising a human IgG1 heavy and light chain sequences selected from SEQ ID NOs: 129, 131, 153 and 130, 132, 154, respectively.
  • the present disclosure provides a FAP x LRRC15 x HYAL trispecific antibody that targets simultaneously proteins linked to FAP-or LRRC15-associated pathways as well as proteins that can have the HA-hydrolyzed activity.
  • FAP, LRRC15 and HA targets have differential expression levels in pathological sites and normal tissues.
  • the present disclosure provides a trispecific antibody that targets and binds to human FAP, LRRC15 and HA simultaneously and is capable of effectively blocking FAP or LRRC15 proteins at the protein level and hydrolyzing the HA polymer.
  • the trispecific antibody binds FAP and LRRC15 proteins or HA polymers and binds to one protein or portion thereof without affecting the binding of the other portion, that is, having the ability to bind FAP, LRRC15 and hydrolyze HA simultaneously.
  • the trispecific antibody disclosed herein inhibits FAP-or LRRC15-mediated disease or disorders, such as the colon carcinoma.
  • Figure 18C illustrate the format of a trispecific antibody disclosed herein.
  • Figure 18C shows a trispecific antibody as indicated under the notations of "first arm” (hyaluronidase enzyme domain hydrolyzing HA) and “second arm” (targeting FAP and LRRC15) .
  • Figure 18C shows that the Fc-hyaluronidase fusion arm (first arm) of the trispecific Ab can comprise a human IgG with an HC, wherein the heavy chain fusion comprises from the N-terminus to the C-terminus: one hyaluronidase enzyme domain or fragment thereof, and an Fc region; and the FAP-LRRC15 binding arm (second arm) of the trispecific Ab can comprise a human IgG with an HC and an LC, wherein the light chain comprises from the N-terminus to the C-terminus: LC variable region VL, and constant light chain CL, and wherein the heavy chain fusion comprises from the N-terminus to the C-terminus: an HC variable region VH, a CH1 domain, an Fc region and a linker and a tandem of two VHOs.
  • the disclosure provides for "first arm” (hyaluronidase) amino acid sequence set forth as SEQ ID NO: 191 in Table 19.
  • the disclosure provides for "second arm” (FAP x LRRC15) amino acid sequence set forth as SEQ ID NO: 192 in Table 19.
  • Targeting CAF cancer-associated fibroblast
  • anti-FAP and anti-LRRC15 antibodies for homing of hyaluronidase into solid tumors is to specifically target and penetrate the dense stroma of the tumor microenvironment, where CAFs are abundant. This can be achieved by engineering hyaluronidase to have a high affinity for the extracellular matrix components, particularly hyaluronic acid, which is a major component of the stroma. Additionally, the dual-targeting strategy aims to minimize off-target effects and maximize the therapeutic efficacy of hyaluronidase within the solid tumors.
  • a trispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 19-128, 149, and 151 and a light chain sequence selected from SEQ ID NOs: 4-5, 9-11, 15-16, 18, 150, and 152, respectively.
  • a trispecific antibody disclosed herein may comprise a first binding arm targeting tumor antigens on cancer cells or CAFs.
  • a trispecific antibody disclosed herein comprises a first binding arm comprising a human hyaluronidase enzyme sequence selected from SEQ ID NOs: 352-358 and 144-148, respectively.
  • a trispecific antibody disclosed herein may comprise a second binding arm targeting FAP and LRRC15.
  • a trispecific antibody disclosed herein comprises a second binding arm comprising a human IgG1 heavy chain and a light chain sequence, targeting FAP, selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, and 151 and 4-5, 9-11, 15-16, 18, 150, and 152, respectively; and at least one VHO or antigen-binding fragment thereof, targeting human LRRC15, selected from SEQ ID NOs: 19-128.
  • the present disclosure provides an EGFR x HYAL bispecific antibody that targets simultaneously proteins linked to EGFR-associated pathways as well as proteins that have the HA-hydrolyzing activity.
  • EGFR and HA targets have differential expression levels in pathological sites and normal tissues.
  • the present disclosure provides a bispecific antibody that targets and binds to human EGFR and HA simultaneously and is capable of effectively blocking EGFR protein at the protein level and hydrolyzing the HA polymers.
  • the bispecific antibody binds EGFR protein or HA polymers and binds to one protein or portion thereof without affecting the binding of the other portion, that is, having the ability to bind EGFR and hydrolyze HA simultaneously.
  • the bispecific antibody disclosed herein inhibits EGFR-mediated diseases or disorders, such as the colon carcinoma.
  • the present disclosure provides a bispecific antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -a hyaluronidase domain or portion thereof-an optional linker A -an IgG heavy chain or an antigen-binding portion thereof, wherein the hyaluronidase domain or portion thereof of the first binding arm comprises a hyaluronidase domain or portion as described herein and can hydrolyze the hyaluronic acid (HA) and; and a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -at least one variable-heavy-chain only (VHO) single domain or an antigen-binding portion thereof, wherein the VHO or antigen-binding portion thereof of the second binding arm comprises an anti-EGFR antibody or antigen binding fragment as described herein and can target an EGFR-associated pathway
  • the present disclosure provides a bispecific antibody that can be generated using well established point mutations in the CH1, CH2, and CH3 domains via controlled Fab arm exchange or via co-expression.
  • all constructs are symmetric so that there is no preference for the selection of point mutations of the respective parental antibodies.
  • Figure 15B illustrate the format of a bispecific antibody disclosed herein.
  • Figure 15B shows a bispecific antibody comprising a "first arm” (hyaluronidase enzyme domain hydrolyzing HA) and a “second arm” (targeting EGFR) .
  • Figure 15B shows that the Fc-hyaluronidase fusion arm (first arm) of the bispecific antibody can comprise a heavy chain fusion, wherein the heavy chain fusion comprises from the N-terminus to the C-terminus: one hyaluronidase enzyme domain or fragment thereof, and an Fc region; and the EGFR binding arm (second arm) of the bispecific antibody can comprise a human heavy chain, wherein the heavy chain comprises from the N-terminus to the C-terminus: a VHO, a linker and an Fc region.
  • the disclosure provides for the "first arm" (hyaluronidase) amino acid sequence set forth as SEQ ID NO: 148 in Table 9.
  • the disclosure provides for the "second arm” (EGFR) amino acid sequence set forth as SEQ ID NO: 165 in Table 10.
  • a bispecific antibody disclosed herein comprises at least one VHO or antigen-binding fragment thereof selected from SEQ ID NOs: 165 and 166.
  • a bispecific antibody disclosed herein comprises a HYAL-Fc fusion chain comprising a sequence selected from SEQ ID NOs: 352-358 and 144-146, respectively.
  • a bispecific antibody disclosed herein may comprise a first binding arm targeting HA.
  • a bispecific antibody disclosed herein comprises a first binding arm comprising a human hyaluronidase enzyme sequence selected from SEQ ID NOs: 352-358 and 144-148, respectively;
  • a bispecific antibody disclosed herein may comprise a second binding arm targeting EGFR.
  • a bispecific antibody disclosed herein comprises a second binding arm comprising at least one (e.g., two, three, or four VHOs in tandem) VHO or antigen-binding fragment thereof, targeting human EGFR, selected from SEQ ID NOs: 165 and 166.
  • the present disclosure provides a bispecific antibody that targets and binds to human FAP and LRRC15 simultaneously and is capable of effectively blocking FAP or LRRC15 proteins at the protein level.
  • the bispecific antibody binds FAP and LRRC15 proteins and binds to one protein or portion thereof without affecting the binding of the other portion, that is, having the ability to bind FAP and LRRC15 simultaneously.
  • the bispecific antibody disclosed herein inhibits FAP-or LRRC15-mediated disease or disorders, such as the glioblastoma.
  • the present disclosure provides a bispecific antibody that can be generated using well established point mutations in the CH1, CH2, and CH3 domains via controlled Fab arm exchange or via co-expression.
  • all constructs are symmetric so that there is no preference for the selection of point mutations of the respective parental antibodies.
  • Figure 17C illustrate a format of a bispecific antibody disclosed herein.
  • Figure 17C shows a bispecific antibody as indicated to target FAP and LRRC15.
  • Figure 17C shows that the bispecific Ab can comprise a FAP-LRRC15 binding arm
  • the bispecific antibody can comprise a human IgG with an HC and an LC, wherein the light chain comprises from the N-terminus to the C-terminus: LC variable region VL and constant light chain CL, and wherein the heavy chain fusion comprises from the N-terminus to the C-terminus: an HC variable region VH targeting FAP, a CH1 domain, an Fc region and a linker and a tandem of two VHOs targeting LRRC15.
  • the disclosure provides for a heavy chain fusion of an FAP x LRRC15 binding arm with amino acid sequence set forth as SEQ ID NO: 192 in Table 19.
  • a bispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 19-128, 149, and 151 and a light chain sequence selected from SEQ ID NOs: 4-5, 9-11, 15-16, 18, 150, and 152, respectively.
  • a bispecific antibody disclosed herein may comprise a binding arm targeting FAP and LRRC15.
  • a bispecific antibody disclosed herein comprises a binding arm comprising a human IgG1 heavy chain and a light chain sequence, targeting human FAP, selected from SEQ ID NOs: 1-3, 6-8, 12-14, 17, 149, and 151 and 4-5, 9-11, 15-16, 18, 150, and 152, respectively; and at least one VHO or antigen-binding fragment thereof, targeting human LRRC15, selected from SEQ ID NOs: 19-128.
  • Trispecific EGFR x cMet x VEGF antibodies selected from SEQ ID NOs: 19-128.
  • the present disclosure provides an EGFR x cMet x VEGF trispecific antibody (see, e.g., Intl. Publ. No. WO 2023/069888 A1) that targets simultaneously proteins linked to EGFR-associated pathways, cMet-associated pathways or VEGF-associated pathways.
  • EGFR and cMet and VEGF targets have differential expression levels in pathological sites and normal tissues.
  • the present disclosure provides a trispecific antibody that targets and binds to human EGFR and cMet and VEGF simultaneously and is capable of effectively blocking EGFR or cMet or VEGF proteins at the protein level.
  • the trispecific antibody binds EGFR or cMet or VEGF proteins and binds to one protein without affecting the binding of the other protein, that is, having the ability to bind EGFR, cMet and VEGF simultaneously.
  • the trispecific antibody disclosed herein inhibits EGFR or cMet or VEGF-mediated disease or disorders, such as the non-small cell lung cancer.
  • the present disclosure provides trispecific antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -VHH -an optional linker B -VHH -an optional linker A -Fc -an optional linker C -a scFv or an antigen-binding portion thereof, wherein the VHOs and scFv or an antigen-binding portions thereof of the first binding arm can target a EGFR-and VEGF-associated pathways, respectively, and comprise an anti-EGFR and an anti-VEGF or antigen binding fragments as described herein; a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, a signal sequence B -an IgG light
  • the Fc heterodimerization can also be realized by Fc mutations to facilitate a Knob-in-Hole strategy (see, e.g., Intl. Publ. No. WO 2006/028936) .
  • An amino acid with a small side chain (hole) is introduced into one Fc domain and an amino acid with a large side chain (knob) is introduced into the other Fc domain.
  • a heterodimer is formed as a result of the preferential interaction of the heavy chain with a "hole” with the heavy chain with a "knob” (Ridgway et al., 1996) .
  • Exemplary Fc mutation pairs forming a knob and a hole are: T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S/L368A/Y407V.
  • the controlled Fab arm exchange can be applied to generate trispecific antibodies from separate transfections and purification of the corresponding parental antibodies.
  • a trispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 161, 163, 165, 166, and a light chain sequence selected from SEQ ID NOs: 162 and 164, respectively.
  • a trispecific antibody disclosed herein may comprise a first binding arms targeting EGFR and VEGF.
  • a trispecific antibody disclosed herein comprises a first binding arm comprising at least one VHO or antigen-binding fragment thereof, targeting human EGFR, selected from SEQ ID NOs: 165 and 166, respectively; and one single-chain fragment variable (scFv) domain or antigen-binding fragment thereof, targeting human VEGF, selected from SEQ ID NOs: 163 and 164, respectively.
  • a trispecific antibody disclosed herein may comprise a second binding arm targeting cMet.
  • a trispecific antibody disclosed herein comprises a second binding arm comprising an IgG heavy chain and a light chain or antigen-binding fragment thereof, targeting human cMet, selected from SEQ ID NOs: 161 and 162.
  • the present disclosure provides an EGFR x cMet bispecific antibody (see, e.g., Intl. Publ. No. WO 2023/069888 A1) that targets simultaneously proteins linked to EGFR-associated pathways and cMet-associated pathways.
  • EGFR and cMet targets have differential expression levels in pathological sites and normal tissues.
  • the present disclosure provides a bispecific antibody that targets and binds to human EGFR and cMet simultaneously and is capable of effectively blocking EGFR or cMet proteins at the protein level.
  • the bispecific antibody binds EGFR or cMet proteins and binds to one protein without affecting the binding of the other protein, that is, having the ability to bind EGFR and cMet simultaneously.
  • the bispecific antibody disclosed herein inhibits EGFR or cMet-mediated disease or disorders, such as the colorectal cancer.
  • the present disclosure provides bispecific antibody comprising: a first binding arm comprising: a first heavy chain fusion protein comprising, from the N- to the C-terminus, an optional signal sequence A -VHH -an optional linker B -VHH -an optional linker A -Fc, wherein the tandem VHOs or antigen-binding portions thereof of the first binding arm can target an EGFR-associated pathways, and comprise an anti-EGFR or antigen binding fragments as described herein; a second binding arm comprising: a second heavy chain fusion protein comprising, from the N-to the C-terminus, an optional signal sequence A -an IgG heavy chain or an antigen-binding portion thereof, and a first light chain protein comprising, from the N-to the C-terminus, an optional signal sequence B -an IgG light chain or an antigen-binding portion thereof, wherein the IgG heavy chain or an antigen-binding portion thereof and the IgG light chain or an antigen-binding portion thereof
  • the Fc heterodimerization can also be realized by Fc mutations to facilitate a Knob-in-Hole strategy (see, e.g., Intl. Publ. No. WO 2006/028936) .
  • An amino acid with a small side chain (hole) is introduced into one Fc domain and an amino acid with a large side chain (knob) is introduced into the other Fc domain.
  • a heterodimer is formed as a result of the preferential interaction of the heavy chain with a "hole” with the heavy chain with a "knob” (Ridgway et al., 1996) .
  • Exemplary Fc mutation pairs forming a knob and a hole are: T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S/L368A/Y407V.
  • the controlled Fab arm exchange can be applied to generate bispecific antibodies from separate transfections and purification of the corresponding parental antibodies.
  • a bispecific antibody disclosed herein comprises a heavy chain sequence selected from SEQ ID NOs: 161, 165, 166, and a light chain sequence of SEQ ID NO: 162, respectively.
  • a bispecific antibody disclosed herein may comprise a first binding arms targeting EGFR.
  • a bispecific antibody disclosed herein comprises a first binding arm comprising at least one VHO or antigen-binding fragment thereof, targeting human EGFR, selected from SEQ ID NOs: 165 and 166.
  • a bispecific antibody disclosed herein may comprise a second binding arms targeting cMet.
  • a bispecific antibody disclosed herein comprises a second binding arm comprising an IgG heavy chain and a light chain or antigen-binding fragment thereof, targeting human cMet, selected from SEQ ID NOs: 161 and 162. Table 12. Sequences of synthetic linkers Table 13. Sequences of Fc Leader Sequences
  • a leader peptide is chosen to drive the secretion of the antibody described in this disclosure into the cell culture supernatant as a secreted antibody protein. Any leader peptide for any known secreted proteins /peptides can be used.
  • leader peptide or “signal peptide” or “leader sequence” includes a short peptide, usually 16-30 amino acids in length, that is present at the N-terminus of most of newly synthesized proteins that are destined towards the secretory pathway.
  • lead peptides are extremely heterogeneous in sequence, and many prokaryotic and eukaryotic lead peptides are functionally interchangeable even between distinct species, the efficiency of protein secretion may be strongly determined by the sequence of the lead /signal peptide.
  • the leader peptide that is from a protein residing either inside certain organelles (such as the endoplasmic reticulum, Golgi, or endosomes) , secreted from the cell, or inserted into most cellular membranes may be used.
  • organelles such as the endoplasmic reticulum, Golgi, or endosomes
  • the leader peptide is from a eukaryotic protein.
  • the leader peptide is from a secreted protein, e.g., a protein secreted outside a cell.
  • the leader peptide is from a transmembrane protein.
  • the leader peptide contains a stretch of amino acids that is recognized and cleaved by a signal peptidase.
  • the leader peptide does not contain a cleavage recognition sequence of a signal peptidase.
  • the leader peptide is a signal peptide for tissue plasminogen activator (tPA) , herpes simplex virus glycoprotein D (HSV gD) , a growth hormone, a cytokine, a lipoprotein export signal, CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD8 ⁇ , CD19, CD28, 4-1BB or GM-CSFR, or S. cerevisiae mating factor ⁇ -1 signal peptide.
  • tPA tissue plasminogen activator
  • HSV gD herpes simplex virus glycoprotein D
  • CD2 CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD8 ⁇ , CD19, CD28, 4-1BB or GM-CSFR
  • S. cerevisiae mating factor ⁇ -1 signal peptide S. cerevisiae mating factor ⁇ -1 signal peptide.
  • a leader sequence as described herein may be a mammalian CD4 or CD8 leader sequence, including but not limited to, e.g., a human CD4 or CD8 leader sequence, a non-human primate CD4 or CD8 leader sequence, a rodent CD4 or CD8 leader sequence, and the like.
  • a CD4 or CD8 leader comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the human CD4 or CD8 leader sequences.
  • the anti-FAP, anti-LRRC15, anti-CD3, and Fc-hyaluronidase fusions and multispecific antibodies may comprise a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a native Fc region, for example, to extend the half-life of the bispecific or trispecific antibody, enhance resistance of the bispecific or trispecific antibody to proteolytic degradation, reduce effector functionality of the bispecific or trispecific antibody, facilitate generation of the bispecific or trispecific antibody by Fc heterodimerization, facilitate the multimerization of the bispecific or trispecific, and/or improve manufacturing and drug stability of the bispecific or trispecific antibody.
  • the modified Fc region comprises at least one amino acid modification relative to a native Fc region, for example, to extend the half-life of the bispecific or trispecific antibody, enhance resistance of the bispecific or trispecific antibody to proteolytic degradation, reduce effector functionality of the bispecific or trispecific antibody, facilitate generation of the bispecific or trispecific antibody by Fc heterodimerization, facilitate the multi
  • the Fc domain is altered to allow for silencing of the Fc domain to minimize effector function activity which can cause immune cell depletion and cytokine release syndrome.
  • anti-FAP, anti-LRRC15, anti-CD3, and Fc-hyaluronidase fusions and multispecific antibodies such as FAP x CD3, LRRC15 x CD3, FAP x LRRC15 x HYAL bispecific or trispecific antibodies as described herein are provided with a modified Fc region wherein a naturally occurring Fc region is modified to extend the half-life of the antibody when compared to the parental native antibody in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
  • Exemplary mutations that may be made singularly or in combination are T250Q, M252Y, I253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R mutations.
  • the present antibodies and Fc-hyaluronidase fusions may comprise an IgG Fc domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an IgG1 Fc sequence such as SEQ ID NO: 172.
  • the present antibodies and Fc-hyaluronidase fusions may comprise an IgG Fc domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an IgG2 Fc sequence such as SEQ ID NO: 173.
  • the present antibodies and Fc-hyaluronidase fusions may comprise an IgG Fc domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an IgG3 Fc sequence such as SEQ ID NO: 174.
  • the present antibodies and Fc-hyaluronidase fusions may comprise an IgG Fc domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an IgG4 Fc sequence such as SEQ ID NO: 175.
  • the S228P mutation may be made into IgG4 antibodies to enhance IgG 4 stability.
  • the antibodies and Fc-hyaluronidase fusions may comprise a modified F c region, wherein the modified F c region comprises at least one amino acid modification relative to a native F c region.
  • the present antibodies and Fc-hyaluronidase fusions are provided with a modified F c region where a naturally-occurring F c region is modified to extend the half-life of the antibody when compared to the parental native antibody in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
  • the extension of half-life can be realized by engineering the M252Y/S254T/T256E mutations, collectively designated as YTE mutations, e.g., in IgG1 F c such as SEQ ID NO: 179, numbering according to the EU Index (Dall'A cqua et al., 2006) .
  • the extension of half-life can also be realized by engineering the M428L/N434S mutations, collectively designated as LS mutations, collectively designated as LS mutations, e.g., in IgG 1 F c such as SEQ ID NO: 180 (Zalevsky et al., 2010) .
  • the extension of half-life can also be realized by engineering the T250Q/M428L mutations in IgG 1 F c (Hinton et al., 2006) .
  • the extension of half-life can also be realized by engineering the N434A mutations in IgG 1 F c (Shields et al., 2001a) .
  • the extension of half-life can also be realized by engineering the T307A/E380A/N434A mutations in IgG 1 F c (Petkova et al., 2006) .
  • the present antibodies and Fc-hyaluronidase fusions are provided with a modified F c region where a naturally-occurring F c region is modified to enhance the antibody resistance to proteolytic degradation by a protease that cleaves the wild-type antibody between or at residues 222-237 (EU numbering) .
  • the resistance to proteolytic degradation can be realized by engineering E233P/L234A/L235A mutations in the hinge region with G236 deleted when compared to a parental native antibody, such as SEQ ID NO: 181, residue numbering according to the EU Index (Kinder et al., 2013) .
  • Such modifications can comprise low or null Fc fucosylation, and/or engineering of Fc mutations such as S239E/I332E/A330L, S239D/I332E/A330L, S239D/I332E, S239D, I332E, S298A/E333A/K334A.
  • the enhancement of effector functions can be realized by engineering F243L/R292P/Y300L/V305I/P396L mutations, collectively designated as LPLIL mutations, when compared to a parental native antibody, such as SEQ ID NO: 182, residue numbering according to the EU Index (Stavenhagen et al., 2008) .
  • the enhancement of effector functions can be realized by engineering F243L/R292P/Y300L/V305I/P396L mutations, collectively designated as LPLIL mutations, and E345R mutation when compared to a parental native antibody, such as SEQ ID NO:184, residues numbering according to the EU Index.
  • the antibodies of the disclosure may be engineered to introduce at least one mutation in the antibody F c that reduces binding of the antibody to an activating F c ⁇ receptor (F c ⁇ R) and/or reduces F c effector functions such as C1q binding, complement dependent cytotoxicity (CDC) , antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP) .
  • F c ⁇ R activating F c ⁇ receptor
  • F c effector functions such as C1q binding, complement dependent cytotoxicity (CDC) , antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP) .
  • the reduction of effector functions can be realized by engineering L234A/L235A mutations, collectively designated as AA mutations, when compared to a parental native antibody, such as SEQ ID NO: 185, residue numbering according to the EU Index.
  • Antibodies of the disclosure further comprising conservative modifications are within the scope of the disclosure.
  • “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences.
  • Conservative modifications include amino acid substitutions, additions and deletions.
  • Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain.
  • Antibodies of the disclosure may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the disclosure.
  • Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein/protein interface interactions that have impact upon the HC and LC pairing, (McNeil et al.) burial of polar and charged residues, (4) H-bonding network for polar and charged residues; and/or (5) surface charge and polar residue distribution among other intra-and inter-molecular forces ( &Plückthun, 2001)
  • Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modelling in certain cases, and the effect of the residues on antibody stability may be tested by generating and evaluating variants harboring mutations in the identified residues.
  • T m thermal transition midpoint
  • Spigel et al. differential scanning calorimetry
  • the antibodies or fusion proteins of the disclosure can be encoded by one or more nucleic acids for protein expression.
  • a FAP x CD3 bispecific antibody of the present disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences that encode the light and heavy chain polypeptides of the antibody) , or by two or more separate nucleic acids, each of which encodes a different part of the parental antibody.
  • the appropriate recombinant DNA is prepared by the DNA recombination techniques and then transfected into mammalian cells, the corresponding proteins, such as anti-FAP, anti-LRRC15, anti-FAP/LRRC15, anti-CD3 antibodies and Fc-hyaluronidase fusions are expressed, purified, identified, and/or screened.
  • the corresponding proteins such as anti-FAP, anti-LRRC15, anti-FAP/LRRC15, anti-CD3 antibodies and Fc-hyaluronidase fusions are expressed, purified, identified, and/or screened.
  • a multispecific such as bispecific or trispecific, antibody can be generated using the controlled Fab arm exchange or other bispecific antibody generation process to produce a multispecific such as bispecific or trispecific antibody which shows biological effects of simultaneous binding to corresponding antigens.
  • FAP x CD3 bispecific antibody can be generated from anti-FAP and anti-CD3 antibodies
  • FAP x LRRC15 x HYAL trispecific antibodies can be generated from anti-FAP x LRRC15 antibodies and HYAL-Fc fusion protein using the controlled Fab arm exchange or other bispecific antibody generation process.
  • Affinity and blocking efficiency of multispecific antibodies, such as bispecific or trispecific antibodies, are identified through in vitro experiments.
  • nucleic acids described herein can be inserted into vectors, e.g., nucleic acid expression vectors and/or targeting vectors.
  • vectors can be used in many ways, e.g., for the expression of an antibody described herein in a cell or transgenic animal.
  • Vectors are typically selected to be functional in the host cell in which the vector will be used.
  • a nucleic acid molecule encoding an antibody described herein may be amplified /expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells.
  • Selection of the host cell will depend in part on whether the antibodies disclosed herein, such as a FAP x CD3 bispecific antibody described herein, is to be post-translationally modified (e.g., glycosylated and/or phosphorylated) . If so, yeast, insect, or mammalian host cells are preferable.
  • post-translationally modified e.g., glycosylated and/or phosphorylated
  • Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a leader or signal sequence is engineered at the N-terminus of the antibodies, e.g., a FAP x CD3 bispecific antibody, described herein to guide its secretion.
  • the secretion of the FAP x CD3 bispecific antibody from a host cell will result in the removal of the signal peptide from the antibody.
  • the mature FAP x CD3 bispecific antibody will lack any leader or signal sequence.
  • the disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the disclosure.
  • the cell can be any type of cell capable of being transformed with the nucleic acid or vector of the disclosure so as to produce a polypeptide encoded thereby.
  • DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • Methods of introducing nucleic acids and vectors into isolated cells and the culture and selection of transformed host cells in vitro include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.
  • the cell After introducing the nucleic acid or vector of the disclosure into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence.
  • the antibody, antigen binding fragment, or portion of the antibody then can be isolated from the cell.
  • two or more vectors that together encode the multispecific proteins such as the anti-FAP, anti-LRRC15, anti-FAP/LRRC15, anti-CD3 antibodies and Fc-hyaluronidase fusions described herein, can be introduced into the cell.
  • an antibody e.g., a FAP x CD3 bispecific antibody, described herein, which is secreted into the cell media
  • purification of an antibody can be accomplished using a variety of techniques including affinity, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high-pressure liquid chromatography.
  • proteins comprising an Fc region may be purified by affinity chromatography with Protein A, which selectively binds the Fc region.
  • Modified forms of the antibodies may be prepared with affinity tags, such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn. ) or Myc (Invitrogen) at either its carboxyl or amino terminus and purified by a one-step affinity column.
  • affinity tags such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn. ) or Myc (Invitrogen) at either its carboxyl or amino terminus
  • affinity tags such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn. ) or Myc (Invitrogen) at either its carboxyl or amino terminus
  • Poly histidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the nickel columns) can be used for purification of Poly histidine-tagged selective binding agents. In some instances, more than one pur
  • the binding of an antibody may be determined by ELISA by immobilizing recombinant or purified antigen, sequestering the antibody with the immobilized antigen and determining the amount of bound antibody. This can also be performed using a surface plasmon resonance or biolayer interferometry instrument for kinetic analysis of binding interactions.
  • the binding of antibody may be determined by flow cytometry by incubating the antibody with cells expressing antigens on the cell surface and determining the amount of antibody bound to the cell surface antigen.
  • the art describes the use of animal models and ex vivo assays to determine the in vivo efficacy of the anti-FAP antibodies, anti-LRRC15 antibodies, and the enzyme activity of hyaluronidase as well as multispecific (e.g., bispecific, trispecific, or tetra-specific) including portions targeting different targets in the present disclosure.
  • the anti-tumor efficacy of these antibodies can be evaluated using xenograft mouse models with human tumor cell lines and patient-derived primary tumor cells.
  • the anti-tumor efficacy of the described antibodies can also be assessed in xenograft models in conjunction with the reconstitution of human peripheral blood mononuclear cells (PBMC) in immunodeficient mice.
  • PBMC peripheral blood mononuclear cells
  • compositions such as a trispecific antibody comprising anti-FAP, anti-LRRC15 and/or HYAL-Fc
  • compositions can be formulated in compositions, especially pharmaceutical compositions, for use in the methods herein.
  • Such compositions comprise a therapeutically or prophylactically effective amount of the antibody described in this disclosure in mixture with a suitable carrier, e.g., a pharmaceutically acceptable agent.
  • a suitable carrier e.g., a pharmaceutically acceptable agent.
  • the antibody described in this disclosure is sufficiently purified for administration to an animal before formulation in a pharmaceutical composition.
  • Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
  • the composition can be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents.
  • compositions can be suitable for parenteral administration.
  • Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal) , intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral, and inhaled routes.
  • the present disclosure provides antibodies for treating various diseases or conditions,
  • the present disclosure provides antibodies such as a trispecific antibody comprising anti-FAP, anti-LRRC15 antibodies and Fc-fusion hyaluronidase, for the treatment or prevention of a disease or condition, e.g., cancer, such as breast, ovarian, gastric, lung, pancreatic, and other cancers.
  • a disease or condition e.g., cancer, such as breast, ovarian, gastric, lung, pancreatic, and other cancers.
  • the present disclosure provides a method for treating a FAP-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the anti-FAP antibody or antigen binding fragment; the anti-LRRC15 antibody or antigen binding fragment; the Fc-fusion hyaluronidase; the multispecific antibodies, and/or the conjugates, or the pharmaceutical composition disclosed herein.
  • the FAP-mediated disease or disorder includes, but is not limited to, cancers such as breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer) , pancreatic cancer, glioblastoma, gastro-intestinal cancers, pancreatic/ampullary cancers, desmoid tumors, thyroid cancers, and colorectal carcinoma.
  • cancers such as breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer) , pancreatic cancer, glioblastoma, gastro-intestinal cancers, pancreatic/ampullary cancers, desmoid tumors, thyroid cancers, and colorectal carcinoma.
  • the present disclosure provides a method for treating an LRRC15-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the anti-FAP antibody or antigen binding fragment; the anti-LRRC15 antibody or antigen binding fragment; the Fc-fusion hyaluronidase; the multispecific antibodies, and/or the conjugates, or the pharmaceutical composition disclosed herein.
  • the LRRC15-mediated disease or disorder includes, but is not limited to, cancers such as glioblastoma, osteosarcoma, sarcomas, melanoma, prostate cancers, breast cancers, head and neck cancers, lung cancers, cervical cancers, and colorectal carcinoma.
  • the present disclosure provides a method for treating an EGFR-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the Fc-fusion hyaluronidases, the multispecific antibodies, and/or the conjugates, or the pharmaceutical composition disclosed herein.
  • the EGFR-mediated disease or disorder includes, but is not limited to, non-small cell lung cancer and colorectal carcinoma.
  • the present disclosure provides a method for treating an HA-mediated disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the anti-FAP antibody or antigen binding fragment; the anti-LRRC15 antibody or antigen binding fragment of any one of claims 4-8; the Fc-fusion hyaluronidases; or the multispecific antibody; and/or the conjugate, or the pharmaceutical composition disclosed herein.
  • the HA-mediated disease or disorder includes, but is not limited to, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, prostate cancer, gastric cancer and pancreatic ductal adenocarcinoma.
  • Example 1 Expression and purification of anti-FAP, anti-LRRC15 and anti-CD3 antibodies
  • Plasmids encoding heavy and light chains for each monoclonal antibody of anti-FAP, anti-LRRC15, and anti-CD3 variants in a human IgG1 backbone were co-transfected into Expi293F cells, following the transfection kit instructions (Thermo Scientific) .
  • the plasmid encoding the VHO heavy chain was transfected into Expi293F cells using the same transfection kit instructions. Subsequently, the cells were spun down five days post-transfection, and the supernatant was passed through a 0.2 ⁇ m filter. The purification of the supernatants was conducted by affinity chromatography over protein A agarose columns (GE Healthcare Life Sciences) .
  • the purified monoclonal antibody underwent buffer exchange into DPBS, pH 7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm. The protein purity was further confirmed by SDS-PAGE or SEC.
  • Example 2 Expression and purification of the HYAL-His6 and HYAL-Fc fusion proteins
  • plasmids encoding fusion chains were transfected into Chinese hamster ovary (CHO) -K1 cell line by electroporation, following the transfection kit instructions (Etta Biotech) . Subsequently, the cells were spun down six days post-transfection, and the supernatant were passed through a 0.2 ⁇ m filter. The purification of the supernatants was conducted by affinity chromatography over Ni-NTA or protein A agarose columns (GE Healthcare Life Sciences) . The purified fusion proteins underwent buffer exchange into DPBS, pH 7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.
  • the enzymatic activity of hyaluronidase enzyme fusing to poly-histidine or Fc was determined using a micro-turbidity assay (Rapport et al., 1950) .
  • One unit of hyaluronidase activity was defined as the amount of enzyme that caused a change in A600 of 0.330 per minute at pH 5.35 at 37 °C in a 2.0 mL reaction mixture (45-minute assay) .
  • the method involved the generation of an insoluble precipitate by the HYAL enzyme when acidified serum albumin interacted with hyaluronic acid.
  • the following reagents were prepared: Hyaluronic Acid Solution (0.03%w/v) , Enzyme Diluent (20 mM Sodium Phosphate with 77 mM Sodium Chloride and 0.01%w/v Bovine Serum Albumin, pH 7.0 at 37 °C) , and Acidic Albumin Solution (24 mM Sodium Acetate, 79 mM Acetic Acid with 0.1%w/v Bovine Serum Albumin, pH 3.75 at 25 °C) .
  • Example 4 Bispecific or trispecific antibodies generation by controlled Fab-arm exchange technology
  • the bispecific or trispecific antibodies described in this disclosure were generated using controlled Fab-arm exchange (cFAE) technology (cFAE) (Labrijn, Meesters, Priem, et al., 2014) . Briefly, the two parental IgG1 antibodies, each containing a single matching point mutation (F450L and K409R) in the CH3 domain, were separately expressed. They were then mixed at a 1: 1 molar ratio under permissive redox conditions (75 mM ⁇ -MEA, Sigma, M9768) in vitro, allowing for the recombination of half-molecules (37°C for 5 hours in a water bath) .
  • cFAE controlled Fab-arm exchange
  • Example 7 FAP x CD3 bispecific antibodies induce T cell activation in U-87 MG cells
  • the anti-FAP hits were further validated for their activity for T cell activation by generating bispecific antibodies paring with various anti-CD3 antibodies.
  • the NFAT luciferase reporter Jurkat cell line expressed firefly luciferase under the control of the NFAT response elements stably integrated into the Jurkat cell genome.
  • Jurkat-NFAT effector cells (Vazyme, DD1302) were used to assess the T cell activation activity. Experiments were conducted following the manufacturer’s instructions. Briefly, target cells (U-87 MG) were seeded into each well of the 96-well white plate and incubated overnight.
  • the medium was removed on the next day, and Jurkat-NFAT effector cells were added into each well for an E: T ratio of 6: 1. Also, antibodies with a serial of three-fold dilution were added into each well. The plate is further incubated at 37°C, 5%CO 2 for 6 h. After incubation, the plate was equilibrated at room temperature for 10 min and then Bio-Lite luciferase assay substrate (Vazyme, DD1201-01) was added into each well for another 10 min in the dark for signal stabilization. The final luminescence signal was measured using a multi-plate reader (Tecan) .
  • FAP x CD3 bispecific antibodies were found to induce T cell activation with presence of U-87 MG cells in a concentration-dependent manner, as depicted in Figure 4.
  • the FAP_MO36_v6 x Cris7_v4 bispecific antibodies exhibited comparable T cell activation activity to the FAP_MO33_v6 x Cris7_v4.
  • the FAP_MO36_v6 x Cris7_v3 bispecific antibodies showed lower T cell activation activity than the FAP_MO33_v6 x Cris7_v3.
  • FAP_MO36_v6 x Cris7_v4 bispecific antibodies displayed comparable T cell activation activity to the FAP_MO36_v7 x Cris7_v4, and higher than other FAP_MO36_v6 x CD3 bispecific antibodies, including anti-CD3 variants like 40G5_CD3 and 1979_CD3.
  • the FAP_4B9 x 40G5_CD3 showed higher T cell activation activity than FAP_MO36_v6 x 40G5_CD3 antibodies.
  • Antibodies that display a range of activities will be valuable in disease specific applications or in specific pairings with other antibodies in a bispecific format.
  • Example 8 FAP x CD3 bispecific antibodies induce T cell cytotoxicity in U-87 MG cells
  • TAVO412 has several mechanisms of action in inhibiting tumor growth: 1 -blocking EGFR and cMet pathways that drive cancer growth and proliferation; 2 -immune-mediated killing of tumor cells using antibody dependent cellular cytotoxicity (ADCC) , antibody dependent cellular phagocytosis (ADCP) , and complement dependent cytotoxicity (CDC) ; and 3 -blocking VEGF to reduce angiogenesis.
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • the bispecific antibody FAP x CD3 that included the anti-CD3 arm 40G5_CD3 and the anti-FAP arm FAP_4B9 was evaluated for its in vivo anti-tumor activity in a FAP positive cell-derived xenograft model of A549 human non-small cell lung cancer.
  • PBMC was engrafted into immunocompromised mice prior to A549 tumor cell inoculation. Once tumors were established, the humanized tumor-bearing mice were randomly assigned to treatment groups and the test molecules were administered intraperitoneally. Subsequently, the antibody-mediated tumor shrinkage was assessed.
  • Example 11 Cell binding of anti-LRRC15 hit antibodies in U-118 MG cells
  • anti-LRRC15_VHO494-504 exhibited higher binding than anti-LRRC15_VHO504-494, indicating a specific permutation preferential binding mode during tandem VHOs design.
  • anti-LRRC15 antibodies'capability for both antigen and cell binding enabling the subsequent assessment of LRRC15-mediated cell functions.
  • Example 12 LRRC15 x CD3 bispecific antibodies induce T cell activation in U-87 MG and U-2 OS cells
  • the NFAT reporter assay procedure used here is same as the T cell activation analysis of anti-FAP x anti-CD3 described in Example 7.
  • LRRC15 x CD3 bispecific antibodies were found to induce T cell activation in these cancer cells in a concentration-dependent manner, as shown in Figure 13.
  • nearly all tandem anti-LRRC15_VHOs demonstrated higher efficacy and potency in activating T cells than the single VHO antibodies, indicating that the bivalent binding of tandem VHOs enhanced the stability of association between target cells and T cells.
  • Example 13 LRRC15 x CD3 bispecific antibodies induce T cell cytotoxicity in U-87 MG cells
  • the T cell cytotoxicity effect of anti-LRRC15 x anti-CD3 antibodies was further confirmed in vitro with the presence of human PBMCs and U-87 MG cancer cells.
  • Bispecific antibodies were combined in increasing concentrations with PBMCs and the target cell line.
  • the PBMC killing assay procedure closely resembled the T cell cytotoxicity analysis of anti-FAP x anti-CD3 described in Example 8.
  • LRRC15_VHO494-499 x huSP34_v1 exhibited the highest killing efficacy among all test antibodies (Figure 14A) .
  • Figure 14B the tandem anti-LRRC15 VHOs x CD3 antibodies demonstrated higher killing potency and efficacy than single anti-LRRC15_VHO x CD3 bispecific antibodies.
  • Experimental limitations such as the restricted coculture time, which resulted in suboptimal killing effects for some variants due to the low-affinity binding of the CD3 arm, should be considered. However, this could be beneficial in specific diseases by reducing cytokine release syndrome while maintaining the killing effect on diseased cells.
  • Example 14 Cell binding of EGFR x HYAL antibodies to HCC827 cells
  • bispecific antibodies comprising a hyaluronidase and an antigen-binding domain
  • a binding titration of proteins including anti-EGFR x HYAL and anti-EGFR x inert arm bispecific antibodies, as well as HYAL-Fc fusion, to EGFR-positive expression cancer cell lines was conducted using flow cytometric analysis.
  • the schematic structures of the tested antibodies are depicted in Figures 15B, 15C, and 15D, respectively.
  • the cell binding assay procedure closely resembled the cell binding analysis of anti-FAP x anti-CD3 described in Example 6.
  • the recombinant soluble human hyaluronidase PH20 (36-482) has been identified as a minimally active domain, as described in US007767429B2.
  • two types of mutations were introduced into the human hyaluronidase PH20 active domain with Fc fusions for different enzyme properties and applications in the treatment of diseases, such as various solid tumors.
  • One type of mutation is based on the "consensus" sequence derived from 100 closest orthologs from primates, mammals, and rodents. Mutations in the hydrophobic core, based on the structural model of PH20 derived from the structure of HYAL1, are excluded.
  • Constructs with these consensus mutations included PH20 mut1, PH20 mut2, and PH20 mut3, all sharing less than 95%identity to the wild-type unmodified sequence.
  • the other type of mutations is designed to make substrate binding pH-dependent by introducing His residues in the active site in place of substrate-binding residues, including Y264H, D310H, Y219H, Y92H, V93H, L342H, and T341H/L342S.
  • the human PH20 hyaluronidase with these mutations is fused to a human IgG1 Fc region (L234A/L235A/K409R, SEQ ID NO: 178) with a GGGGS flexible linker (SEQ ID NO: 167) .
  • Expression, purification, and enzyme activity determination of PH20 hyaluronidase variants are summarized in Table 16. All of these Fc-hyaluronidase fusion variants have an expression titer of 2-26 mg/L in CHO expression systems and a good monomeric purity of more than 90%on average by SEC-HPLC analysis.
  • the wild-type PH20 (36-482) exhibited the highest catalytic activity of 80,000 Units/mg, followed by PH20 V93H (30,000 Units/mg) , PH20 mut1 and PH20 mut2 (20,000 Units/mg) , PH20 L342H (15,000 Units/mg) , PH20 T341H/L342S (2,000 Units/mg) , PH20 Y92H (500 Units/mg) , and PH20 Y264H and Y219H (200 Units/mg) .
  • the hyaluronidase variants displayed a range of activities, which will be valuable in disease-specific applications or in specific pairings with other antibodies in an Fc-fusion format.
  • the mutations described above can indeed be further combined to create hyaluronidase variants with even more diverse enzyme properties. By combining different mutations, it is possible to generate a wide range of hyaluronidase variants with unique characteristics tailored for specific applications in the treatment of diseases. This approach allows for the customization of enzyme properties to meet the specific needs of different therapeutic strategies and disease targets. Table 16. Screening of hyaluronidase variants
  • Example 17 Cell binding of FAP x LRRC15 bispecific antibodies in U-87 MG and IMR-90 cells
  • Hyaluronidases are enzymes that break down hyaluronic acid (HA) in the stroma, leading to the remodeling of the tumor microenvironment and potentially enhancing the therapeutic benefits.
  • HA hyaluronic acid
  • it is essential to find tumors with a high level of HA in the stroma.
  • the levels of HA in the stroma were confirmed by conducting HA staining on formalin-fixed paraffin-embedded (FFPE) samples derived from different cancer cell line-derived xenograft (CDX) tumor models. This was achieved using a specific biotin-conjugated HA binding protein (HABP, Amsbio, AMS.
  • FFPE formalin-fixed paraffin-embedded
  • CDX cancer cell line-derived xenograft
  • mice Female Balb/c nude mice were subcutaneously implanted with tumor cells, and treatments commenced when the mean tumor volume reached 100-200 mm3.
  • the testing antibodies were administered intraperitoneally (i. p. ) at a twice-weekly dosing regimen. Tumor growth and body weight were monitored twice weekly until the endpoint, and tumor volume is calculated using the formula length x width2 x 0.5.
  • Example 20 In vivo anti-tumor activity of FAP x LRRAC15 x HYAL antibody in combination with 5T4 x CD3 in an RKO colorectal carcinoma xenograft model
  • Example 21 In vivo anti-tumor activity of FAP x LRRAC15 x HYAL antibody in combination with EGFR x cMET (TAVO412A) in an RKO colorectal carcinoma xenograft model
  • TAVO412A has several mechanisms of action in inhibiting tumor growth: 1 -blocking EGFR and cMet pathways that drive cancer growth and proliferation; 2 -immune-mediated killing of tumor cells using antibody dependent cellular cytotoxicity (ADCC) , antibody dependent cellular phagocytosis (ADCP) , and complement dependent cytotoxicity (CDC) .
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Example 22 Structural designs for trispecific antibodies targeting FAP, LRRC15, and HA
  • Figure 21 depicts formats for FAP x LRRC15 x HYAL trispecific antibodies.
  • Figure 21A shows a first heavy chain with FAP and LRRC15 binding arms, and a second chain comprising a N-terminal hyaluronidase fused to an Fc region
  • Figure 21B shows a first heavy chain with FAP binding arm, and a second chain comprising a N-terminal hyaluronidase and a C-terminal LRRC15 binding arm fused to an Fc region, respectively
  • Figure 21C shows a first heavy chain with FAP and LRRC15 binding arms, and a second chain comprising a N-terminal hyaluronidase and a C-terminal LRRC15 binding arm fused to an Fc region, respectively
  • Figure 21D shows a first heavy chain with FAP and LRRC15 binding arms, and a second chain comprising a C-terminal hyaluronidase fused to an Fc region
  • Figure 21E shows
  • Antibodies in these different formats may have different binding and functional properties.
  • Complement is activated by IgG hexamers assembled at the cell surface. Science, 343 (6176) , 1260-1263. https: //doi. org/10.1126/science. 1248943 Donelan, W., Dominguez-Gutierrez, P.R., &Kusmartsev, S. (2022) . Deregulated hyaluronan metabolism in the tumor microenvironment drives cancer inflammation and tumor-associated immune suppression [Systematic Review] . Frontiers in Immunology, 13. https: //doi. org/10.3389/fimmu.
  • nlm. nih. gov/pubmed/16365427 Hussain, A., Voisin, V., Poon, S., Karamboulas, C., Bui, N.H.B., Meens, J., Dmytryshyn, J., Ho, V.W., Tang, K.H., Paterson, J., Clarke, B.A., Bernardini, M.Q., Bader, G.D., Neel, B.G., &Ailles, L.E. (2020) . Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21. J Exp Med, 217 (8) . https: //doi.

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Abstract

L'invention concerne des anticorps et des fragments ciblant FAP, LRRC15 et AH. L'invention concerne également des anticorps multispécifiques qui ciblent de multiples cibles comprenant, mais sans s'y limiter, CD3, EGFR, FAP, LRRC15 et AH. Les anticorps de l'invention peuvent traiter des cancers et/ou d'autres maladies, troubles et états dans lesquels la pathogenèse peut être traitée avec des modalités de redirection de lymphocytes T ou de remodelage de stroma.
PCT/CN2025/084379 2024-03-25 2025-03-24 Anticorps ciblant fap et lrrc15 et protéines de fusion ciblant l'hyaluronane et leurs utilisations Pending WO2025201242A1 (fr)

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