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WO2025059037A1 - Molécules de fusion d'anticorps bispécifiques ciblant b7-h4 et cd3 et leurs procédés d'utilisation - Google Patents

Molécules de fusion d'anticorps bispécifiques ciblant b7-h4 et cd3 et leurs procédés d'utilisation Download PDF

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WO2025059037A1
WO2025059037A1 PCT/US2024/045986 US2024045986W WO2025059037A1 WO 2025059037 A1 WO2025059037 A1 WO 2025059037A1 US 2024045986 W US2024045986 W US 2024045986W WO 2025059037 A1 WO2025059037 A1 WO 2025059037A1
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amino acid
seq
acid sequence
antibody
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WO2025059037A9 (fr
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Sonali DHINDWAL
Mohosin SARKAR
Jeremy S. MYERS
Eric M. Tam
Guixian JIN
Oksana SERGEEVA
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Evolveimmune Therapeutics Inc
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Evolveimmune Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2827—Immunoglobulins [IGs], 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 B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to pharmaceutical compositions of bispecific antibodies that bind B7-H4 and CD3 and their use. This invention also relates to a method and kits for detecting a predisposition to, determining risk of, and guiding therapy for cancers.
  • B7-H4 (B7-H4, V-set domain containing T cell activation inhibitor 1 or VTCN1) is a member of the B7 family of proteins, which family comprises cell-surface protein ligands that bind to receptors on lymphocytes.
  • the B7 family plays an important role in the regulation of immune responses.
  • B7-H4 negatively regulates T cell-mediated immune responses by inhibiting T cell activation, proliferation, cytokine production and cytotoxic activity (Prasad et al., 2003, Immunity 18: 863-873).
  • B7-H4 is a type I transmembrane protein that includes a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with an IgV-and an IgC-like domain with four conserved cysteine residues and seven sites for N-linked glycosylation.
  • B7-H4 expression is very limited, whereas B7-H4 expression is found on tumor cells in numerous cancer tissues (Kaur and Janakiram, 2019, ESMO Open
  • B7-H4 expression is correlated with advanced stages of cancer, poor prognosis, and decreased overall patient survival.
  • B7-H4 is an attractive target for breast cancer due to its frequent and robust expression in breast cancers and relatively low expression in normal tissues.
  • the present disclosure provides a bispecific antibody comprising a first antigen binding region that binds to CD3 and a second antigen binding region that binds to B7-H4, wherein the first antigen binding region that binds to CD3 comprises three heavy chain complementarity determining regions (CDRH1, CDRH2, CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2, CDRL3), wherein a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 34; CDRH3 comprises the amino acid sequence of SEQ ID NO: 37; CDRL1 comprises the amino acid sequence of SEQ ID NO: 42; CDRL2 comprises the amino acid sequence of SEQ ID NO: 43; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 45; or b)CDRHl comprises the amino acid sequence of SEQ ID NO: 30;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 34;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 37;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 42;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 43;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 45; or c) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 34; CDRH3 comprises the amino acid sequence of SEQ ID NO: 40; CDRL1 comprises the amino acid sequence of SEQ ID NO: 42; CDRL2 comprises the amino acid sequence of SEQ ID NO: 43; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 47; or d) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; CDRH2 comprises the amino acid sequence of SEQ ID NO: 35; CDRH3 comprises the amino acid sequence of SEQ ID NO: 38; CDRL1 comprises the amino acid sequence of SEQ ID NO: 42; CDRL2 comprises the amino acid sequence of SEQ ID NO: 43; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 47; or e) CDRH1 comprises the amino acid sequence of SEQ ID NO:
  • the first antigen binding region that binds to CD3 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: a) VH comprises the amino acid sequence of SEQ ID NO: 17, and VL comprises the amino acid sequence of SEQ ID NO: 22; or b) VH comprises the amino acid sequence of SEQ ID NO: 19, and VL comprises the amino acid sequence of SEQ ID NO: 26; or c) VH comprises the amino acid sequence of SEQ ID NO: 18, and VL comprises the amino acid sequence of SEQ ID NO: 26; or d) VH comprises the amino acid sequence of SEQ ID NO: 20, and VL comprises the amino acid sequence of SEQ ID NO: 26; and wherein the second antigen binding region that binds to B7-H4 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: i) VH comprises the amino acid sequence of SEQ ID NO: 248, and VL comprises the amino acid sequence of SEQ
  • the bispecific antibody has the following structure: a first heavy chain polypeptide (Hl) comprising a variable region (VH1), and a constant region (CHI) having a constant region 1 domain (CHIHI), a hinge region (H1H), a constant region 2 domain (CHlm) and a constant region 3 domain (CHIHS); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1); and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant
  • the bispecific antibody comprises: i) the CH1H3 and/or the CH2H3 has an A at position 297 (EU numbering); ii) the CH1H3 and/or the CH2H3 has a G at position 297 (EU numbering); or iii) the CH 1H3 and/or the CH2H3 has a S at position 297 (EU numbering). In some embodiments, the CH1H3 and/or the CH2H3 has an S at position 331 (EU numbering).
  • the first antigen binding region that binds to CD3 comprises a heavy chain (HC) and a light chain (LC), wherein: a) HC comprises the amino acid sequence of SEQ ID NO: 131, and LC comprises the amino acid sequence of SEQ ID NO: 125; or b) HC comprises the amino acid sequence of SEQ ID NO: 204, and LC comprises the amino acid sequence of SEQ ID NO: 123; or c) HC comprises the amino acid sequence of SEQ ID NO: 152, and LC comprises the amino acid sequence of SEQ ID NO: 123; or d) HC comprises the amino acid sequence of SEQ ID NO: 205, and LC comprises the amino acid sequence of SEQ ID NO: 123; and wherein the second antigen binding region that binds to B7-H4 comprises a heavy chain (HC) and a light chain (LC), wherein: i) HC comprises the amino acid sequence of SEQ ID NO: 195, and LC comprises the amino acid sequence of SEQ ID NO: 195, and
  • This disclosure provides a method for treating a human subject having breast cancer comprising the steps of: a) obtaining a biological sample from a human subject having breast cancer; b) detecting the expression levels of B7-H4 (VTCN1); c) comparing the detected expression level of B7-H4 (VTCN1) with a control level of expression; d) identifying the subject as a responder when the detected expression level of B7-H4 (VTCN1) is greater than the control level of expression; and e) administering an anti-B7-H4 antibody or fragment thereof in an amount sufficient to alleviate a symptom of breast cancer when the subject is identified as a responder.
  • VTCN1 B7-H4
  • the method comprises: i) detecting the expression levels of HER2 (ERBB2), ER(ESRl) and/or PR (PGR); ii) comparing the detected expression levels of HER2 (ERBB2), ER(ESRl) and/or PR (PGR) to a control level of expression; iii)
  • SUBSTITUTE SHEET (RULE 26) determining if the expression is classified as HER2-, ER- and/or PR-; iv) identifying the subject as a responder when the expression is classified as HER2+, HER2- ER+, HER2- ER- or PR- (triple negative).
  • the anti-B7-H4 antibody or fragment thereof is any one of the bispecific antibody of the disclosure.
  • the subject in need thereof has breast cancer, ovarian cancer, uterine cancer and/or cervical cancer.
  • the breast cancer is estrogen receptor (ER) negative, progesterone receptor (PR) negative, HER2 negative or a combination thereof.
  • the breast cancer is a triple negative cancer.
  • the breast cancer is DCIS, invasive (other), metastatic, medullary, invasive ductal, SCC, invasive lobular, invasive papillary or mucinous.
  • the subject is simultaneously administered or previously administered with a therapeutically effective amount of an additional therapeutic agent.
  • the additional therapeutic agent comprises a selective estrogen receptor degrader (SERD) and wherein the SERD is fulvestrant, elacestrant, camizestrant or a combination thereof.
  • the additional therapeutic agent comprises a CDK4/6 inhibitor, wherein the CDK4/6 inhibitor is palbociclib, ribociclib, abemaciclib or a combination thereof.
  • the additional therapeutic agent is a CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand or a cytokine.
  • FIGS. 1A-1E depicts schematic diagrams of antibodies with charged pair mutations, disulfide bond repositioning and knob into hole mutations.
  • Grey shaded domains represent a first heavy chain polypeptide (Hl) having a heavy chain variable region (VH1), having a constant region 1 domain (CHIHI), a hinge region (H1H), a constant region 2 domain (CHIHZ) and a constant region 3 domain (CHIHS); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1).
  • White shaded domains represent a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2HS); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • H2HI constant region 1 domain
  • H2H hinge region
  • second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • the + and - symbols between the antigen binding domains represent the charged pair mutations. Lines between the CHIHI and CL1 domain and CH2HI and CL2 domain
  • SUBSTITUTE SHEET (RULE 26) represent disulfide bonds, where a solid line represents an endogenous disulfide bond and a dashed line represents a repositioned disulfide bond.
  • the protrusion and dent between the CH1H3 and CH2H3 domain represent knob into hole mutations.
  • Charged pair mutations, disulfide bond repositioning and knob into hole mutations provide increased heavy chain and light chain heterodimerization, which is advantageous for production and purification of bispecific antibodies of the disclosure.
  • FIG. IE depicts a schematic diagram of an exemplary bispecific antibody fusion molecule comprising an anti-CD3 arm, an anti-B7-H4 arm and a CD58 peptide fused to the CH1H3 domain.
  • FIGS. 2A-2C are a series of graphs depicting chromatograms obtained from analytical size exclusion chromatography of light chain pairing bispecific antibodies.
  • FIG. 2A shows EIP1046.
  • FIG. 2B shows EIP1047.
  • FIG. 2C shows EIP1060.
  • FIG. 3 is a graph depicting differential scanning calorimetry analysis of light chain pairing bispecific antibodies.
  • FIGS. 4A-4B are a series of graphs depicting binding of light chain pairing bispecific antibodies to recombinant B7-H4.
  • FIG. 4A shows human B7-H4.
  • FIG. 4B shows cyno B7- H4.
  • FIGS. 5A-5B are a series of depicting cytokine release assays of light chain pairing bispecific antibodies and positive and negative controls.
  • FIG. 5A shows IL2.
  • FIG. 5B shows IL6.
  • FIG. 5C shows IFNg.
  • FIG. 5D shows TNFa.
  • FIGS. 6A-6B are a series of line graphs depicting B7-H4 levels in a variety of breast cancer cell lines.
  • FIG. 6A shows triple negative and HER2+ subtypes.
  • FIG. 6B shows HER2- HR+ subtypes.
  • FIGS. 7A-7E are a series of graphs depicting functional evaluations of bispecific antibodies (EIP 1046 and EIP 1060) compared to controls (EIP0909 [Crossmab surrogate of PF-0437] and EIP1053 [negative isotype control]) in a tumor cell and PBMC co-culture assay.
  • Various GFP-Luc tumor cells were treated with PBMCs at a 15: 1 E:T ratio with a serial dilution of bispecific antibody for 72h. Tumor cells % was calculated by GFP imaging and/or luminescence readout.
  • FIG. 7A shows T-47D.
  • FIG. 7B shows HCC-1954.
  • FIG. 7C shows CAMA-1.
  • FIG. 7D shows ZR-75-1.
  • FIG. 7E shows HCC-1569.
  • FIGS. 8A-8E are a series of graphs depicting functional evaluations of bispecific antibodies (EIP 1046 and EIP 1060) compared to controls (EIP0909 [Crossmab surrogate of PF-0437] and EIP1053 [negative isotype control]) in a tumor cell and activated T cell co-
  • FIG. 8A shows T-47D.
  • FIG. 8B shows HCC- 1954.
  • FIG. 8C shows CAMA-1.
  • FIG. 8D shows ZR-75-1.
  • FIG. 8E shows HCC-1569.
  • FIGS. 9A-9H are a series of graphs depicting gene expression of breast cancer subtypes. Patients were clustered based on their Her2 (ERBB2), ER (ESRI) or PR (PGR) gene expression into three subtypes: Her2+, Her2- HR+, and triple negative. Each of these genes are graphed (FIG. 9A, 9E for Her2; FIG. 9B, 9F for ER; and, FIG. 9C, 9G for PR) along with B7-H4 (VTCN1) (D, F) gene expression for either the TCGA (A-D) or SCAN-B (E-H) datasets.
  • ERBB2 Her2
  • ESRI ER
  • PR PR
  • the TCGA dataset was clustered into 106 Her2+, 763 Her2- HR+, and 231 triple negative patients while the SCAN-B dataset was clustered into 26 Her2+, 678 Her2- HR+, and 1178 triple negative patients.
  • ANOVA with post hoc testing was performed with * is p ⁇ 0.05, ** is p ⁇ 0.01, *** is p ⁇ 0.001, and **** is p ⁇ 0.0001.
  • FIGS. 10A-10H are a series of graphs depicting gene expression of breast cancer subtypes in the TCGA dataset.
  • FOXA1 (FIG. 10A) and GATA3 (FIG. 10B) are shown as luminal markers
  • KRT5 (FIG. 10C) and KRT6A (FIG. 10D) are shown as basal markers
  • CD8 (FIG. 10E)
  • granzyme B (FIG. 10F)
  • PDL1 FIG. 10G
  • PD1 FIGS. 10A-10H are shown as markers of immune infiltration.
  • FIGS. 11A-11H are a series of graphs depicting gene expression of breast cancer subtypes in the SCAN-B dataset. For ease of comparison, the Her2+ category was removed because it had so few samples (1% of the full dataset).
  • FOXA1 (FIG. 11A) and GATA3 (FIG. 11B) are shown as luminal markers
  • KRT5 (FIG. 11C) and KRT6A (FIG. 11D) are shown as basal markers
  • CD8 FIG. HE
  • granzyme B FIG. HF
  • PDL1 FIG. HG
  • PD1 FIGS. 11A-11H are shown as markers of immune infiltration.
  • FIGS. 12A-12C are a series of graphs depicting immunohistochemistry data for B7- H4 in three independent breast cancer tumor microarrays.
  • FIG. 12A shows percent of each core that has at least 1+ staining.
  • FIG. 12B shows B7-H4 H-score in three subtypes of breast cancer.
  • FIG. 12C shows H-scores for types of breast cancers.
  • FIGS. 13A-13B are a series of graphs depicting B7-H4 levels when cell lines are treated with endocrine therapies (fulvestrant, letrozole, tamoxifen) or CDK4/6 inhibitors (palbociclib) for 96 h.
  • FIG. 13A shows the HER2- HR+ cell line T-47D.
  • FIG. 13B shows the TNBC cell line MFM-223.
  • FIGS. 14A-14B are a series of graphs depicting B7-H4 levels on the T-47D cell line with specific gene knock outs. Using CRISPR/Cas9, T-47D cell lines were knocked out for the control (AAVS1), estrogen receptor (ESRI), or FOXA1 and then allowed to recover for 1 week.
  • FIG. 14A shows B7-H4 levels after knockout at baseline.
  • FIG. 14B shows B7-H4 levels of the same edited cell lines after treatment of fulvestrant, palbociclib, or a combination of fulvestrant and palbociclib.
  • FIGS. 15A-15C are a series of graphs depicting functional evaluations of bispecific antibodies compared to controls in a tumor cell and PBMC co-culture assay after treatment with either control (DMSO) or a combination of Fulvestrant and Palbociclib.
  • T-47D GFP- Luc tumor cells were treated with PBMCs at a 15:1 E:T ratio with a serial dilution of bispecific antibody and either treatment for 72h. Tumor cells % was calculated by GFP imaging and/or luminescence readout.
  • FIG. 15A shows EIP1046.
  • FIG. 15B shows EIP1060.
  • FIG. 15C shows EIP0909 (PF-0437 surrogate).
  • FIGS. 16A-16B are a series of homology models.
  • FIG. 16A depicts the EIP 1060 bispecific antibody;
  • FIG. 16B depicts the EIP 1046 bispecific antibody.
  • FIGS. 17A-17B are a series of size exclusion chromatograms obtained from protein A affinity and size exclusion chromatography tandem purification of representative EIP1060 humanized variants (FIG. 17A) or EIP1046 re-engineered variants (FIG. 17B).
  • FIGS. 18A-18F are a series of graphs depicting chromatograms obtained from analytical size exclusion chromatography of light chain pairing bispecific antibodies.
  • FIG. 18A shows EIP1300.
  • FIG. 18B shows EIP1303.
  • FIG. 18C shows EIP1358.
  • FIG. 18D shows EIP1416.
  • FIG. 18E shows EIP1450.
  • FIG. 18F shows EIP1451.
  • FIGS. 19A-19B are a series of line graphs depicting antigen binding of EIP 1060 and its humanized variants via sandwich ELISA where antibodies were captured on the plate coated with recombinant human B7-H4 protein (FIG. 19A) or cyno B7-H4 protein (FIG.
  • FIGS. 20A-20B are a series of line graphs depicting antigen binding of EIP 1046 and its re-engineered variants via sandwich ELISA where antibodies were captured on the plate coated with recombinant human B7-H4 protein (FIG. 20A) or cyno B7-H4 protein (FIG.
  • FIG. 21 is a graph depicting differential scanning calorimetry analysis of humanized EIP 1060 variants.
  • FIG. 22 is a graph depicting differential scanning calorimetry analysis of reengineered EIP 1046 variants.
  • FIGS. 23A-23H are a series of graphs depicting cytokine release assays of humanized EIP 1060 variants.
  • the top panel includes both positive and negative controls, while the bottom panel depicts the zoomed in view of these graphs after removing the positive controls.
  • FIGS. 23A, 23B depict IFNg.
  • FIGS. 23C, 23D depict IL6.
  • FIGS. 23E, 23F depict IL2.
  • FIGS. 23G, 23H depict TNFa.
  • FIG. 25 is a graph depicting the functional evaluations of bispecific antibodies (reengineered EIP 1046 series and humanized EIP 1060 series) compared to negative control EIP0614 in a tumor cell and PBMC co-culture assay.
  • MDA-MB468 GFP-Luc tumor cells were treated with PBMCs at a 15:1 E:T ratio with a serial dilution of bispecific antibodies for 48h.
  • Tumor cells % was calculated by GFP imaging and/or luminescence readout.
  • FIGS. 26A-26D depict a series of graphs of cytokine measurements as assessed by ELISA (IFNg (FIG. 26A), IL6 (FIG. 26B), IL2 (FIG. 26C), and TNFa (FIG. 26D)) of PBMC/tumor co-culture assays in FIG. 25 using exemplary antibodies (re-engineered EIP1046 series and humanized EIP1060 series) and negative control (EIP0614).
  • FIGS. 27A-27B are a series of graphs depicting the functional evaluations of exemplary re-humanized EIP 1046 (FIG. 27A) and humanized EIP 1060 (FIG. 27B) bispecific antibodies at different CD3 affinities compared to Genmab and Pfizer biosimilars and negative control EIP0614.
  • MDA-MB-468 tumor cells were co-cultured with PBMCs at a 15:1 E:T ratio with a serial dilution of bispecific antibodies for 4 days.
  • Four different CD3 affinities were used from intermediate to lower affinity: CD3-A6, CD3-A8, CD3-A9, and CD3-A10. (Tumor cells % was calculated by GFP imaging and/or luminescence readout.
  • FIGS. 28A-28B are a series of graphs depicting the tumor and T cell binding of exemplary bispecific antibodies (re-engineered EIP1046 series and humanized EIP1060 series) at two different CD3 affinities (CD3-A6 and CD3-A9) compared to Genmab and
  • FIG. 28A shows binding to MDA-MB-468 tumor cells while FIG. 28B shows binding to T cells.
  • FIGS. 29A-29C are a series of graphs depicting the binding of bispecific antibodies (EIP1450 from the re-engineered EIP1046 series and EIP1357 from the humanized EIP1060 series) compared to Genmab and Pfizer biosimilars for constructs in which both IgV and IgC domains are derived from B7-H4 (FIG. 29A), both IgV and IgC domains are derived from B7-H3 (FIG. 29B), and IgV domain is derived from B7-H4 and IgC domain is derived from B7-H3 (FIG. 29C).
  • FIGS. 30A-30D are a series of graphs showing mass spectrometry analysis of intact and reduced masses of one of the representative B7-H4 targeted bispecific antibodies using Xevo TQ-XS Triple Quadrupole Mass Spectrometer by Waters.
  • FIG. 30A depicts intact mass EIP1046 determined after PNGAse F deglycosylation in non-reduced condition and chromatographic separation using reverse phase C18 column.
  • FIG. 30B depicts intact mass EIP 1060 determined after PNGAse F deglycosylation in non-reduced condition and chromatographic separation using reverse phase C18 column.
  • FIGS. 31A-31C are a series of images and a graph depicting B7-H4 levels on the T- 47D cell line with specific gene knock outs or after small molecule treatment.
  • FIG. 31A shows a series of western blots from T-47D cells knocked out for AAVS1 (non-targeting negative control), ESRI, FOXA1, or GATA3 and then lysed, separated on SDS-PAGE, and blotted with antibodies against B7-H4, ER, FOXA1, GATA3, and GAPDH.
  • FIG. 31B shows quantification of western blots such as in A over 2-3 experiments. Average and SEM are shown.
  • FIGS. 32A-32B are a series of graphs depicting functional evaluations of an exemplary bispecific antibody (EIP1046) compared to a non-targeting control (EIP1053) in a tumor cell and PBMC co-culture assay after co-treatment with 2 ug/mL of either control
  • FIG. 32A shows results from co-culture assay with one healthy PBMC donor.
  • FIG. 32B shows results from co-culture assay with a different healthy PBMC donor.
  • T cell retargeting (or T cell redirecting) bispecific antibodies are a novel class of therapeutics, capable of recruiting T cells to tumor cells and inducing tumor- specific (but MHC-independent) activation of T cell effector activities.
  • the present disclosure is directed toward T cell retargeting bispecific antibodies containing an antigen binding domain that targets the CD3 portion of the T cell receptor protein complex for T cell recruitment and an antigen binding domain that targets T cells to a B7-H4 antigen.
  • This targeting design promotes the recruitment of T cell and positions it in close contact with a B7-H4-expressing cell, resulting in the formation of an immunological synapse, local T cell activation and the subsequent destruction of the target cell, such as but not limited to a cancer cell, by perforin and granzyme released from T cell cytotoxic granules into the target cell.
  • the present invention also relates to the generation of a panel of antibodies that display different binding affinities.
  • the affinity of the CD3 arm of a bispecific antibody can significantly modify the functional activity of the bispecific antibody.
  • bispecific antibodies disclosed herein may have a cytokine or costimulatory molecule fusion peptide that acts as antagonist to inhibit or block deleterious interactions or as an agonist to mimic or enhance physiological responses.
  • Physiological responses include but are not limited to T-cell activation, T-cell proliferation, T-cell persistence and prevention of T-cell exhaustion. These properties are advantageous over conventional CD3-bispecific antibodies or tumor targeted co- stimulatory receptor agonists which do not optimally activate T-cells and induce (or promote) T-cell dysfunction.
  • cytokine and/or costimulatory fusion peptides are advantageous for enhancing the therapeutic potential of bispecific antibodies.
  • the costimulatory molecule of the anti-B7-H4/anti-CD3 bispecific antibodies of the present disclosure is CD58,
  • SUBSTITUTE SHEET (RULE 26) or a fragment thereof.
  • T-cell retargeting bispecific and multispecific antibodies share the druglike properties of human monoclonal antibodies. Further, T-cell retargeting bispecific and multispecific antibodies are advantageous over other existing therapies (e.g. CAR-T therapies) because it provides an off-the-shelf product with a high safety profile (e.g. mitigation of cytokine release syndrome and reduced levels of tonic signaling leading to T- cell dysfunction) and the possibility of dose titration and escalation.
  • CAR-T therapies e.g. CAR-T therapies
  • the present disclosure provides an antibody comprising the following domain structure: a) a first heavy chain polypeptide (Hl) comprising a variable region (VH1), and a constant region (CHI) having a constant region 1 domain (CHI HI), a hinge region (H1H), a constant region 2 domain (CH Im) and a constant region 3 domain (CH Im); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1), and
  • SUBSTITUTE SHEET (RULE 26) b) a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2HS); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • the term “antibody” refers to an immunoglobulin (Ig) molecule and immunologically active portions of an immunoglobulin molecule, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • immunologically active portions of an immunoglobulin molecule i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • specifically bind” or “immunoreacts with” “or directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Ka > IO -6 ).
  • Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, IgG4 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Accordingly, in one embodiment, the antibody disclosed herein is an IgG antibody.
  • Antibodies may be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • antibody fragment as used herein is intended to include without limitation,
  • SUBSTITUTE SHEET (RULE 26) Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies.
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
  • Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the disclosure (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • epitopope refers to the site on an antigen that is recognized by the antibodies and fragments disclosed herein.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 micromolar; e.g., ⁇ 100 nM, preferably ⁇ 10 nM and more preferably ⁇ 1 nM.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different antigens.
  • the present disclosure provides a bispecific antibody having a first antigen binding region that binds to a first antigen (e.g. CD3) and a second antigen binding region that binds to a second antigen (e.g. disease associated antigen).
  • a first antigen e.g. CD3
  • a second antigen binding region that binds to a second antigen (e.g. disease associated antigen).
  • Antibodies with more than two valencies are also contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the heavy chain heterodimerization, light chain heterodimerization, binding affinity, and/or other biological
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).
  • Amino acids may be grouped according to common side-chain properties:
  • Functional variants of the antibody or antigen-binding fragments described herein are also encompassed by the present disclosure.
  • the term "functional variant” as used herein includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptides or nucleic acid molecules disclosed herein in substantially the same way.
  • functional variants of polypeptides disclosed herein include, without limitation, conservative amino acid substitutions.
  • a "conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue that change an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size).
  • Variants of polypeptides also include additions and deletions to the polypeptide sequences disclosed herein.
  • variant nucleotide sequences include analogs and derivatives thereof.
  • a variant of the binding proteins disclosed herein include proteins that bind to the same antigen or epitope as the binding proteins.
  • the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid.
  • the naturally occurring charged amino acid residue is an arginine, a lysine, a histidine, a glutamic acid or an aspartic acid.
  • the first heavy chain polypeptide (Hl) has a strong preference for binding with the first light chain polypeptide (LI) relative to the second light chain polypeptide (L2); and the second heavy chain polypeptide (H2) has a strong preference for binding with the second light chain polypeptide (L2) relative to first light chain polypeptide (LI).
  • the first heavy chain polypeptide (Hl) and the second heavy chain polypeptide (H2) have a stronger preference for heterodimerization than homodimerization (i.e. heavy chain heterodimerization).
  • Antibody variants having one or more amino acid substitutions are provided herein.
  • Exemplary substitutional mutagenesis sites include the charged substitution pairs shown in Tables 1-6.
  • Wild type (WT) indicates the natural amino acid at the indicated position
  • Wild type (WT) indicates the natural amino acid at the indicated position
  • anti-CD3/anti-B7-H4 bispecific antibodies of the present disclosure comprise antibody variants comprise substitutions in the variable heavy, variable light, constant heavy or constant light chain domains of the anti-CD3 or the anti-B7-H4 arms or both, as discussed in PCT Application No. PCT/US2023/064728 and PCT Publication No. W02019/104075 Al, which are incorporated by reference herein in their entirety.
  • the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of the anti-CD3 arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CHIHI is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; Hi) the amino acid at position 173 (EU numbering) of the CHIHI is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain and light chain of the anti-B7-H
  • the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of the anti-B7-H4 arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CHIHI is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; Hi) the amino acid at position 173 (EU numbering) of the CHIHI is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain and light chain of the anti-CD
  • SUBSTITUTE SHEET (RULE 26) numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2HI is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.
  • an antibody disclosed herein with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC complement- mediated cell killing and antibody-dependent cellular cytotoxicity
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371 ,826). See also Duncan & Winter,
  • antibodies may comprise a substitution mutation in the Fc region that reduces effector function.
  • the substitution mutation is an aglycosylation site mutation.
  • the aglycosylation site mutation is at amino acid residue 297 and amino acid substitutions at residues 234, 235, 265 and 331 (EU numbering) to disrupt the Fc receptor binding interface.
  • the aglycosylation site mutation reduces effector function of the antibody.
  • the H1H and/or the H2H has an A at positions 234 and 235 (EU numbering); or ii) the H1H and/or the H2H has an A at positions 234, 235 and 237 (EU numbering) iii) the H1H and/or the H2H has an A at positions 234 and 235 and G at position 329 (EU numbering).
  • a “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the heteromultimeric antibody, and thereby favor heteromultimeric antibody formation over homomultimeric antibody formation, for example.
  • the protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance.
  • nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original amino acid residue in the interface of the first polypeptide.
  • SUBSTITUTE SHEET (RULE 26) and corresponding import residue.
  • the upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.
  • the preferred import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine.
  • the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.
  • Exemplary amino acid substitutions in the CH1H3 or CH2H3 domain for forming the protuberance include without limitation the T366W substitution.
  • a “cavity” refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide.
  • the cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue.
  • Exemplary amino acid substitutions in the CH1H3 or CH2H3 domain for generating the cavity include without limitation the T366S, L368A, Y407A, Y407T and Y407V substitutions.
  • the knob half-antibody comprises T366W substitution
  • the hole half-antibody comprises the T366S/L368A/Y407V substitutions.
  • the antibody variant comprises the following substitutions: the CH1H3 has a C at position 349, an S at position 366, an A at position 368 and a V at
  • SUBSTITUTE SHEET (RULE 26) position 407 (EU numbering); and the CH2H3 has a C at position 354 and a W at position 366 (EU numbering).
  • the antibody variant comprises the following substitutions: the CH2H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 354 and a W at position 366 (EU numbering).
  • the antibody variant comprises the following substitutions: the CH1H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3 has a C at position 349 and a W at position 366 (EU numbering).
  • the antibody variant comprises the following substitutions: the CH2H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 349 and a W at position 366 (EU numbering).
  • the present disclosure provides a bispecific antibody comprising a first antigen binding domain that binds to a CD3 expressed on a T-cell, and a second antigen binding domain that binds to a B7-H4 antigen on the surface of a cancer cell.
  • Antibodies of the invention are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing B7-H4).
  • CD3 cluster of differentiation 3
  • mammals such as primates (e.g. humans, cynomolgus monkey) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3e, CD3y, CD3a, and CD3P chains.
  • CD3 is a cell surface complex expressed on T cells in association with the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes.
  • CD3 gamma chain one CD3 delta chain
  • CD3 epsilon chains which associate with each other to form a CD3 epsilon/gamma heterodimer, and a CD3 epsilon/delta heterodimer.
  • the two CD3 heterodimers, together with the T cell receptor (TCR) and the signal-transducing zeta chain homodimer form the T cell receptor complex.
  • the term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3e or CD3y), as well as any form of CD3 that results from processing in the
  • CD3 includes, for example, human CD3e protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length.
  • the invention provides isolated antibodies that bind to CD3. In some embodiments, the invention provides antibodies that bind to CD3e. In some instances, the anti-CD3e antibody binds to a human CD3e polypeptide or a cynomolgus monkey (cyno) CD3e polypeptide.
  • the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3e polypeptide (MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGS EILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFY LYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGA GAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 419)) or a cyno CD3e polypeptide (MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSE AQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVC ENCMEMDVMAVATIV
  • the anti-CD3 antibody binds to an epitope within a fragment of CD3e (e.g., human CD3e) consisting of amino acid residues 1-26 or amino acid residues 1-27 of human CD3e (SEQ ID NO: 419).
  • a fragment of CD3e e.g., human CD3e
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • SUBSTITUTE SHEET (RULE 26) [00119] In some embodiments, alanine scanning mutagenesis was performed on the “SP34” anti-CD3e antibody to produce affinity modulated anti-CD3e antibodies of the invention.
  • the first antigen binding region that binds to CD3 comprises any one of the VH and VL sequences listed in Table 7.
  • Table 7 the underlined sequences are CDR sequence according to Kabat and the bolded sequences are CDR sequences according to Chothia.
  • the first antigen binding region comprises a VH region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 257, or 258.
  • the first antigen binding region comprises a VL region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 22, 23, 24, 25, 26, 27, and 28.
  • an anti-CD3 antibody of the disclosure comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDRI), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDRS); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDRI), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDRS).
  • VH heavy chain variable region
  • VHCDRI VH complementarity determining region 1
  • VHCDR2 VH complementarity determining region 2
  • VHCDRS VH complementarity determining region 3
  • Tables 8 and 9 provide exemplary of CDR sequences of the anti-CD3 antibodies provided herein.
  • bispecific antibodies comprising a first antigen binding domain that binds a first antigen (e.g. CD3) and a second antigen binding domain that binds to a second antigen (e.g. B7-H4).
  • the bispecific antibody has the following structure: a first heavy chain polypeptide (Hl) comprising a variable region (VH1), and a constant region (CHI) having a constant region 1 domain (CHI HI), a hinge region (H1H), a constant region 2 domain (CH Im) and a constant region 3 domain (CH Im); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • Hl first heavy chain polypeptide
  • CHI constant region having a constant region 1 domain (CHI HI), a hinge region (H1H),
  • the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3) comprising any one of the VH1 and VL1 sequences listed in Table 7.
  • a first antigen binding domain e.g. binding to CD3
  • the underlined sequences are CDR sequence according to Kabat and the bolded sequences are CDR sequences according to Chothia.
  • the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3s) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VHICDRI), a VH complementarity determining region 2 (VH1CDR2) and a VH complementarity determining region 3 (VHICDRS); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLICDRI), a VL complementarity determining region 2 (VL1CDR2) and a VL complementarity determining region 3 (VLICDRS).
  • VH1 VH complementarity determining region 1
  • VH1CDR2 VH complementarity determining region 2
  • VHICDRS VH complementarity determining region 3
  • VLICDRI VL complementarity determining region 1
  • VL1CDR2 VL complementarity determining region 2
  • VLICDRS VL complementar
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS
  • VH heavy chain variable region
  • SUBSTITUTE SHEET (RULE 26) comprising the amino acid sequence of SEQ ID NO: 38.
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, in) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 39.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 30, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 35, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 38.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 40.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 41.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 31, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable region
  • the first antigen binding region comprises a heavy chain variable region (VH) comprising i) a VHCDRI comprising the amino acid sequence of SEQ ID NO: 29, ii) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 34, iii) a VHCDRS comprising the amino acid sequence of SEQ ID NO: 39.
  • VH heavy chain variable region
  • the first antigen binding region comprises a light chain variable region (VL) comprising i) a VL complementarity determining region 1 (VLCDRI) comprising the amino acid sequence of SEQ ID NO: 42, ii) a VL complementarity
  • VLCDR2 determining region 2
  • VLCDRS VL complementarity determining region 3
  • the first antigen binding region comprises a light chain variable region (VL) comprising i) a VLCDRI comprising the amino acid sequence of SEQ ID NO: 42, ii) a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 43, iii) a VLCDRS comprising the amino acid sequence of SEQ ID NO: 46.
  • VL light chain variable region
  • the first antigen binding region comprises a light chain variable region (VL) comprising i) a VLCDRI comprising the amino acid sequence of SEQ ID NO: 42, ii) a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 44, iii) a VLCDRS comprising the amino acid sequence of SEQ ID NO: 45.
  • VL light chain variable region
  • the first antigen binding region comprises a light chain variable region (VL) comprising i) a VLCDRI comprising the amino acid sequence of SEQ ID NO: 42, ii) a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 43, iii) a VLCDRS comprising the amino acid sequence of SEQ ID NO: 47.
  • VL light chain variable region
  • the first antigen binding region comprises a light chain variable region (VL) comprising i) a VLCDRI comprising the amino acid sequence of SEQ ID NO: 42, ii) a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 43, iii) a VLCDRS comprising the amino acid sequence of SEQ ID NO: 48.
  • VL light chain variable region
  • the bispecific antibody comprises any one of the anti-CD3 antibodies of the disclosure.
  • Exemplary anti-CD3 antibodies of the invention include CD3- 01, CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12 and CD3-A13.
  • the bispecific antibody comprises a “CD3-01” anti-CD3 antibody.
  • the bispecific antibody comprises a “CD3-A6” anti-CD3 antibody.
  • the binding affinity (KD) of the first antigen binding region of the bispecific antibody that binds to CD3 is about 0.001 nM to about 5000 nM.
  • the binding affinity to CD3 is about 0.001 nM to about 0.01 nM, about 0.01 to about 0.1 nM or about 0.1 to about InM.
  • the binding affinity is about 1 nM to about 1000 nM, about 10 nM to about 1000 nM or about lOOnM to about 1000 nM.
  • the binding affinity is about 50 nM to about 5000 nM, about 50 nM to about 4000 nM, about 50 nM to about 3000 nM, about 50 nM to about 2000 nM, about 50 nM to about 1000 nM, about 50 nM to about 900 nM, about 50 nM to about 800 nM, about 50 nM to about 700 nM, about 50 nM to about 600 nM, about 50 nM to about 500 nM, about 50 nM to about 400 nM, about 50 nM to about 300 nM, about 50 nM to about 200 nM, about 50 nM to about 100 nM or about 50 nM to about 500 nM. In some embodiments, the binding affinity is about 50 nM to about 200 nM.
  • the binding affinity is about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 110 nM, about 110 nM to about 120 nM, about 120 nM to about 130 nM, about 130 nM to about 140 nM, about 150 nM to about 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, or about 190 nM to about 200 nM.
  • the binding affinity is less than about 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 275 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9.5 nM, 9nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1
  • the present disclosure provides a bispecific antibody comprising a first antigen binding domain that binds to a CD3 expressed on a T-cell, and a second antigen binding domain that binds to a B7-H4 antigen on the surface of a cancer cell.
  • Antibodies of the invention are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing B7-H4).
  • B7-H4 (B7-H4, V-set domain containing T cell activation inhibitor 1 or VTCN1) is a member of the B7 family of proteins, which family comprises cell-surface protein ligands that bind to receptors on lymphocytes.
  • the B7 family plays an important role in the regulation of immune responses.
  • B7S1, the mouse B7-H4 homolog negatively regulates mouse T cell- mediated immune responses by inhibiting T cell activation, proliferation, cytokine production and cytotoxic activity (Prasad et al., 2003, Immunity 18: 863-873).
  • Human B7-H4 is a type I
  • SUBSTITUTE SHEET (RULE 26) transmembrane protein that includes a short intracellular domain, a hydrophobic transmembrane domain, and an extracellular domain with an IgV-and an IgC-like domain with four conserved cysteine residues and seven sites for N-linked glycosylation (Sica et al., 2003, Immunity 18: 849-861). To date, no receptor for B7-H4 has been identified.
  • B7-H4 expression is very limited, whereas B7-H4 expression is found on tumor cells in numerous cancer tissues (Kaur and Janakiram, 2019, ESMO Open 4:e000554). In cancer, B7-H4 expression is correlated with advanced stages of cancer, poor prognosis, and decreased overall patient survival.
  • B7-H4 binding antibodies are in development for cancer therapy.
  • FPA150 is an afucosylated human antibody that relieves the B7-H4- mediated suppression of T cell activation and exhibits antibody dependent cellular cytotoxicity (ADCC) activity (Wainberg et al., 2019, Annals of Oncology 30, Suppl. 5, v489 (1198P). It is currently in early clinical trials as a monotherapy or in combination with pembrolizumab in advanced solid tumors.
  • B7-H4 refers to a protein entitled B7-H4, which is also referred to as: B7-H4; V- set domain containing T cell activation inhibitor 1; or VTCN1.
  • B7-H4 is a member of the B7 family of proteins, which family comprises cell-surface protein ligands that bind to receptors on lymphocytes.
  • B7-H4 proteins are known from various species, such as human (Homo sapiens) B7-H4 (Uniprot accession no. Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7-H4 transcript 1 (Uniprot accession no.
  • B7-H4 Uniprot accession no. F1P8R9
  • rabbit (Oryctolagus cuniculus) B7-H4 Uniprot accession no. G1TQE8
  • rat rattus norvegicus
  • mouse mouse
  • mus musculus B7-H4
  • pig sus scrofa
  • B7-H4 and B7-H4 antigen are used interchangeably herein, and include any variants, isoforms and species homologs of human B7-H4 which are naturally expressed by cells or are expressed on cells transfected with the B7-H4 gene. Binding of an antibody of the invention to the B7-H4 antigen mediate the killing of cells expressing B7-H4 (e.g., a tumor cell) by inactivating B7-H4. The killing of the cells expressing B7-H4 may occur by one or more of the following mechanisms: Cell death/apoptosis induction, ADCC and CDC.
  • anti-B7-H4 antibody is an antibody that binds specifically to B7-H4 antigen.
  • two types of anti-B7-H4 antibodies can be distinguished according to Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S sharp et al., Blood 101 (2003) 1045-1052.
  • B7-H4 may be expressed in low copy number on the target cell (e.g. tumor cell).
  • B7-H4 is expressed or present at less than 35,000 copies per target cell.
  • the low copy number cell surface B7-H4 is present between 100 and 35,000 copies per target cell; between 100 and 30,000 copies per target cell; between 100 and 25,000 copies per target cell; between 100 and 20,000 copies per target cell; between 100 and 15,000 copies per target cell; between 100 and 10,000 copies per target cell; between 100 and 5,000 copies per target cell; between 100 and 2,000 copies per target cell; between 100 and 1,000 copies per target cell; or between 100 and 500 copies per target cell.
  • Copy number of the cell surface B7-H4 can be determined, for example, using a standard Scratchcard plot.
  • This disclosure provides a bispecific antibody comprising a first antigen binding region that binds CD3 and a second binding region that binds B7-H4.
  • the bispecific antibody has the following structure: a first heavy chain polypeptide (Hl) comprising a variable region (VH1), and a constant region (CHI) having a constant region 1 domain (CHIHI), a hinge region (H1H), a constant region 2 domain (CH Im) and a constant region 3 domain (CH Im); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2HS); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • Hl first heavy chain polypeptide
  • CHI constant region having a constant region 1 domain (CHIHI), a hinge region (H1H), a constant
  • the second binding region that binds to B7-H4 can be derived from the binding regions of an anti-B7-H4 antibody.
  • anti-B7-H4 antibodies include but are not limited to alsevalimab FPA150 (Amgen), and those described in PCT Publications WO 2009/073533, WO 2008/067283, each of which are incorporated herein in their entireties.
  • a second antigen binding region that binds B7-H4 comprises any one of the VH and VL sequences listed in Table 10. In Table 10, the underlined
  • SUBSTITUTE SHEET (RULE 26) sequences are CDR sequence according to Kabat and the bolded sequences are CDR sequences according to Chothia.
  • the second binding region that binds to B7-H4 comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDRI), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDRS); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDRI), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDRS).
  • VH heavy chain variable region
  • VHCDRI VH complementarity determining region 1
  • VHCDR2 VH complementarity determining region 2
  • VHCDRS VH complementarity determining region 3
  • Tables 11 and 12 provide exemplary of CDR sequences of the anti-B7-H4 antibodies.
  • the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDRI) comprising the amino acid sequence of SEQ ID NO: 67, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 79, iii) a VH complementarity determining region 3 (VHCDRS) comprising the amino acid sequence
  • VH heavy chain variable region
  • VHCDRI VH complementarity determining region 1
  • VHCDR2 VH complementarity determining region 2
  • VHCDRS VH complementarity determining region 3
  • VL light chain variable region
  • VLCDRI VL complementarity determining region 1
  • VLCDR2 VL complementarity determining region 2
  • VLCDRS VL complementarity determining region 3
  • the second antigen binding region comprises a VH region comprising a VHCDRI comprising the amino acid sequence of SEQ ID NO: 68, a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VHCDRS comprising the amino acid sequence of SEQ ID NO: 91; and a VL region comprising a VLCDRI comprising the amino acid sequence of SEQ ID NO: 96, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 101, and a VLCDRS comprising the amino acid sequence of SEQ ID NO: 107.
  • the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 114 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 113.
  • the second antigen binding region comprises a VH region comprising a VHCDRI comprising the amino acid sequence of SEQ ID NO: 69, a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 92; and a VL region comprising a VLCDRI comprising the amino acid sequence of SEQ ID NO: 96, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 101, and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 108.
  • the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 116 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 115.
  • the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 118 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 117.
  • the second antigen binding region comprises a VH region comprising a VHCDRI comprising the amino acid sequence of SEQ ID NO: 71, a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VHCDRS comprising the amino acid sequence of SEQ ID NO: 94; and a VL region comprising a VLCDRI comprising the amino acid sequence of SEQ ID NO: 99, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a VLCDRS comprising the amino acid sequence of SEQ ID NO: 110.
  • the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 120 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 119.
  • the second antigen binding region comprises a VH region comprising a VHCDRI comprising the amino acid sequence of SEQ ID NO: 72, a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 95; and a VL region comprising a VLCDRI comprising the amino acid sequence of SEQ ID NO: 100, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 105, and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 109.
  • the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 122 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 121.
  • bispecific antibodies comprising a first antigen binding domain that binds a first antigen (e.g. CD3e) and a second antigen binding domain that binds to a B7- H4.
  • the bispecific antibody has the following structure: a first heavy chain polypeptide (Hl) comprising a variable region (VH1), and a constant region (CHI) having a constant region 1 domain (CHIHI), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CHIHS); and a first light chain polypeptide (LI) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2HI), a hinge region (H2H), a constant region 2 domain (CH2H2)
  • SUBSTITUTE SHEET (RULE 26) and a constant region 3 domain (CH2HS); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).
  • the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3e) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VHICDRI), a VH complementarity determining region 2 (VH1CDR2) and a VH complementarity determining region 3 (VHICDRS); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLICDRI), a VL complementarity determining region 2 (VL1CDR2) and a VL complementarity determining region 3 (VLICDRS); and a second antigen binding domain (e.g. binding to CD3e) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VHICDRI), a VH complementarity determining region 2 (VH1CDR2) and a VH complementarity determining region 3 (VHICD
  • binding to B7-H4) comprising: a) a heavy chain variable region (VH2) comprising a VH complementarity determining region 1 (VH2CDRI), a VH complementarity determining region 2 (VH2CDR2) and a VH2 complementarity determining region 3 (VH2CDRS); and b) a light chain variable region (VL2) comprising a VL complementarity determining region 1 (VL2CDRI), a VL complementarity determining region 2 (VL2CDR2) and a VL complementarity determining region 3 (VL2CDR3).
  • Tables 8 and 9 provide exemplary of CDR sequences of the anti-CD3e antibodies provided herein.
  • Tables 11 and 12 provide exemplary of CDR sequences of the anti-B7-H4 antibodies provided herein.
  • the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3e) comprising any one of the VH1 and VL1 sequences listed in Table 7 and a second antigen binding domain (e.g. binding to B7-H4) comprising any one of the VH2 and VL2 sequences listed in Table 10.
  • a first antigen binding domain e.g. binding to CD3e
  • a second antigen binding domain e.g. binding to B7-H4 comprising any one of the VH2 and VL2 sequences listed in Table 10.
  • the bispecific antibody of the disclosure comprises a first heavy chain polypeptide (Hl) and a first light chain polypeptide (LI); and a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2) comprising any one of the sequences listed in Table 13 and Table 16.
  • the italicized sequences are the heavy chain variable regions and the light chain variable regions.
  • the underlined sequences are CDRs according to Kabat and the bolded sequences are CDRs according to Chothia.
  • the bispecific antibody of the disclosure provided herein comprises a Hl that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.
  • the Hl comprises a C-terminal lysine (K) residue.
  • the bispecific antibody of the disclosure provided herein comprises a LI that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.
  • the bispecific antibody of the disclosure provided herein comprises a H2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.
  • the Hl comprises a C-terminal lysine (K) residue.
  • the bispecific antibody of the disclosure provided herein comprises a L2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.
  • the Hl amino acid sequence is numbered in accordance with SEQ ID NO: 126.
  • the LI amino acid sequence is numbered in accordance with SEQ ID NO: 125.
  • the H2 amino acid sequence is numbered in accordance with SEQ ID NO: 122.
  • the L2 amino acid sequence is numbered in accordance with SEQ ID NO: 121.
  • Exemplary, CD3e x B7-H4 bispecific antibodies of the invention include EIP0832, EIP0833, EIP0834, EIP0835, EIP1234, EIP1260, EIP1261, EIP1262, EIP1263, EIP1221, EIP 1222, EIP 1223 and EIP 1224.
  • Bispecific antibody EIP0832 comprises a first antigen binding domain that binds CD3s which comprising a VH1, comprising a VHICDRI having the amino acid sequence of 51
  • SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 35; and a VHICDRS having the amino acid sequence of SEQ ID NO: 38; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 47.
  • Bispecific antibodies EIP0833, EIP1221, EIP1222, EIP1223 and EIP1224 comprise a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 30; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 45.
  • Bispecific antibody EIP0834 comprises a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 39; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 47.
  • Bispecific antibody EIP0835 comprises a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 44; and a VLICDRS having the amino acid sequence of SEQ ID NO: 45.
  • Bispecific antibodies EIP1234, EIP1260, EIP1261, EIP1262 and EIP1263 comprise a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VLICDRI having the amino acid sequence of
  • SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 45.
  • Bispecific antibodies EIP0832, EIP0833, EIP0834 and EIP0835 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 67; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 79; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 90; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 96; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 101; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 106.
  • Bispecific antibodies EIP1234 and EIP1221 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 68; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 80; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 91; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 96; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 101; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 107.
  • Bispecific antibody EIP1260 comprises a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 69; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 81; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 92; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 96; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 101; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 108.
  • Bispecific antibodies EIP1261 and EIP1222 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 70; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 82; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 93; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 98; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 103; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 109.
  • Bispecific antibodies EIP1262 and EIP1223 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 71; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 83;
  • SUBSTITUTE SHEET (RULE 26) and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 94; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 99; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 104; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 110.
  • Bispecific antibodies EIP1263 and EIP1224 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 72; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 84; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 95; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 100; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 105; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 109.
  • the bispecific antibody EIP0832 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0832 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 124, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0833 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0833 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 126, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0834 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 16, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0834 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 127, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0835 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 27, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0835 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 128, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP1234 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 58, and a VL2 comprising the amino acid sequence of SEQ ID NO: 57.
  • the bispecific antibody EIP1234 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 114, and a L2 comprising the amino acid sequence of SEQ ID NO: 113.
  • the bispecific antibody EIP1260 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL2 comprising the amino acid sequence of SEQ ID NO: 59.
  • the bispecific antibody EIP1260 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 116, and a L2 comprising the amino acid sequence of SEQ ID NO: 115.
  • the bispecific antibody EIP1261 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL2 comprising the amino acid sequence of SEQ ID NO: 61.
  • the bispecific antibody EIP1261 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 118, and a L2 comprising the amino acid sequence of SEQ ID NO: 117.
  • the bispecific antibody EIP1262 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 64, and a VL2 comprising the amino acid sequence of SEQ ID NO: 63.
  • the bispecific antibody EIP1262 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 120, and a L2 comprising the amino acid sequence of SEQ ID NO: 119.
  • the bispecific antibody EIP1263 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 66, and a VL2 comprising the amino acid sequence of SEQ ID NO: 65.
  • the bispecific antibody EIP1263 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 129, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 122, and a L2 comprising the amino acid sequence of SEQ ID NO: 121.
  • the bispecific antibody EIP1221 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 58, and a VL2 comprising the amino acid sequence of SEQ ID NO: 57.
  • the bispecific antibody EIP1221 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 126, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 114, and a L2 comprising the amino acid sequence of SEQ ID NO: 113.
  • the bispecific antibody EIP1222 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL2 comprising the amino acid sequence of SEQ ID NO: 61.
  • the bispecific antibody EIP1222 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 126, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 118, and a L2 comprising the amino acid sequence of SEQ ID NO: 117.
  • the bispecific antibody EIP1223 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 64, and a VL2 comprising the amino acid sequence of SEQ ID NO: 63.
  • the bispecific antibody EIP1223 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 126, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 120, and a L2 comprising the amino acid sequence of SEQ ID NO: 119.
  • the bispecific antibody EIP1224 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 66, and a VL2 comprising the amino acid sequence of SEQ ID NO: 65.
  • the bispecific antibody EIP1224 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 126, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 122, and a L2 comprising the amino acid sequence of SEQ ID NO: 121.
  • any one of the bispecific antibodies shown above in Table 13 can be further modified by substituting any one of the anti-CD3e antigen binding regions with any one of the anti-CD3e binding regions shown in Tables 7-9.
  • the anti-CD3e antigen binding regions of bispecific antibody “EIP1224” can be substituted with any one of the anti- CD3e binding regions shown in Tables 7-9 to produce the bispecific antibodies of the invention.
  • Exemplary antibodies are shown in Table 13.
  • an antibody e.g. monospecific antibody or bispecific antibody
  • a fusion peptide fused to the N-terminus or the C-terminus of the first heavy chain polypeptide or the second heavy chain polypeptide.
  • T cell activation occurs when the T cell receptor engages a cell that displays a foreign or mutated protein fragment or antigen in a specific protein complex called the Major Histocompatibility Complex I (MHCI).
  • MHCI Major Histocompatibility Complex I
  • the activation of the T cell receptor is by itself both activating and auto-regulatory to T cells. Strong binding of the TCR to an MHCI complex creates chronic activation of the TCR. This form of signal is associated with T cells that are reactive to self-antigens. T cells are programed to inactivate when they experience this form activation. T cells with TCR that bind weaker, but sufficient for activation, experience acute signaling with the potential to remain active and differentiate into memory T cells. This is emerging as important consideration in the design the T cell therapeutics.
  • T cell cytokine activation is important in T cell transitions, either from non-dividing to a state of rapid cell division or from one phenotypic state to another.
  • T cell cytokine receptors bind to cytokines that are produced by immune and non- immune cells and depending on the cytokine and the state of the T cell at the time of receiving the cytokine signal can induce cell proliferation, can sustain vitality, or can induce differentiation of T cells into a specialized cell state appropriate for sustained activation or inactivation following infection.
  • cytokines which can induce naive T cells to proliferate and promote T cell differentiation into memory T cells.
  • cytokines include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-21.
  • Costimulatory receptor activation referred to as signal 2 provides a context specific cell-to-cell reinforcement of T activation.
  • the most recognized form of costimulation occurs when T cells interact with activated antigen presenting cells through the T cell costimulatory receptor CD28 with CD80 and CD86 ligands found on APCs. These interactions can “prime” specific T cells armed with T cell receptors responsive to pathogen or cancer proteins.
  • costimulation induced at the site of infection and malignancies This includes costimulation that acts through CD2 and NKG2D receptors responsive to ligands like CD58 and UL16 binding proteins (e.g. ULBP2/5/6) that are induced in immune cells and epithelial cells upon viral infection. These signals provide not only reinforcement of T activation, but confirmation that the T cell’s lethal effector activities are targeted with single cell accuracy. While many costimulatory receptors have been discovered, the importance of each receptor’s specific context and the impact of concurrent signaling of
  • Costimulatory ligands include but are not limited to CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL or ICOSLG.
  • CD58 is advantageous over other costimulatory ligands in that it is the primary costimulatory pathway available at the tumor site as tumor infiltrating T lymphocytes often lose expression of other costimulatory receptors like CD28, or due to the low immunogenicity of tumor cells, tumor cells do not sufficiently activate T cell, thus limiting the potential of inducible costimulatory receptors like 41BB.
  • the anti-CD3s antibodies of the disclosure induce varying levels of T cell receptor activation that confer alteration in T cell vitality and cytokine production. Accordingly, a fusion of the costimulatory ligand CD58 to the anti-CD3e bispecific antibody provides integrated costimulatory T cell activation for optimal T cell activation.
  • the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (Hl). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (Hl). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 or portions thereof.
  • Exemplary peptides include but are not limited to CD48, CD58, CD86, TNFSF9, OX40L, 4- 1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG or portions thereof.
  • Exemplary peptide sequences that are fused to the bispecific antibodies include but are not limited to those listed in Table 14.1 and Table 14.2.
  • the polypeptide is fused directly to the bispecific antibody. In some embodiments, the polypeptide is fused indirectly through a linker. In some embodiments, the bispecific antibody fused with a peptide comprises a linker sequence. Exemplary linker sequences include but are not limited to those listed in Table 15.1 and Table 15.2.
  • the CD3 x B7-H4 bispecific antibodies of the invention have a CD58 fusion peptide (SEQ ID NO: 49) fused indirectly at the C-terminus of the first heavy chain polypeptide (Hl) using linker-2 (SEQ ID NO: 53).
  • the CD3 x B7-H4 bispecific antibodies of the invention have a CD58v* fusion peptide (SEQ ID NO: 50) fused indirectly at the C-terminus of the first heavy chain polypeptide (Hl) using linker-2 (SEQ ID NO: 53).
  • Exemplary, CD3e x B7-H4 bispecific antibodies with a C-terminus fusion peptide of the invention include EIP0836, EIP0837, EIP0838, EIP0839, EIP0945, EIP0946, EIP0947, EIP0951, EIP0952, EIP1046, EIP1047, EIP1060, EIP1061, EIP1354, EIP1551, EIP1552, EIP1553, EIP1554, EIP1555, EIP1556, EIP1300, EIP1557, EIP1558, EIP1270, EIP1269, EIP1559, EIP1560, EIP1299, EIP1303, EIP1355, EIP1356, EIP1357, EIP1561, EIP1301, EIP1358, EIP1562, EIP1359, EIP1360, EIP1563, EIP1302, EIP1564, EIP1416, EIP1417, EIP1418, EIP1419, EIP1424, EIP14
  • Bispecific antibody EIP0836 comprises a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 35; and a VHICDRS having the amino acid sequence of SEQ ID NO: 38; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 47.
  • Bispecific antibodies EIP0837, EIP1046, EIP1047, EIP1060 and EIP1061 comprise a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 30; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VLICDRI having the amino acid sequence of
  • Bispecific antibody EIP0838 comprises a first antigen binding domain that binds CD3e which comprising a VH1, comprising a VHICDRI having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VHICDRS having the amino acid sequence of SEQ ID NO: 39; and a VL1, comprising a VLICDRI having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VLICDRS having the amino acid sequence of SEQ ID NO: 47.
  • SUBSTITUTE SHEET (RULE 26) sequence of SEQ ID NO: 68; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 80; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 91; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 96; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 101; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 107.
  • Bispecific antibodies EIP0947 and EIP1047 comprise a second antigen binding domain that binds B7-H4 comprising a VH2, comprising VH2CDRI having the amino acid sequence of SEQ ID NO: 70; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 82; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 93; and a VL2, having VL2CDRI having the amino acid sequence of SEQ ID NO: 98; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 103; and a VL2CDR3 having the amino acid sequence of SEQ ID NO: 109.
  • the bispecific antibody EIP0836 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0836 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 130, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0837 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0837 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0838 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 16, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0838 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 132, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0839 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 27, a VH2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL2 comprising the amino acid sequence of SEQ ID NO: 55.
  • the bispecific antibody EIP0839 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 133, a LI comprising the amino acid sequence of SEQ ID NO: 128, a H2 comprising the amino acid sequence of SEQ ID NO: 112, and a L2 comprising the amino acid sequence of SEQ ID NO: 111.
  • the bispecific antibody EIP0945 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 58, and a VL2 comprising the amino acid sequence of SEQ ID NO: 57.
  • the bispecific antibody EIP0945 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 133, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 114, and a L2 comprising the amino acid sequence of SEQ ID NO: 113.
  • the bispecific antibody EIP0946 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL2 comprising the amino acid sequence of SEQ ID NO: 59.
  • the bispecific antibody EIP0946 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 133, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 116, and a L2 comprising the amino acid sequence of SEQ ID NO: 115.
  • the bispecific antibody EIP0947 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL2 comprising the amino acid sequence of SEQ ID NO: 61.
  • the bispecific antibody EIP0951 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 133, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 120, and a L2 comprising the amino acid sequence of SEQ ID NO: 119.
  • the bispecific antibody EIP0952 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 66, and a VL2 comprising the amino acid sequence of SEQ ID NO: 65.
  • the bispecific antibody EIP0952 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 133, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 122, and a L2 comprising the amino acid sequence of SEQ ID NO: 121.
  • the bispecific antibody EIP1046 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 58, and a VL2 comprising the amino acid sequence of SEQ ID NO: 57.
  • the bispecific antibody EIP1046 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 114, and a L2 comprising the amino acid sequence of SEQ ID NO: 113.
  • the bispecific antibody EIP1047 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL2 comprising the amino acid sequence of SEQ ID NO: 61.
  • the bispecific antibody EIP1060 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 64, and a VL2 comprising the amino acid sequence of SEQ ID NO: 63.
  • the bispecific antibody EIP1060 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 120, and a L2 comprising the amino acid sequence of SEQ ID NO: 119.
  • the bispecific antibody EIP1061 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 122, and a L2 comprising the amino acid sequence of SEQ ID NO: 121.
  • the bispecific antibody EIP1354 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 180, and a L2 comprising the amino acid sequence of SEQ ID NO: 156.
  • the bispecific antibody EIP1551 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 181, and a L2 comprising the amino acid sequence of SEQ ID NO: 157.
  • the bispecific antibody EIP1552 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 182, and a L2 comprising the amino acid sequence of SEQ ID NO: 158.
  • the bispecific antibody EIP1553 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 183, and a L2 comprising the amino acid sequence of SEQ ID NO: 159.
  • the bispecific antibody EIP1554 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 184, and a L2 comprising the amino acid sequence of SEQ ID NO: 160.
  • the bispecific antibody EIP1555 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 185, and a L2 comprising the amino acid sequence of SEQ ID NO: 156.
  • the bispecific antibody EIP1556 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 206, and a L2 comprising the amino acid sequence of SEQ ID NO: 161.
  • the bispecific antibody EIP1300 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 161.
  • the bispecific antibody EIP1557 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 187, and a L2 comprising the amino acid sequence of SEQ ID NO: 162.
  • the bispecific antibody EIP1558 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 188, and a L2 comprising the amino acid sequence of SEQ ID NO: 163.
  • the bispecific antibody EIP1270 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 189, and a L2 comprising the amino acid sequence of SEQ ID NO: 164.
  • the bispecific antibody EIP1269 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 190, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1559 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 191, and a L2 comprising the amino acid sequence of SEQ ID NO: 166.
  • the bispecific antibody EIP1560 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 192, and a L2 comprising the amino acid sequence of SEQ ID NO: 167.
  • the bispecific antibody EIP1299 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 193, and a L2 comprising the amino acid sequence of SEQ ID NO: 168.
  • the bispecific antibody EIP1303 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 180, and a L2 comprising the amino acid sequence of SEQ ID NO: 161.
  • the bispecific antibody EIP1355 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 190, and a L2 comprising the amino acid sequence of SEQ ID NO: 161.
  • the bispecific antibody EIP1356 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 156.
  • the bispecific antibody EIP1357 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 239, and a VL2 comprising the amino acid sequence of SEQ ID NO: 216.
  • the bispecific antibody EIP1357 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1561 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 192, and a L2 comprising the amino acid sequence of SEQ ID NO: 156.
  • the bispecific antibody EIP1301 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 192, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1358 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 192, and a L2 comprising the amino acid sequence of SEQ ID NO: 161.
  • the bispecific antibody EIP1562 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 192, and a L2 comprising the amino acid sequence of SEQ ID NO: 164.
  • the bispecific antibody EIP1359 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 190, and a L2 comprising the amino acid sequence of SEQ ID NO: 162.
  • the bispecific antibody EIP1360 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 189, and a L2 comprising the amino acid sequence of SEQ ID NO: 162.
  • the bispecific antibody EIP1563 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 180, and a L2 comprising the amino acid sequence of SEQ ID NO: 162.
  • the bispecific antibody EIP1302 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 190, and a L2 comprising the amino acid sequence of SEQ ID NO: 164.
  • the bispecific antibody EIP1564 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 189, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1416 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 194, and a L2 comprising the amino acid sequence of SEQ ID NO: 169.
  • the bispecific antibody EIP1417 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 248, and a VL2 comprising the amino acid sequence of SEQ ID NO: 221.
  • the bispecific antibody EIP1417 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 195, and a L2 comprising the amino acid sequence of SEQ ID NO: 170.
  • the bispecific antibody EIP1418 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 196, and a L2 comprising the amino acid sequence of SEQ ID NO: 171.
  • the bispecific antibody EIP1419 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 197, and a L2 comprising the amino acid sequence of SEQ ID NO: 172.
  • the bispecific antibody EIP1424 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 198, and a L2 comprising the amino acid sequence of SEQ ID NO: 173.
  • the bispecific antibody EIP1425 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 199, and a L2 comprising the amino acid sequence of SEQ ID NO: 174.
  • the bispecific antibody EIP1426 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 200, and a L2 comprising the amino acid sequence of SEQ ID NO: 175.
  • the bispecific antibody EIP1427 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 201, and a L2 comprising the amino acid sequence of SEQ ID NO: 176.
  • the bispecific antibody EIP1428 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 202, and a L2 comprising the amino acid sequence of SEQ ID NO: 177.
  • the bispecific antibody EIP1438 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 198, and a L2 comprising the amino acid sequence of SEQ ID NO: 174.
  • the bispecific antibody EIP1449 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 202, and a L2 comprising the amino acid sequence of SEQ ID NO: 178.
  • the bispecific antibody EIP1433 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 131, a LI comprising the amino acid sequence of SEQ ID NO: 125, a H2 comprising the amino acid sequence of SEQ ID NO: 203, and a L2 comprising the amino acid sequence of SEQ ID NO: 179.
  • the bispecific antibody EIP1529 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 19, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 248, and a VL2 comprising the amino acid sequence of SEQ ID NO: 221.
  • the bispecific antibody EIP1529 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 204, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 195, and a L2 comprising the amino acid sequence of SEQ ID NO: 170.
  • the bispecific antibody EIP1535 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 248, and a VL2 comprising the amino acid sequence of SEQ ID NO: 221.
  • the bispecific antibody EIP1535 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 152, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 195, and a L2 comprising the amino acid sequence of SEQ ID NO: 170.
  • the bispecific antibody EIP1536 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 20, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 248, and a VL2 comprising the amino acid sequence of SEQ ID NO: 221.
  • the bispecific antibody EIP1536 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 205, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 195, and a L2 comprising the amino acid sequence of SEQ ID NO: 170.
  • the bispecific antibody EIP1526 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 19, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 239, and a VL2 comprising the amino acid sequence of SEQ ID NO: 216.
  • the bispecific antibody EIP1526 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 204, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1527 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 239, and a VL2 comprising the amino acid sequence of SEQ ID NO: 216.
  • the bispecific antibody EIP1527 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 152, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 165.
  • the bispecific antibody EIP1528 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 20, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 239, and a VL2 comprising the amino acid sequence of SEQ ID NO: 216.
  • the bispecific antibody EIP1528 comprises a Hl comprising the amino acid sequence of SEQ ID NO: 205, a LI comprising the amino acid sequence of SEQ ID NO: 123, a H2 comprising the amino acid sequence of SEQ ID NO: 186, and a L2 comprising the amino acid sequence of SEQ ID NO: 165. Any one of the anti-CD3/anti-B7-
  • H4 bispecific antibodies shown above in Table 16 can be further modified by substituting any one of the anti-CD3e antigen binding regions with any one of the anti-CD3e binding regions shown in Tables 7-9.
  • the anti-CD3e antigen binding regions of bispecific antibody “EIP1061” can be substituted with any one of the anti-CD3e binding regions shown in Tables 7-9 to produce the bispecific antibodies of the invention.
  • Exemplary antibodies are shown in Table 13 and Table 16.
  • Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • the antibodies of the invention are monoclonal antibodies.
  • Monoclonal antibodies are generated, for example, by using the procedures set forth in the Examples provided herein.
  • Antibodies are also generated, e.g., by immunizing BALB/c mice with combinations of cell transfectants expressing high levels of a given target on their surface. Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to the selected target.
  • Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • SUBSTITUTE SHEET (RULE 26) [00344] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a nonimmunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • Monoclonal antibodies of the invention include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies)
  • SUBSTITUTE SHEET (RULE 26) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization is performed, e.g., by following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539).
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies also comprise, e.g., residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody includes substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.
  • Monoclonal antibodies can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • XenomouseTM is a mouse termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.
  • scFv single chain Fv
  • SUBSTITUTE SHEET (RULE 26) a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • One method for producing an antibody of interest is disclosed in U.S. Patent No. 5,916,771.
  • This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • the antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.
  • Vectors can include vectors, liposomes, naked DNA, adjuvant-assisted DNA. gene gun, catheters, etc.
  • Vectors include chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g., a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vector (e.g., a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., a protamine), plasmids, phage, etc.
  • the vectors can be chromosomal, non-chromosomal or synthetic.
  • Retroviral vectors include moloney murine leukemia viruses.
  • DNA viral vectors are preferred.
  • These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.
  • HSV herpes simplex I virus
  • Pox viral vectors introduce the gene into the cell’ s cytoplasm.
  • Avipox virus vectors result in only a short-term expression of the nucleic acid.
  • Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors.
  • the particular vector chosen will depend upon the target cell and the condition being treated.
  • the introduction can be by standard techniques, e.g., infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, (Ca) 2 (PO 4 ) 3 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.
  • the vector can be employed to target essentially any desired target cell.
  • stereotaxic injection can be used to direct the vectors (e.g., adenovirus, HSV) to a desired location.
  • the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System.
  • icv intracerebroventricular
  • a method based on bulk flow, termed convection has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell.
  • convection A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell.
  • Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for a first target such as CD3e or any fragment thereof.
  • the second binding target is a disease associated antigen such as B7-H4 or any fragment thereof.
  • bispecific antibodies Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished
  • Bispecific and/or monovalent antibodies of the invention can be made using any of a variety of art-recognized techniques, including those disclosed in co-pending application WO 2012/023053, filed August 16, 2011, the contents of which are hereby incorporated by reference in their entirety.
  • the methods described in WO 2012/023053 generate bispecific antibodies that are identical in structure to a human immunoglobulin.
  • This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and second light chain variable region fused to a constant Lambda domain. Each combining site displays a different antigen specificity to which both the heavy and light chain contribute.
  • the light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively. This is preferred in order to avoid the generation of non-natural polypeptide junctions.
  • bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity.
  • the bispecific antibodies described in WO 2012/023053 are referred to as IgGrA antibodies or “K bodies,” a new fully human bispecific IgG format.
  • This KA- body format allows the affinity purification of a bispecific antibody that is undistinguishable from a standard IgG molecule with characteristics that are undistinguishable from a standard monoclonal antibody and, therefore, favorable as compared to previous formats.
  • An essential step of the method is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain.
  • Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).
  • Fully human antibodies are antibody molecules in which the sequence of both the light chain and the heavy chain, including the CDRs 1 and 2, arise from human genes.
  • the CDR3 region can be of human origin or designed by synthetic means. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.
  • Human monoclonal antibodies can be
  • SUBSTITUTE SHEET (RULE 26) prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983.
  • Monoclonal antibodies are generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof.
  • the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells.
  • a variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which is hereby incorporated by reference in its entirety.
  • the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen.
  • This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”).
  • phage displayed library e.g., bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles
  • a library can be prepared in yeast as protein or peptide fusions to a cell wall protein on the surface of yeast cells and encoding DNA sequences contained within the yeast cells (i.e. “yeast display library”).
  • Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to the target antigen.
  • Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • SUBSTITUTE SHEET (RULE 26) antigens is highly unlikely. Indeed, in most cases the heavy chain contributes largely to the antigen binding surface and is also the most variable in sequence. In particular the CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost invariably carry different heavy chain variable domains.
  • SUBSTITUTE SHEET (RULE 26) expressed at different levels and/or do not assemble with the same efficiency. Therefore, a means to modulate the relative expression of the different polypeptides is used to compensate for their intrinsic expression characteristics or different propensities to assemble with the common heavy chain. This modulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) featuring different efficiencies or other types of regulatory elements that can act at transcriptional or translational levels as well as acting on mRNA stability.
  • IRS internal ribosome entry sites
  • Different promoters of different strength could include CMV (Immediate- early Cytomegalovirus virus promoter); EFl- la (Human elongation factor la- subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simian virus 40 promoter).
  • CMV Intermediate- early Cytomegalovirus virus promoter
  • EFl- la Human elongation factor la- subunit promoter
  • Ubc Human ubiquitin C promoter
  • SV40 Synimian virus 40 promoter
  • IRES have also been described from mammalian and viral origin. (See e.g., Hellen CU and Sarnow P. Genes Dev 2001 15: 1593-612). These IRES can greatly differ in their length and ribosome recruiting efficiency. Furthermore, it is possible to further tune the activity by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541).
  • the modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions.
  • the Examples provided herein demonstrate that controlling the relative expression of the different chains is critical for maximizing the assembly and overall yield of the bispecific antibody.
  • the co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody.
  • the latter has to be purified from the mixture to obtain the molecule of interest.
  • the method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the invention.
  • antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface includes at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • bispecific antibodies can be prepared using chemical linkage.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • SUBSTITUTE SHEET (RULE 26) This method can also be utilized for the production of antibody homodimers.
  • the “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigenbinding sites.
  • Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti- antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • SUBSTITUTE SHEET (RULE 26)
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J.
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2, 4- dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyri
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026).
  • Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • the preferred binding is, however, covalent binding.
  • Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.
  • MBS M-maleimidobenzoyl-N- hydroxysuccinimide ester
  • linkers include: (i) EDC (l-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido] hexanoate (Pierce Chem.
  • linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties.
  • sulfo-NHS sulfo-NHS
  • SUBSTITUTE SHEET (RULE 26) esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.
  • Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo-NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • any of the bispecific antibodies of the disclosure may be used in therapeutic methods.
  • a bispecific antibody of the disclosure may be for use as a medicament is provided.
  • a bispecific antibody of the disclosure may be for use in treating or delaying progression of a cell proliferative disorder (e.g., cancer).
  • a bispecific antibody of the disclosure may be for use in a method of treatment is provided.
  • the invention provides a bispecific antibody for use in a method of treating an individual having a cell proliferative disorder comprising administering to the individual an effective amount of the bispecific antibody.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below.
  • the invention provides a bispecific antibody for use in enhancing
  • the invention provides a bispecific antibody for use in a method of enhancing immune function in an individual having a cell proliferative disorder comprising administering to the individual an effective of the bispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell population, and/or kill a target cell (e.g., target tumor cell).
  • effector cells e.g., T cells, e.g., CD8+ and/or CD4+ T cells
  • An "individual" according to any of the above embodiments may be a human.
  • the invention provides for the use of bispecific antibodies of the disclosure (e.g. anti-CD3e and anti-B7-H4 antibody; anti-CD3e and anti-B7-H4 antibody with CD58 fusion peptide) in the manufacture or preparation of a medicament.
  • the medicament is for treatment of a cell proliferative disorder (e.g., cancer, e.g., esophageal cancer or an adenocarcinoma).
  • the medicament is for use in a method of treating a cell proliferative disorder or an autoimmune disorder comprising administering to an individual having a cell proliferative disorder or an autoimmune disorder an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below.
  • the medicament is for activating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expanding (increasing) an effector cell population, reducing a target cell (e.g., a cell expressing B7-H4) population, and/or killing target cells (e.g., target tumor cells) in the individual.
  • effector cells e.g., T cells, e.g., CD8+ and/or CD4+ T cells
  • expanding (increasing) an effector cell population reducing a target cell (e.g., a cell expressing B7-H4) population
  • killing target cells e.g., target tumor cells
  • the medicament is for use in a method of enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder comprising administering to the individual an amount effective of the medicament to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing B7-H4) population, and/or kill a target cell (e.g., target tumor cell).
  • effector cells e.g., T cells, e.g., CD8+ and/or CD4+ T cells
  • expand (increase) an effector cell population e.g., a cell expressing B7-H4) population
  • kill a target cell e.g., target tumor cell.
  • An "individual" according to any of the above embodiments may be a human.
  • the invention provides a method for treating a cell proliferative disorder (e.g., cancer).
  • a cell proliferative disorder e.g., cancer
  • the method comprises administering to an individual having such a cell proliferative disorder an effective amount of an bispecific antibody.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as
  • the invention provides a method for enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder in an individual having a cell proliferative disorder or an autoimmune disorder.
  • the method comprises administering to the individual an effective amount of a bispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing B7-H4) population, and/or kill a target cell (e.g., target tumor cell).
  • effector cells e.g., T cells, e.g., CD8+ and/or CD4+ T cells
  • a target cell e.g., a cell expressing B7-H4 population
  • kill a target cell e.g., target tumor cell
  • the invention provides a method for treating a cancer that expresses B7-H4.
  • the method of treating a cancer comprises administering an effective amount of the bispecific antibody of the disclosure (e.g. anti-CD3e and anti-B7-H4 antibody; anti-CD3e and anti-B7-H4 antibody with CD58 fusion peptide).
  • the cancer expresses B7-H4 at a high level.
  • the cancer is at least one of a lung cancer, a breast cancer, a head and neck cancer, an ovarian cancer (e.g., serous adenocarcinoma), uterine cancer (e.g., carcinoma and corpus endometrioid carcinoma), cervical cancer, or an endometrial cancer.
  • the cancer consists of a lung cancer, a breast cancer, a head and neck cancer, an ovarian cancer (e.g., serous adenocarcinoma), uterine cancer (e.g., carcinoma and corpus endometrioid carcinoma), cervical cancer and an endometrial cancer.
  • the breast cancer is a triple negative cancer, an ER+ cancer or a HER2+ cancer. In some embodiments, the breast cancer is a triple negative cancer. In some embodiments, any of the types of cancers provided herein can be treated with a B7-H4 antibody.
  • the B7-H4 expression is induced by the addition of an additional therapeutic agent.
  • the additional therapeutic agent is a selective estrogen receptor degrader (SERD).
  • SEED selective estrogen receptor degrader
  • Exemplary SERDs include but are not limited to fulvestrant, elacestrant and camizestrant.
  • the additional therapeutic agent is a CDK4/6 inhibitor.
  • Exemplary CDK4/6 inhibitors include but are not limited to palbociclib, ribociclib and abemaciclib.
  • the additional therapeutic agent is a SERM (selective estrogen receptor modulator).
  • SERMs selective estrogen receptor modulator
  • Exemplary SERMs include but are not limited to tamoxifen.
  • the additional therapeutic agent is an aromatase inhibitor (AIs).
  • AIs include but are not limited to letrozole.
  • the additional therapeutic agent is fulvestrant. In some embodiments, the additional therapeutic agent is palbociclib. In some embodiments, the additional therapeutic agent is fulvestrant and palbociclib.
  • a method of identifying a subject to receive a B7-H4 therapy comprises testing a cancer sample for a high level of expression of B7-H4.
  • the cancer is one or more of triple negative breast cancer, ovarian cancer, uterine cancer, breast cancer (PR+), breast cancer (PR-), breast cancer (ER+), or breast cancer (HER2+).
  • the breast cancer is ductal carcinoma in situ (DCIS), invasive (other), metastatic, medullary, invasive ductal, SCC, invasive lobular, invasive papillary or mucinous.
  • the method further comprises comparing a level of B7-H4 expression in the cancer sample to a level of B7-H4 expression in healthy/control/reference tissue to determine if the level of B7-H4 expression in the cancer sample is higher. If the level of B7-H4 expression is higher in the cancer sample, the subject receives a B7-H4 antagonist.
  • the B7-H4 antagonist is one or more of the antibodies provided herein.
  • any method for measuring the level of B7-H4 can be employed. In some embodiments, this is any amount greater than a negative amount in a staining assay.
  • this is any amount above a level present in surrounding healthy/control/reference tissue or corresponding tissue from a healthy/control/reference subject.
  • high levels of expression of B7-H4 is defined in comparison to an expression level of B7-H4 in a non-cancer sample.
  • the expression level of B7-H4 in the cancer sample is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% higher than the expression level of B7-H4 in the non-cancer sample. In some embodiments, the expression level of B7- H4 in the cancer sample is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% higher than the expression level of B7-H4 in the non-cancer sample.
  • the expression level of B7-H4 in the cancer sample is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475% or 500% higher than the expression level of B7-H4 in the non-cancer sample.
  • the expression level of B7-H4 in the cancer sample is at least 2 fold, 3, fold, 4 fold, 5, fold, 6
  • the method further comprises comparing a level of HER2 (ERBB2), ER(ESRl) and/or PR (PGR) expression in the cancer sample to a level of HER2 (ERBB2), ER(ESRl) and/or PR (PGR) expression in healthy/control/reference tissue to determine if the level of HER2 (ERBB2), ER(ESRl) and/or PR (PGR) expression in the cancer sample is higher.
  • the subject receives a B7-H4 antagonist.
  • the B7-H4 antagonist is one or more of the antibodies provided herein.
  • any method for measuring the level of HER2 (ERBB2), ER(ESRl) and/or PR (PGR) can be employed.
  • the method further comprises comparing levels of FOXA1, ESRI, GATA3, KRT5, KRT6, KRT7, KRT14 and KRT20 to determine luminal versus basal- squamous molecular featured where basal- squamous tumors histology is correlated with T cell infiltration and is used to identify responders to immunotherapies that include immune checkpoint inhibitors (anti-PDl antibodies: pembrolizumab and Nivolumab; anti-PDLl antibodies: atezolizumab); T cell engager biologies including CD3-bispecific and target costimulatory agonists.
  • immune checkpoint inhibitors anti-PDl antibodies: pembrolizumab and Nivolumab
  • anti-PDLl antibodies atezolizumab
  • T cell engager biologies including CD3-bispecific and target costimulatory agonists.
  • anti-FOXAl, anti-ESRl or anti-GATA3 cancer expresses FOXA1, ESRI or GATA3 at a 1+, 2+ or 3+ level defines a non-responder to immunotherapy.
  • a positive cancer is a cancer that expresses FOXA1, ESRI or GATA3 according to a reverse-transcriptase PCR (RT-PCR) that detects FOXA1, ESRI or GATA3.
  • the RT-PCR is quantitative and indicates a signal of expression that is greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
  • the method may use RNA from a commercially available luminal cancer cell line to determine the relative level of FOXA1, ESRI, GATA3 signal when normalized by a housekeeping gene including the 553 genes used in Aran et al., Nat Commun 8, 1077 (2017) which
  • SUBSTITUTE SHEET (RULE 26) provide house keeping genes with no increase or decrease in normal adjacent tumor tissues when compared to normal tissues from healthy donors, or the 3804 genes indicated in Eisenberg et al., Trends Genet., 257: 569-574 (2013).
  • the healthy/control/reference sample is a sample from a normal tissue.
  • the normal tissue is a tissue that is an adjacent tissue to the cancer in the subject.
  • the levels of the protein are above those understood to be “negative” to one of skill in the art for the particular assay.
  • Exemplary assays include but are not limited to RNA based assays, FACS, and IHC, each with their appropriate levels of positive and negative results. Any method of detecting the level of a protein in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of proteins being detected.
  • Nonlimiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc.
  • any method of detecting the level of an mRNA in a sample is contemplated.
  • One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of mRNAs being detected.
  • Nonlimiting exemplary such methods include RT-PCR, quantitative RT-PCR and microarray-based methods, etc.
  • a B7-H4-positive cancer may be, e.g., B7-H4-positive breast cancer (including a B7-H4-positive triple negative (ER-/PR-/Her2-) breast cancer), B7-H4-positive ovarian cancer, and B7-H4-positive endometrial cancer.
  • an B7-H4-positive cancer is a cancer that receives an anti-B7-H4 immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than “0,” which corresponds to very weak or no staining in >90% of tumor cells.
  • IHC immunohistochemistry
  • ISH in situ hybridization
  • a B7-H4-positive cancer expresses B7-H4 at a 1+, 2+ or 3+ level.
  • a B7- H4-positive cancer is a cancer that expresses B7-H4 according to a reverse-transcriptase PCR (RT-PCR) assay that detects B7-H4 mRNA.
  • RT-PCR reverse-transcriptase PCR
  • the RT-PCR is quantitative RT-PCR.
  • the invention provides pharmaceutical formulations comprising any of the bispecific antibodies provided herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent, for example, as described herein.
  • an antibody of the invention can be used either alone or in combination with other agents in a therapy.
  • an antibody of the invention may be co-administered with at least one additional therapeutic agent.
  • an additional therapeutic agent is a chemotherapeutic agent, growth inhibitory agent, cytotoxic agent, agent used in radiation therapy, anti-angiogenesis agent, apoptotic agent, anti-tubulin agent, or other agent, such as a epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TarcevaTM), platelet derived growth factor inhibitor (e.g., GleevecTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferon, cytokine, antibody other than the anti-CD3 antibody of the invention, such as an antibody that bind to one or more of the following targets
  • EGFR epidermal growth
  • the invention provides a method wherein the additional therapeutic agent is a glucocorticoid.
  • the glucocorticoid is dexamethasone.
  • the additional therapeutic agent is a monoclonal anti-B7-H4 antibody.
  • exemplary anti-B7-H4 antibodies include but are not limited to alsevalimab FPA150 (Amgen), and those described in PCT Publications WO 2009/073533, WO 2008/067283, each of which are incorporated herein in their entireties.
  • the additional therapeutic agent for the particular indication being treated preferably those with complementary activities that do not adversely affect each other.
  • Avastin® bevacizumab
  • B7-H4-positive cancer such as B7-H4-positive breast cancer (including B7-H4-positive triple negative (ER-/PR-/Her2-) breast cancer), B7- H4-positive ovarian cancer or B7-H4-positive endometrial cancer.
  • the additional therapeutic agent is a checkpoint inhibitor.
  • the term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor.
  • an activity e.g., an activity of, e.g., PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or
  • SUBSTITUTE SHEET (RULE 26) TGFR beta, of at least 5%, 10%, 20%, 30%, 40% or more is included by this term. The level of inhibition need not be 100%.
  • the checkpoint inhibitor is a PD-1 inhibitor.
  • the PD-1 inhibitor is an anti-PDl antibody.
  • the PD-1 inhibitor is an anti PD-1 monoclonal antibody.
  • Exemplary anti-PD-1 monoclonal antibodies include, but are not limited to cemiplimab (Libtayo), nivolumab (Opdivo), pembrolizumab (Keytruda).
  • the checkpoint inhibitor is a PD-L1 inhibitor.
  • Exemplary PD-L1 inhibitors include but are not limited to avelumab (Bavencio), durvalumab (Imfinzi) and atezolizumab (Tecentriq).
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents.
  • administration of the bispecific antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.
  • Bispecific antibodies of the disclosure can also be used in combination with radiation therapy.
  • the additional therapeutic agent is a chimeric antigen receptor (CAR) T cell therapy.
  • the CAR-T cell therapy specifically binds CD 19.
  • Exemplary CAR-T cell therapies that specifically bind CD 19 include but are not limited to BREYANZI® (lisocabtagene maraleucel), TECARTUSTM (brexucabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), YESCARTATM (axicabtagene ciloleucel), ABECMA® (idecabtagene vicleucel), or CARVYKTITM (ciltacabtagene autoleucel).
  • An antibody of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the antibody is administered by subcutaneous administration.
  • an anti-CD3e antibody administered by subcutaneous injection exhibits a less toxic response in a patient than the same anti-CD3e antibody administered by intravenous injection. Dosing can be by any suitable route, for example, by injections, such as intravenous
  • SUBSTITUTE SHEET (RULE 26) or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an antibody of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • the therapeutically effective amount of the bispecific anti- CD3e and anti-B7-H4 antibody administered to human will be in the range of about 0.01 to about 100 mg/kg of patient body weight whether by one or more administrations.
  • the antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.
  • a bispecific antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg,
  • SUBSTITUTE SHEET (RULE 26) about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles.
  • the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg kg, or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of the bispecific antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the methods may further comprise an additional therapy.
  • the additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DMA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti- metastatic agent.
  • the additional therapy is the administration of sideeffect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more of the therapeutic agents described above.
  • SUBSTITUTE SHEET (RULE 26) Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration.
  • the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration.
  • Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for
  • Therapeutic formulations of the invention are used to treat or alleviate a symptom associated with a cancer, such as, by way of non-limiting example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung & bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity & pharynx cancer, uterine corpus cancer, and/or melanoma
  • a therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods.
  • Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers a clinical benefit.
  • Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.
  • ELISA enzyme linked immunosorbent assay
  • Antibodies directed against a target such as CD3e, B7-H4, or a combination thereof (or a fragment thereof), may be used in methods known within the art relating to the localization and/or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies specific any of these targets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).
  • An antibody of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
  • Antibodies of the invention (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (z.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolumine scent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • Antibodies of the invention may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject.
  • An antibody preparation preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target.
  • Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target.
  • Administration of the antibody may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds.
  • a therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
  • Antibodies or a fragment thereof of the invention can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
  • the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • an agent that enhances its function such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,
  • SUBSTITUTE SHEET (RULE 26) hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and y ethyl-L-glutamate non- degradable ethylene- vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylenevinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • an antibody according to the invention can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample.
  • the antibody contains a detectable label.
  • Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof e.g., F a b, scFv, or F( a b)2) is used.
  • the term “labeled”, with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological
  • SUBSTITUTE SHEET sample in vitro as well as in vivo.
  • in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E.
  • in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are primarily those that are used in the preparation of a pharmaceutically acceptable salt.
  • SUBSTITUTE SHEET (RULE 26) prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • SUBSTITUTE SHEET (RULE 26) oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
  • B7-H4-positive cancer refers to a cancer comprising cells that express B7-H4 on their surface.
  • expression of B7-H4 on the cell surface is determined, for example, using antibodies to B7-H4 in a method such as immunohistochemistry, FACS, etc.
  • B7-H4 mRNA expression is considered to correlate to B7-H4 expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).
  • B7-H4-positive cell refers to a cell that expresses B7-H4 on its surface.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and nonHodgkin's lymphoma), blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including triple negative (ER-/PR-/Her2-) breast cancer), colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer,
  • SUBSTITUTE SHEET (RULE 26) vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer
  • reference sample denotes a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic.
  • a reference sample can be used as a positive or negative indicator.
  • a reference sample can be used to establish a level of protein and/or mRNA that is present in, for example, healthy tissue, in contrast to a level of protein and/or mRNA present in the sample with unknown characteristics.
  • the reference sample comes from the same subject, but is from a different part of the subject than that being tested.
  • the reference sample is from a tissue area surrounding or adjacent to the cancer.
  • the reference sample is not from the subject being tested, but is a sample from a subject known to have, or not to have, a disorder in question (for example, a particular cancer or B7-H4 related disorder).
  • the reference sample is from the same subject, but from a point in time before the subject developed cancer.
  • the reference sample is from a benign cancer sample (for example, benign breast cancer sample), from the same or a different subject.
  • a negative reference sample is used for comparison, the level of expression or amount of the molecule in question in the negative reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is no and/or a low level of the molecule.
  • the level of expression or amount of the molecule in question in the positive reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is a level of the molecule.
  • a “disease” or “disorder” as used herein refers to a condition where treatment is needed and/or desired.
  • tumor cell refers to a cell (or cells) exhibiting an uncontrolled growth and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.
  • cancer and “tumor” encompass solid and hematological/lymphatic cancers and also encompass malignant, pre-malignant, and benign
  • SUBSTITUTE SHEET (RULE 26) growth such as dysplasia.
  • cells having abnormal proliferation that is not impeded e.g immune evasion and immune escape mechanisms
  • the immune system e.g. virus infected cells.
  • Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
  • the term cancer can encompass: a lung cancer, a breast cancer, a head and neck cancer, an ovarian cancer, and/or an endometrial cancer.
  • a lung cancer a breast cancer, a head and neck cancer, an ovarian cancer, and/or an endometrial cancer.
  • the difference between a “cancer” and a “cancer cell” can be denoted by the use of the explicit use of the phrase “cancer cell”; however, the term “cancer” will encompass concepts such as the subject having cancer, and multicellular tumors, as well as single cancer cells.
  • an “increase or decrease” refers to a statistically significant increase or decrease respectively.
  • “modulating” can also involve effecting a
  • SUBSTITUTE SHEET (RULE 26) change (which can either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of an antibody, bispecific or multispecific polypeptide agent.
  • This can be determined in any suitable manner and/or using any suitable assay known per se or described herein, depending on the target involved.
  • an immune response is meant to encompass cellular and/or humoral immune responses that are sufficient to inhibit or prevent onset or ameliorate the symptoms of disease (for example, cancer or cancer metastasis).
  • An immune response can encompass aspects of both the innate and adaptive immune systems.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • Treatment covers any administration or application of a therapeutic for disease in a mammal, including a human.
  • beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
  • treatment is a reduction of pathological consequence of a proliferative disease.
  • the methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder.
  • “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a B7-H4 antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.
  • biological sample means a quantity of a substance from a living thing or formerly living thing.
  • substances include, but are not limited to, blood, (for example,
  • SUBSTITUTE SHEET (RULE 26) whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.
  • control refers to a composition known to not contain an analyte (“negative control”) or to contain analyte (“positive control”).
  • a positive control can comprise a known concentration of analyte.
  • Control “positive control,” and “calibrator” may be used interchangeably herein to refer to a composition comprising a known concentration of analyte.
  • a “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (for example, analytes).
  • Predetermined cutoff and “predetermined level” refer generally to an assay cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked or associated with various clinical parameters (for example, severity of disease, progression/nonprogression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (for example, antibodies employed, etc.).
  • inhibitors refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.
  • “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This
  • SUBSTITUTE SHEET (RULE 26) delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms “reduce”, “inhibit”, or “prevent” do not denote or require complete prevention over all time.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
  • antibody binding region refers to a region of the antigen, which comprises the epitope to which the antibody binds.
  • An antibody binding region may be determined by epitope binning using biolayer interferometry, by alanine scan, or by domain shuffle assays (using antigen constructs in which regions of the antigen are exchanged with that of another species and determining whether the antibody still binds to the antigen or not).
  • the amino acids within the antibody binding region that are involved in the interaction with the antibody may be determined by hydrogen/deuterium exchange mass spectrometry and/or by crystallography of the antibody bound to its antigen.
  • the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • immunoglobulin immunoglobulin molecules
  • an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Ka > KF 6 ).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F a b, F a b’ and F( a b')2 fragments, scFvs, and an F a b expression library.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • SUBSTITUTE SHEET (RULE 26) includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, IgG4 and others.
  • the light chain may be a kappa chain or a lambda chain.
  • the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
  • MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
  • antigen binding region or “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains.
  • V N-terminal variable
  • H heavy
  • L light
  • hypervariable regions Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”.
  • FR refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”
  • CDRs complementarity-determining regions
  • the Kabat numbering system See Kabat, E.A., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)
  • the IMGT numbering system See IMGT®, the international ImMunoGeneTics information
  • IMGT numbering system is routinely used and accepted as a reliable and accurate system in the art to determine amino acid positions in coding sequences, alignment of alleles, and to easily compare sequences in immunoglobulin (IG) and T-cell receptor (TR) from all vertebrate species.
  • IG immunoglobulin
  • TR T-cell receptor
  • the accuracy and the consistency of the IMGT data are based on IMGT-ONTOLOGY, the first, and so far unique, ontology for immunogenetics and immunoinformatics (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).
  • IMGT tools and databases run against IMGT reference directories built from a large repository of sequences.
  • the IG V-DOMAIN and IG C-DOMAIN are delimited taking into account the exon delimitation, whenever appropriate. Therefore, the availability of more sequences to the IMGT database, the IMGT exon numbering system can be and “is used” by those skilled in the art reliably to determine amino acid positions in coding sequences and for alignment of alleles. Additionally, correspondences between the IMGT unique numbering with other numberings (i.e., Kabat) are available in the IMGT Scientific chart (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).
  • hypervariable region refers to the amino acid residues of an antibody that are typically responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region” or "CDR" (e.g., around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • CDR complementarity determining region
  • residues from a "hypervariable loop” e.g., residues 24- 34 (LI), 50-56 (L2) and 89-97 (L3) in the V L , and 26-32 (HI), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol.
  • the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (HI),
  • epitopic determinants include any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide.
  • An antibody is the to specifically bind an antigen when the dissociation constant is ⁇ 1 pM; e.g., ⁇ 100 nM, preferably ⁇ 10 nM and more preferably ⁇ 1 nM.
  • immunological binding refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (Kon) and the “off rate constant” (K o ff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)).
  • the ratio of K o ff /K on enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • An antibody of the present invention is the to specifically bind to its target, when the equilibrium binding constant (Kd) is ⁇ 1 pM, e.g., ⁇ 100 nM, preferably ⁇ 10 nM, and more preferably ⁇ 1 nM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
  • Kd equilibrium binding constant
  • isolated polynucleotide shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in
  • SUBSTITUTE SHEET (RULE 26) which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • Polynucleotides in accordance with the invention include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules, and nucleic acid molecules encoding the light chain immunoglobulin molecules described herein.
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus.
  • Polypeptides in accordance with the invention comprise the heavy chain immunoglobulin molecules, and the light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
  • operably linked refers to positions of components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • control sequence refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated.
  • the nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • polynucleotide as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
  • Examples of unconventional amino acids include: 4 hydroxyproline, y-carboxyglutamate, e-N,N,N-trimethyllysine, e -N- acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, o-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic - hydroxyl side chains is serine and threonine
  • a group of amino acids having amide- containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is
  • SUBSTITUTE SHEET (RULE 26) lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • SUBSTITUTE SHEET (RULE 26) computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, in In, 125 1, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups,
  • radioisotopes or radionuclides e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, in In, 125 1, 131 I
  • fluorescent labels e.g., FITC, rhodamine, lanthanide phosphors
  • enzymatic labels e.g., horseradish peroxidase,
  • SUBSTITUTE SHEET predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • patient includes human and veterinary subjects.
  • FIG. IE A schematic of the anti-CD3/anti-B7-H4 targeted bispecific antibodies of the present disclosure is represented in FIG. IE and is composed of the following: (1) a first antigen binding region that binds CD3 comprising SP34 antibody variants (“anti-CD3” or “anti-CD3 arm”), (2) a second antigen binding region that binds B7-H4 (“anti-B7-H4” or “anti-B7-H4 arm”), (3) knob-in-hole mutations in the heavy chain as described in US 63/368,852, (4) mutations that enforce light chain heterodimerization, (5) a protein linker, and (6) full length CD58 peptide fused to the C-terminal of the heavy chain of the CD3 binding arm.
  • bispecific or multispecific antibodies were designed to include (i) knob-into-hole mutations to promote efficient heavy chain heterodimerization and (ii) charged pairing (between VH1 and VL1 interface and/or VH2 and VL2 interface, and CHI HI and CL1 interface and/or CH2HI and CL2 interface) and disulfide stabilization mutations (between CHI HI and CL1 interface and/or CH2HI and CL2 interface), which together promote correct cognate pairing of heavy chain and light chains. Correct pairing of heavy and light chains is advantageous for efficient large scale antibody production and purification.
  • FIGS. 1A-1E Exemplary bispecific antibodies of the disclosure are shown in FIGS. 1A-1E.
  • FIG. IE shows a bispecific antibody with a first antigen binding region that binds CD3 and a second antigen binding region that binds B7-H4 and a CD58 peptide fused the C-terminal of the first heavy chain.
  • Exemplary antibodies, EIP1046, EIP1047, EIP1060, EIP 1061 comprise the mutations shown in Table 16. This structural design of the antibody provides improved specificity and complete cognate pairing, which is advantageous for functional antibody expression and purification.
  • DNA sequences corresponding to antibody heavy and light chains were synthesized in pDT5 vector (ATUM, Newark CA). Plasmid DNA (1 pg/ml) were transfected into Expi293 cells (ThermoFisher) according to manufacturer’s protocols. Cells were grown in flasks with rotation (125 rpm) at 37°C with 8% CO2. Five days post-transfection, the conditioned media was harvested from the cells by centrifugation (3000 x g) for 30 minutes and filtered using 0.45 pm filter.
  • Antibodies were then purified using an AKTA Avant chromatography system (Cytiva) and a tandem purification method using HiTrap Mabselect Protein A and HiLoad Superdex 200 pg columns (Cytiva). Antibodies were stored in PBS, pH 7.4 at 4°C following purification and prior to analysis.
  • Anti-CD3/anti-B7-H4 targeted bispecific antibodies and bispecific fusions were produced in transient Expi293 system.
  • the preparative size exclusion chromatogram of exemplary bispecific antibodies (EIP1046, EIP1047, and EIP1060) of the present disclosure following protein A purification is shown in FIG. 2.
  • FIG. 3 shows differential scanning calorimetry data for exemplary bispecific antibodies (EIP1046, EIP1047, and EIP1060).
  • Exemplary bispecific antibodies were advanced for further analysis via surface plasma resonance (SPR) for binding to recombinant human and cyno CD3e/d heterodimer and to human B7-H4.
  • SPR surface plasma resonance
  • the kinetic constants association, dissociation and equilibrium are shown in Table 17 for human and cyno CD3e/d and Table 18 for human B7-H4.
  • the binding kinetics were carried out at 25 °C or 37 °C and the association contact and dissociation times used were 60-120 seconds and 120-420 seconds.
  • the chip surface was regenerated by injecting 10 mM glycine, pH 1.5, at a flow rate of 30 pL/min for 60 sec.
  • the kinetic and affinity constants are determined by fitting the data to a 1:1 binding model using the Biacore Insight
  • the bispecific variants were further analyzed by sandwich ELISA for binding to human and cyno B7-H4. Maxisorp plates (Nunc) were coated with proteins at a concentration of 1 pg/ml in PBS, pH 7.2 for 16 hours at 4 °C. All subsequent steps were performed at room temperature. The plates were then blocked with blocking buffer (PBS, pH 7.2, 3% BSA) for 1 hour. The plates were then washed using PBS pH 7.2, 0.1% Tween. Bispecific antibodies were diluted in blocking buffer at a 1 in 3 dilution starting at 100 nM and incubated with blocked wells for 1 hour.
  • SUBSTITUTE SHEET (RULE 26)
  • the exemplary bispecific antibodies were further checked for their superagonism activity.
  • Target-independent cytokine release was assessed by plating 200K PBMCs of three different donors and adding bispecific antibodies at a concentration of 500 nM. Supernatant was taken at 24h and 48 h and assessed for various cytokines (IL6, IL2, TNFa, IFNg) using conventional ELISA.
  • Staphylococcal enterotoxin B (SEB) and the CD3 antibody clone OKT3 were used as positive controls while EIP0120 and EIP 1053 were used as negative controls.
  • SEB Staphylococcal enterotoxin B
  • OKT3 were used as positive controls while EIP0120 and EIP 1053 were used as negative controls.
  • 5A-5D show cytokine measurements (IL2, IL6, IFNg, and TNFa) of exemplary antibodies (EIP1046, EIP1047, and EIP1060) and positive and negative controls for 3 different PBMC donors. While EIP 1046 and EIP 1060 do not show any superagonism activity, cytokine release was seen for EIP 1047.
  • FIG. 6A shows B7-H4 levels on triple negative and Her2 amplified cell lines while FIG. 6B shows levels on hormone receptor positive cell lines.
  • antigen binding capacity was further measured using reference beads (Bangs Laboratories) incubated with the same anti-B7-H4 antibody and calculated in Excel. The quantification is shown in Table 19.
  • T cells were isolated from healthy human PBMCs donors using StemCell T-cell isolation kit and resuspended in RPMI was supplemented with 10% FBS, IX penicillin/streptomycin.
  • Dynabeads coated with ocCD3 and ocCD28 antibodies were added to the T cells at a ratio of 25 p L beads per million cells and incubated at 37°C in 5% CO2 for 48 hours.
  • T cells were incubated with IL-7 (5 ng/ml) and IL- 15 (5 ng/ml) in supplemented in media for an additional 7 days.
  • cytokines IL7 and IL15
  • Human tumor cells were seeded at 10,000 cells per well in 96- well tissue culture plates (Perkin Elmer) and incubated for 24 hours at 37 °C with 5 % CO2. The following day, naive or activated human T cells (50,000) or PBMCs (150,000) were added to tumor cells in the presence or absence of antibodies and incubated for up to 3-7 days. Cytolysis of 157
  • SUBSTITUTE SHEET (RULE 26) Green Fluorescent Protein engineered T-47, HCC-1954, MDA-MB-468, CAMA-1, ZR-75- 1, or HCC-1569 (ATCC) cells were visualized using fluorescent plate reader (Perkin Elmer Ensight). At times, luminescence measurements were utilized by using BrightGlo (Promega) and luminescence plate reading (PerkinElmer Ensight). Tumor cytotoxicity curves were generated by plotting tumor cell counts versus concentration using Prism software (GraphPad).
  • Exemplary bispecific antibodies had superior killing to the benchmark (EIP0909; crossmab surrogate of PF-0437) in either PBMC co-culture assays (FIG. 7) or activate T cell co-culture assays (FIG. 8) in all 5 example cell lines (FIG. 7/8A: T-47D, FIG. 7/8B: HCC1954, FIG. 7/8C: CAMA-1, FIG. 7/8D: ZR-75-1, FIG. 7/8E: HCC-1569).
  • EIP1053 was used as a negative control and showed no tumor killing for any of the cell lines with either PBMCs or activated T cells. EC50s of killing are shown in Table 19. As expected, generally, IC50s are lower when B7-H4 receptors (as measured by antigen binding capacity) are higher.
  • EXAMPLE 3 VTCN1 gene expression and B7-H4 protein expression in patient data [00511] Bioinformatic analysis of patient data
  • TCGA cBioPortal
  • SCAN-B NCBI GEOsets
  • TCGA cBioPortal
  • SCAN-B NCBI GEOsets
  • Z-scores within each gene were used for the ease of analysis in identifying patient groups.
  • Patients were sorted by their receptor (ERBB2: Her2; ESRI: estrogen receptor; and PGR: progesterone) gene expression into 3 groups: Her2 amplified (Her2+), Her2- and estrogen and/or progesterone positive (Her2- HR+), and triple negative (TN).
  • ERBB2 Her2
  • ESRI estrogen receptor
  • PGR progesterone
  • Her2 amplified Her2+
  • Her2- HR+ Her2- and estrogen and/or progesterone positive
  • TN triple negative
  • the normalized RSEM for TCGA
  • TPM for SCAN-B
  • SUBSTITUTE SHEET (RULE 26) shown in FIG. 11, the gene expression of ERBB2, ESRI, and PGR corresponds well with the identified groups in both datasets.
  • the B7-H4 (VTCN1) levels were also plotted for each of the groups and show significantly higher VTCN1 expression in triple negative patients followed next by Her2- HR+ patients (FIGS. 9D, 9H).
  • Each TCGA patient category also corresponds well with luminal (FOXA1, GATA3) and basal (KRT5, KRT6A) markers (FIGS. 10A-10D) as indicated by gene expression of these particular molecular subtypes determined by genes previously reported as luminal and basal markers (Kamoun et al, .77:420-433 (2019)).
  • the immune infiltration levels as assessed by CD8, Granzyme B, PDL1, or PD1 are significantly increased in triple negative patients over Her2+ or Her2- HR+ subtype patients. This suggests more immune cells in the tumor microenvironment of triple negative subtype over other breast cancer subtypes.
  • the SCAN-B dataset also showed similar patterns of luminal (FOXA1, GATA3) and basal (KRT5, KRT6A) markers (FIGS. 11A-11D), and markers of tumor infiltration (FIGS. 11E-11G).
  • the Her2+ subset was underrepresented in this dataset (only 1%) so it was removed from the comparison due to its small sample size.
  • Triple negative patients had statistically significantly lower levels of luminal genes FOXA1 and GATA3, statistically significantly higher levels of expression of basal genes when compared to median expression of all samples, and higher levels of Granzyme B, PDL1, or PD1, genes associated with CD8 T cell, indicating more immune infiltration in triple negative samples than other samples in this dataset.
  • TMAs breast cancer tissue microarrays
  • tissue Array Three independent breast cancer tissue microarrays (TMAs) were procured (Tissue Array) and then stained with rabbit anti-human B7-H4 primary antibody using the Leica BOND III automated staining platform.
  • TMAs low pH Leica BOND epitope retrieval solution was applied while heating before proteinase K digestion.
  • TMAs were scored for plasma membrane staining with 0, 1+ (weak), 2+ (medium), 3+ (high) for a percentage of cells (0-100) in each core for a comprehensive H-score of 0 to 300.
  • the at least 1+ staining percentage for each core of all 3 TMAs was plotted as a histogram (FIG. 12A) with 30% of cores having more than 10% of cells with at least 1+ staining, and about 50% of cores having more than 1% of cells with at least 1+ staining.
  • the B7-H4 H-score was plotted in
  • SUBSTITUTE SHEET (RULE 26) categories of Her2+, Her2- HR+, or TNBC (FIG. 12B). As with the gene expression, TNBC had the highest B7-H4 expression and Her2+ had the lowest by H-score. The patient cancer subtype was also assessed for B7-H4 H-score. DCIS, invasive, and metastatic had the highest H-score with mucinous having the lowest (FIG. 12C).
  • EXAMPLE 4 Modulation of B7-H4 by pharmacological and genetic perturbations [00518] Small molecule treatment
  • Cell lines (T-47D or MFM223) were plated at 100K per well in a 24-well plate. 24 h after seeding, cells were treated with serial dilutions of fulvestrant, tamoxifen, letrozole, and palbociclib. Levels of B7-H4 were assessed by flow cytometry 4 days after the start of treatment. Fulvestrant is a selective estrogen receptor degrader, tamoxifen is a selective estrogen receptor modulator, letrozole is an aromatase inhibitor, and Palbociclib is a CDK4/6 inhibitor; all are used as single agents or combinations for standard of care treatments for Her2- HR+ breast cancers.
  • CDK4/6 inhibitors ribociclib and abemaciclib also increased B7-H4 levels in a dose-dependent manner to a similar magnitude as palbociclib with EC50s of 0.93 nM and 0.13 nM, respectively, compared to palbociclib’s 12 nM. Therefore, other SERDs and CDK4/6 inhibitors have a similar and even more potent effect on B7-H4 increase.
  • sgRNAs synthesized by IDT were added to IDT Recombinant CRISPR Cas9 Alt-R S.p. HiFi Nuclease V3 in a 2.5 to 1 ratio and incubated for 15 minutes at RT.
  • T-47D cells were washed twice in RPMI, then resuspend in Lonza SE Cell Buffer before being added to the RNP Complexes.
  • a Lonza 4D-Nucleofector was used to electroporate the T cells using the EN-104 program. After electroporation, the cells were
  • T-47D GFP-Luc tumor cells were treated with PBMCs at a 15:1 E:T ratio with a serial dilution of bispecific antibody.
  • a combination of Fulvestrant (4 nM) and Palbociclib (100 nM) was included at a constant concentration as a co-treatment.
  • tumor cells % was calculated by GFP imaging and end point readout was done with cell line luminescence.
  • Fulvestrant and Palbociclib combination treatment is an approved standard of care therapy of Her2- HR+ breast cancer.
  • Both exemplary bispecifics (EIP 1046 and EIP 1060) show significant increases (5 and 8-fold, respectively) in EC50 with standard of care treatment as compared to DMSO control while the crossmab surrogate of PF-0437 (EIP0909) showed an increase of 3-fold (FIGS. 15A-15C).
  • the increase of efficacy is a result of the increased B7-H4 expression induced by the treatment and recognized by the anti-B7-H4/anti-CD3 bispecifics.
  • EXAMPLE 6 Humanization of EIP1060 and re-engineering efforts of EIP1046: molecule design, results and discussion:
  • the homology model of variable region of EIP1046 antibody and EIP1060 antibody was generated using “Antibody structure prediction” tool in BioLuminate software (Schrodinger, LLC, New York, NY, 2021).
  • PDB ID: 4KAQ and for EIP1046, PDB ID: 7N3G were selected as template based on the closest percentage sequence similarity from the curated antibody structure database.
  • the templates for the loop building were automatically determined based on sequence similarity. The generated models were visualized and refined for resolving any structural problems like steric
  • EIP 1046 was re-engineered by replacing the framework by CDR grafting, using BioLuminate (Schrodinger). Briefly, the EIP1046 homology model was compared against a human antibody structure database to identify antibody framework with highest level of sequence and structural similarity to the parent antibody. The CDR loops were then grafted onto the most similar human frameworks. Structural guided mutations were introduced to eliminate steric clashes and maximize the stem geometry original geometry of the CDR regions. In total, ten re-engineered variable heavy chains and eleven re-engineered variable light variants were created and combined to generate 14 re-engineered EIP1046 antibody variants.
  • Tumor cells MDA-MB-4678 or isolated Donor T cells were washed and counted.
  • a total of 50K/well of tumor cells or lOOK/well of T cells were used for binding assays.
  • Therapeutic antibodies were serially diluted in FACS buffer and added to the cells. After 30 minutes, cells were washed and anti-human APC (Jackson Laboratories 109-605-170) secondary antibody was added at a dilution of 1:400. After 15 minutes, cells were washed and analyzed on flow cytometry for APC fluorescence.
  • SUBSTITUTE SHEET (RULE 26) (Uniprot accession no Q5ZPR3-1 ; SEQ ID NO: 260), chimeric molecule with IgV domain of B7H4 and the IgC domain of B7H3 (B7H4-IgV/B7H3-igC; SEQ ID No: 261). These molecules were expressed by transiently transfecting the HEK cells by these constructs. Plasmids carrying B7H3, B7H4, and chimeric proteins were electroporated in CHO-K1 cells along with a plasmid carrying the sleeping beauty transposase (SB100X). Transfected cells were selected with puromycin. After two passages of expansion, cells were assessed for antibody binding as described previously. Commercially available B7H4 (MIH43 clone) and
  • B7H3 (MIH42 clone) antibodies were used to check proper expression of the constructs.
  • SUBSTITUTE SHEET (RULE 26) samples (lOpg) were deglycosylated first using rapid PNGase F enzyme (New England Biolabs) in reducing and non-reducing conditions following supplier’s protocols. Reaction mixture was diluted to 1:3 in water and was injected into LC-MS. The total ion chromatogram and m/z data of the proteins were acquired with gradient run of 30 to 50 % HPLC grade acetonitrile containing 0.1% formic acid over 10 min. Mass of the protein samples was deconvoluted from total ion chromatogram using BYOS software from Protein Metrics.
  • EIP1060 is a murine monoclonal antibody that recognizes the human B7-H4. To reduce the potential of immunogenicity when used in human patients, efforts for humanizing EIP 1060 antibody were initiated. On the other hand, EIP 1046 is a humanized antibody but needed improvement in biophysical properties, especially due to its low thermal stability.
  • SUBSTITUTE SHEET (RULE 26) human and cyno B7-H4 protein.
  • the binding for humanized EIP 1060 variants and reengineered EIP 1046 variants with human and cyno B7-H4 is summarized in Table 20 & Table 21 and FIGS. 19A-19B & FIGS. 20A-20B.
  • Table 20 EC50 values for binding of humanized EIP 1060 variants to recombinant human and cyno B7-H4.
  • Table 21 EC50 values for binding of humanized EIP 1046 variants to recombinant human and cyno B7-H4.
  • SUBSTITUTE SHEET (RULE 26) shows the humanized EIP 1060 variants and their closest human identity for Variable domain for both heavy and light chain. This was obtained from IMGT/DomainGapAlign.
  • Table 22 Percentage identity to closest human germline sequence for humanized EIP1060 variants for both heavy and light chain.
  • Exemplary bispecific antibodies were advanced for further analysis via surface plasma resonance (SPR) for binding to recombinant human CD3e/d heterodimer at 25°C and human B7-H4 at 37°C.
  • SPR surface plasma resonance
  • the kinetic constants association, dissociation, and equilibrium are shown in Table 23 for human CD3e/d and human B7-H4.
  • Table 23 Biacore surface plasma resonance values for binding to human CD3e/d and B7-H4.
  • Table 24 Differential scanning calorimetry analysis for re-engineered EIP 1060 variants. The tabulated Tm values are provided for each variant.
  • Table 25 Differential scanning calorimetry analysis for humanized EIP 1046 variants. The tabulated Tm values are provided for each variant.
  • EIP 1060 and EIP 1046 antibodies were humanized and re-engineered, respectively, and expanded into a series of molecules. These molecules were first tested by cytokine release assays. These assays combined PBMCs and biologies for 24h and then read out any cytokine release, most likely due to superagonism properties of the molecules, using SEB and OKT3 as positive controls and EIP0120 and EIP0614 as negative controls. Only two molecules from the EIP1060 series (EIP1358, EIP1360) showed low level of cytokine release, consistent with some evidence of superagonism (FIG. 23). For the EIP 1046 series, another two molecules (EIP1416, EIP1451) showed potential superagonism with low levels of cytokine release (FIG. 24). Therefore, we continued to functional assays with fewer molecules.
  • EIP1417 and EIP1529 bind stronger to B7-H4 than EIP1357 and EIP1526.
  • FIG. 28B shows the binding of these variants to T cells of a human PBMC donor. While EIP1417 and EIP1357 bind stronger to T cells as compared to Pfizer and Genmab biosimilars, EIP 1529 and EIP 1526 bind weaker. This is expected due to the attenuation of the CD3 arm of these molecules. While the efficacy of some variants (EIP1529, EIP1526) is similar to Pfizer and Genmab biosimilars,
  • variant EIP1357 and EIP1450 shows specific binding to B7H4 and not towards B7H3.
  • FIG. 29C shows that IgV domain of B7H4 is involved in binding of EIP1450 which is a variant from EIP1046 series, whereas, EIP1357, one of the EIP1046 variant didn’t show any binding for this chimera signifying that as opposed to EIP1046 variants, it binds to the IgC domain of B7-H4.
  • EIP 1046 variants and EIP 1060 variants bind to different domains of B7-H4 which may be reason for their difference in potency in tumor killing assays.
  • Mass spectrometry was used to assess the degree of mispairing of peptide chains for each bispecific fusion.
  • the expected mass for a correctly paired bispecific fusion was observed for EIP 1046 (FIG 30A) and EIP 1060 (FIG. 30B) by non-reduced intact antibody mass analysis.
  • Four distinct polypeptide chains corresponding to two light chains and two heavy chains of the expected mass were also observed upon reduction for EIP 1046 (FIG. 30C) and EIP1060 (FIG. 30D).
  • EXAMPLE 7 B7-H4 increases are specific to ER modulation in vitro and in patients [00549] T-47D cells were genetically edited by CRISPR-Cas9 as previously described. Guides for AAVS1 (negative control), ESRI, FOXA1, and GATA3 were used. After culturing, cells were lysed for western blotting. Antibodies against B7-H4 (ab209242), ESR1/ER (Thermo MA5-14501), FOXA1 (Thermo PA5-27157), GATA3 (CST #5852T), and GAPDH (Sigma G8795) were used.
  • T47D cells treated with small molecule inhibitors as previously described were lysed and run on western blots using antibodies against B7-H4, ER, and GAPDH. For 170
  • VTCN1 gene levels were significantly increased in patients treated with the SERD fulvestrant (GSE71791) or the SERM tamoxifen (GSE147271) as compared to paired pre-treated samples (Table 26).
  • GSE71791 the SERM tamoxifen
  • GSE147271 the SERM tamoxifen
  • significant differences were only seen for responders as compared to non-responders, suggesting that ER decreases in the patient drive VTCN1, and therefore B7-H4, increases. This suggests that the pharmacologic perturbations we showed in cell lines (lower ER, higher B7-H4 expression) may also hold true for patients being treated with ER-modulating therapies.
  • Table 26 Analysis of treated patient data for VTCN1 expression
  • EXAMPLE 8 Increased bispecific antibody efficacy in cotreatment with checkpoint inhibitor pembrolizumab
  • T-47D GFP-Luc tumor cells were treated with PBMCs at a 15:1 E:T ratio with a serial dilution of bispecific antibody.
  • Either IgG4 control or pembrolizumab was included at a constant concentration as a co-treatment.
  • tumor cells % was calculated by GFP imaging and end point readout was done with cell line 171
  • SUBSTITUTE SHEET (RULE 26) luminescence.
  • Pembrolizumab combination treatment is an approved standard of care therapy for triple negative breast cancer.
  • the exemplary bispecific EIP 1046 showed a 2- fold increase in EC50 with pembrolizumab as compared to standard of care treatment for two different donors (FIG. 32A and FIG. 32B).
  • the increase of efficacy is a result of the combination of two different mechanisms of action (bispecific T cell engager and checkpoint inhibitor) of the two biologies.

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Abstract

La présente divulgation concerne des anticorps bispécifiques ou des fragments de liaison à l'antigène de ceux-ci qui se lient spécifiquement à CD3 et B7-H4. Les anticorps sont éventuellement fusionnés avec un peptide CD58 ou des parties de celui-ci. L'invention concerne des procédés de fabrication et d'utilisation des anticorps bispécifiques pour le traitement de cancers qui expriment B7-H4. La présente divulgation concerne également des procédés et des kits permettant de détecter une prédisposition, de déterminer le risque et d'orienter la thérapie pour les troubles liés à la protéine B7-H4 pour détecter une prédisposition à, déterminer le risque et guider une thérapie pour des troubles liés à B7-H4.
PCT/US2024/045986 2023-09-11 2024-09-10 Molécules de fusion d'anticorps bispécifiques ciblant b7-h4 et cd3 et leurs procédés d'utilisation Pending WO2025059037A1 (fr)

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