EGFRxCD28 multispecific antibody

Abstract

The present invention provides multispecific antibodies (EGFRxCD28) that bind to EGFR and CD28, as well as anti-EGFR antibodies. Such antibodies can be combined with additional therapeutic agents, such as anti-PD1 antibodies. Methods for treating cancer (e.g., EGFR-expressing cancers) by administering such antibodies (and their combination with anti-PD1 antibodies, for example) are also provided. The EGFRxCD28 antibodies of the present invention embody a tumor-targeted immunotherapy modality combined with PD-1 blockade. These bispecific antibodies bind to a tumor-specific antigen (TSA) (EGFR) with one arm and to the T cell costimulatory receptor, CD28, with the other arm. Combination therapy with PD-1 inhibitors clearly enhanced intratumoral T cell activation while promoting an effector memory-like T cell phenotype without systemic cytokine release in various syngeneic and human tumor xenograft models. Combining this type of CD28 costimulatory bispecific antibody with clinically proven anti-PD-1 therapy provides a well-tolerated antibody therapy with significantly enhanced antitumor efficacy.

Description

[Technical field]

[0001] Related Applications This application is related to and claims the benefit of U.S. Provisional Patent Application No. 62 / 822,124, filed March 22, 2019, the entire contents of which are expressly incorporated herein by reference.

[0002] The present invention relates to antibodies that bind to the EGF receptor and CD28, and methods of their use, for example, to treat or prevent cancer.

[0003] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on Mar. 19, 2020, is named 118003-10620_SL.txt, and is 63,481 bytes in size. [Background technology]

[0004] The ability of T cells to recognize and kill their cellular targets, e.g., virus-infected or tumor cells, depends on a series of coordinated interactions, including recognition and binding of the target cell by the T cell receptor (TCR) complex (containing associated CD3γ, δ, ε, and ζ chains), also referred to as “signal 1” of T cell activation. The TCR recognizes viral or tumor peptides presented in the groove of MHC proteins expressed on the surface of the target cell. Such binding is generally of low affinity, so successful induction of “signal 1” requires the clustering of many TCR complexes along the interface between the T cell and its target cell. This interface has been termed the “immune synapse.” T cell activation can be further promoted by additional interactions. For example, T cells have a molecule on their surface called CD28, which can provide a costimulatory “signal 2” to augment activation via the TCR complex. T cells recognize target cells via their own TCR complexes, and then further engage with "signal 2" via CD28 binding to its cognate ligand on the target cell, enhancing T cell activation. Similar to "signal 1," CD28-mediated "signal 2" is also thought to occur via co-clustering at the immune synapse.

[0005] Agonistic anti-CD28 mAbs can be applied for sustained ex vivo expansion of cultured T cells, but the use of antibodies against CD28 has been halted following a series of acute and severe adverse events in a Phase I clinical trial testing a superagonistic anti-CD28 mAb systemically (Hunig, Nature Reviews Immunology. 2012; 12: 317-318). The use of localized or targeted anti-CD28 mAbs can be used with low risk for promoting antitumor immunity. Jung et al., Int J Cancer. 2001 Jan 15; 91(2): 225-30.

[0006] Various families of growth factors and growth factor receptors have been shown to be involved in the autonomous growth of cancer cells. Among these, the epidermal growth factor receptor (EGFR) and the EGF family of peptide growth factors play a central role in the pathogenesis and progression of various carcinoma types. EGFR belongs to a receptor family that includes three additional proteins, ErbB-2, ErbB-3, and ErbB-4. These proteins and the EGF family of growth factors form an integrated system in which signals that hit individual receptor types are often transmitted to other receptors in the same family.

[0007] Monoclonal antibodies (mAbs) targeting T cell activation are in clinical development as antitumor therapeutics. However, most current therapies struggle to overcome the inhibitory nature of the tumor microenvironment and fail to generate efficient tumor-specific T cell activation and subsequent tumor cell killing. Several blocking mAbs targeting checkpoint inhibitors, such as cytotoxic T lymphocyte-associated protein (CTLA)-4 and programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1), have been clinically approved for melanoma, renal cell carcinoma, non-small cell lung cancer, and advanced metastatic cutaneous squamous cell carcinoma. Although blocking PD-1 generates a break in T cell activation, its efficacy as a single agent has often been insufficient to achieve tumor clearance or sustained antitumor responses. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hunig,Nature Reviews Immunology.2012;12:317-318 [Non-Patent Document 2] Jung et al.,Int J Cancer.2001 Jan 15;91(2):225-30 Summary of the Invention [Means for solving the problem]

[0009] The present invention provides an immunotherapeutic modality using bispecific antibodies (EGFRxCD28 or CD28xEGFR) targeting Cluster of Differentiation 28 (CD28) and epidermal growth factor receptor, which, when combined with a PD-1 blocking antibody, induce long-term antitumor immunity and promote stable intratumoral T cell activation without signs of systemic cytokine secretion in both syngeneic and human tumor xenograft models. Toxicity studies in genetically modified humanized immunocompetent mice and cynomolgus monkeys demonstrated that these bispecific antibodies exhibit some single-agent activity and no toxicity by themselves or in combination with anti-PD-1 antibodies. Collectively, these data suggest that the combination of this CD28-based bispecific antibody material with PD-1 blockade provides a safe biological solution with significantly enhanced, specific, and synergistic antitumor activity.

[0010] In one embodiment, the present invention provides a method for producing a cellulose acetate ester having a molecular weight of about 10 as measured by surface plasmon resonance at 25° C. -6 K under M D and a first antigen-binding domain that binds to human CD28 at about 10 as measured by surface plasmon resonance at 25° C. -9 K under M D and a second antigen-binding domain that specifically binds to human epidermal growth factor receptor (EGFR) on a target tumor cell.

[0011] In another aspect, the isolated bispecific antigen binding molecule exhibits a costimulatory effect when used in conjunction with an anti-Mucine 16 (MUC16)X CD3 bispecific antibody and tested against target cells expressing EGFR. In one embodiment, the costimulatory effect is demonstrated by one or more of the following: (a) the ability to activate and induce human T cells to kill target cells expressing EGFR, (b) the ability to upregulate PD-1 on T cells, (c) the ability to increase the release of the cytokines IFN gamma and TNF from PBMCs, (d) the ability to deplete tumor cells, or (f) the ability to enhance tumor clearance. In another embodiment, the costimulatory effect is further demonstrated by one or more of the following: (g) activation of NFκB activity in a T cell / APC luciferase-based reporter assay, or (h) activation of primary CD4 + Measurement of IL-2 cytokine production using a T cell / APC functional assay.

[0012] The present invention provides multispecific (e.g., bispecific) antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) that bind to EGFR and CD28, comprising: (1) an EGFR-binding arm comprising: (a) an immunoglobulin chain comprising an HCDR of a heavy chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 2, 30, 40, and 50, or a variant thereof, and / or (b) an immunoglobulin chain comprising an LCDR of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof, and a CD28-binding arm; or (2) a CD28-binding arm comprising: (c) an immunoglobulin chain comprising an HCDR of a heavy chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 10, 59, and 63, or a variant thereof, and / or (d) an immunoglobulin chain comprising an LCDR of a light chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 16 and 67, or a variant thereof, and an EGFR-binding arm.

[0013] For example, in one embodiment of the invention, a multispecific (e.g., bispecific) antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to EGFR and CD28 comprises: (1) (a) a heavy chain immunoglobulin, or a variable region thereof, that comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2, 24, 30, 40, and 50, or a variant thereof, and / or (b) a light chain immunoglobulin, or a variable region thereof, that comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 16 and 28, or a variant thereof. or (2) (c) a heavy chain immunoglobulin comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 10, 26, 59, and 63, or a variant thereof, or a variable region thereof, and / or (d) a light chain immunoglobulin comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 16, 28, and 67, or a variable region thereof, and a CD28 binding arm comprising an EGFR binding arm.

[0014] In certain embodiments of the invention, a multispecific antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds EGFR and CD28 comprises: (1) an EGFR-binding antigen comprising: (a) a heavy chain immunoglobulin, or a variable region thereof, comprising an HCDR1, HCDR2, and HCDR3 of a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:2, 30, 40, or 50, and an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO:2, 30, 40, or 50, respectively; and / or a light chain immunoglobulin, or a variable region thereof, comprising an LCDR1, LCDR2, and LCDR3 of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:16, and an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:16, respectively. or (2)(c) a heavy chain immunoglobulin comprising an HCDR1, HCDR2 and HCDR3 of a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 10, 59 or 63, or a variable region thereof, having an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, 59 or 63, respectively; and / or (d) a light chain immunoglobulin comprising an LCDR1, LCDR2 and LCDR3 of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 16 or 67, or a variable region thereof, having an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 or 67, respectively; and a CD28-binding arm comprising an EGFR-binding arm.

[0015] In one embodiment of the invention, the multispecific (e.g., bispecific) antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to EGFR and CD28 comprises: (1) CDR-H1 comprising the amino acid sequence: GDSIITFY (SEQ ID NO: 4, or a variant thereof), CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof), and CDR-H3 comprising the amino acid sequence: ARVSEDSYFHYGMDV (SEQ ID NO: 8, or a variant thereof), and an EGFR binding arm comprising a CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof), a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof), CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof), and CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof), and CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); a CD28 binding arm comprising a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); (2) CDR-H1 comprising the amino acid sequence: GFTFSTFI (SEQ ID NO: 32, or a variant thereof), CDR-H2 comprising the amino acid sequence: ISSNGGTI (SEQ ID NO: 34, or a variant thereof), and CDR-H3 comprising the amino acid sequence: TRGGDFWSGYYPFDY (SEQ ID NO: 36, or a variant thereof), CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); an EGFR binding arm comprising a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); and CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); and a CD28 binding arm comprising a CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof), a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); (3) an EGFR binding arm comprising a CDR-H1 comprising the amino acid sequence: GFSFRDAW (SEQ ID NO: 42, or a variant thereof), a CDR-H2 comprising the amino acid sequence: IRNKIDGGTT (SEQ ID NO: 44, or a variant thereof), and a CDR-H3 comprising the amino acid sequence: TTDIWNYVLFYYYGLDV (SEQ ID NO: 46, or a variant thereof), and a CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof), a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); a CD28 binding arm comprising a CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof), a CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof), and a CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof), and a CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof), a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); (4) an EGFR binding arm comprising a CDR-H1 comprising the amino acid sequence: DDSIISYY (SEQ ID NO: 52, or a variant thereof), a CDR-H2 comprising the amino acid sequence: IYYSGRT (SEQ ID NO: 54, or a variant thereof), and a CDR-H3 comprising the amino acid sequence: ARVSEDSYYHYGMDV (SEQ ID NO: 56, or a variant thereof), and a CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof), a CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof), and a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); A CD28 binding arm comprising: CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); and CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); and CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof).

[0016] In one embodiment of the invention, the multispecific (e.g., bispecific) antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to EGFR and CD28 comprises: (1) an EGFR binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:16 (or a variant thereof), and a CD28 binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:16 (or a variant thereof); (2) an EGFR-binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); and a CD28 binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (3) an EGFR-binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); and a CD28 binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (4) an EGFR-binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); and A CD28-binding arm comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof) and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof).

[0017] In one embodiment of the invention, the multispecific (e.g., bispecific) antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to EGFR and CD28 comprises: (1) an EGFR binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), and a CD28 binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); (2) an EGFR-binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); and a CD28 binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); (3) an EGFR binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And a CD28 binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); (4) an EGFR binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And A CD28-binding arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof) and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof).

[0018] In some embodiments of the present invention, the multispecific (e.g., bispecific) antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) is REGN7075 (also referred to herein as bsAb7075), REGN6321 (also referred to herein as bsAb6321), REGN6322 (also referred to herein as bsAb6322), REGN6323 (also referred to herein as bsAb6323). Other bispecific antibodies may be generated using the parent EGFR antibodies described herein in Tables 9A, 9B and 9C, whereby the sequence of the HCVR arm of the parent EGFR antibody may be the sequence found in WO2014 / 004427, and the HCVR amino acid sequence of the parent CD28 antibody may be the sequence found in Table 3 herein. Any EGFR HCVR arm may be paired with any of the CD28 HCVR arms described herein.

[0019] The present invention also provides an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that specifically binds to an epidermal growth factor receptor, comprising: (i) a heavy chain immunoglobulin or a variable region thereof comprising HCDR1, HCDR2, and HCDR3 of the heavy chain immunoglobulin or variable region thereof comprising the amino acid sequence set forth in SEQ ID NO: 2, 24, 30, 38, 40, 48, 50 or 58 or a variant thereof, and / or (ii) a light chain immunoglobulin or a variable region thereof comprising LCDR1, LCDR2, and LCDR3 of the light chain immunoglobulin or variable region thereof comprising the amino acid sequence set forth in SEQ ID NO: 16 or 28 or a variant thereof. In certain embodiments of the invention, the antigen binding protein is multispecific (e.g., bispecific).

[0020] The present invention also provides a multispecific antigen binding protein as described herein that is bound to an EGFR polypeptide, or an antigenic fragment thereof (e.g., on the surface of a tumor cell), and a CD28 polypeptide, or an antigenic fragment thereof (e.g., on the surface of an immune cell such as a T cell).

[0021] The invention also provides a method of making a multispecific (e.g. bispecific) antigen binding protein as described herein comprising: (b) introducing into a host cell (e.g. a CHO cell) one or more polynucleotides encoding the immunoglobulin chains of the antigen binding protein, (c) optionally isolating the antigen binding protein or immunoglobulin chains from the host cell and / or from the medium in which the host cell is grown. Any antigen binding protein or immunoglobulin chain that is the product of the method is part of the invention.

[0022] The present invention also provides polynucleotides encoding one or more of the immunoglobulin chains of the multispecific (e.g. bispecific) antigen binding proteins of the invention. Vectors comprising the polynucleotides of the invention are also part of the invention, as are host cells (e.g. CHO cells) comprising the polynucleotides, vectors or antigen binding proteins of the invention.

[0023] The invention also provides compositions or kits comprising one or more of the multispecific antigen binding proteins of the invention in association with one or more other substances or products, and optionally in association with a further therapeutic agent (e.g. a PD-1 inhibitor, an anti-PD-1 antibody or antigen binding fragment thereof, a PD-L1 inhibitor, an anti-PD-L1 antibody or antigen binding fragment thereof, the platinum-based anti-cancer chemotherapeutic agents paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, oxaliplatin, the anti-cancer antibodies or antigen binding fragments thereof pembrolizumab, nivolumab, trastuzumab, cetuximab, bevacizumab, and / or cemiplimab). Pharmaceutical formulations comprising the multispecific antigen binding proteins of the invention and a pharma- ceutically acceptable carrier or excipient, and optionally a further therapeutic agent, are also part of the invention.

[0024] The invention also provides containers and injection devices (e.g., syringes, pre-filled syringes, or autoinjectors) comprising the multispecific antigen binding proteins, compositions and / or formulations of the invention.

[0025] The invention also provides a method for administering a multispecific antigen binding protein of the invention, a composition or formulation thereof to a subject (e.g., a human), the method comprising injecting the antigen binding protein, composition or formulation into the body of the subject, such as with a syringe, a pre-filled syringe or an autoinjector. Optionally, a further therapeutic agent (e.g., cemiplimab, pembrolizumab, or nivolumab) is also administered to the subject.

[0026] The invention also provides a method for treating or preventing a hyperproliferative disease (e.g., an EGFR-expressing cancer) in a subject (e.g., a human) in need thereof, the method comprising administering (e.g., subcutaneously, intravenously or intramuscularly) an effective amount of a multispecific antigen binding protein or composition or formulation, optionally in association with a further therapeutic agent (e.g., cemiplimab, pembrolizumab, or nivolumab). In certain embodiments of the invention, the EGFR-expressing cancer is esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain tumor, glioblastoma multiforme, breast cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer or ovarian cancer. [Brief description of the drawings]

[0027] [Figure 1A] Figures 1A-1E show that in cancer cell lines with endogenous TSA, EGFRxCD28 bispecific antibody (REGN7075) enhances T cell activation in the presence of TCR stimulation with TSAxCD3 (MUC16xCD3). In Figures 1A and 1B, flow cytometry analysis shows that EGFRxCD28 bispecific antibody binds to CD28+ cells and EGFR+ cells. (Figure 1A) Jurkat cells (CD28+ cells). [Figure 1B] (Figure 1B) PEO1 cells (EGFR+ cells). [Figure 1C] In Figures 1C-1E, human T cells were cultured for 96 hours with cancer target cells (ovarian cancer cell line PEO1) expressing endogenous MUC16 and EGFR, and the indicated bispecifics. (Figure 1C) Tumor cell killing, % of viable cells. [Figure 1D] (FIG. 1D) Frequency of CD25+ T cells (% of CD2). [Figure 1E] (FIG. 1E) Supernatants from the cytotoxicity assay were analyzed using a Cytometric Bead Array (CBA) kit. Released IFN is plotted as pg / ml. [Figure 2A] Figures 2A-2D show that expression of CD28 ligand (CD86) on tumor cells synergizes with anti-PD1 treatment to induce CD8-dependent anti-tumor immunity. MC38 tumor cells were transduced with CD86, a ligand for CD28 (MC38 / CD86), or an empty vector control (MC38 / EV). WT C57BL6 mice were initially implanted with 1x106 tumor cells per mouse and treated with PD-1 mAb or rIgG2a isotype control at 5 mg / kg on days 0, 3, 7, 10, and 14 after tumor implantation. Figure 2A. Mean tumor volume over time. Error bars represent ± SEM. Statistical significance was determined by two-way ANOVA and Tukey's multiple comparison test. [Figure 2B] Figure 2B. Survival over time (percentage of mice bearing tumors <2000 mm3). Statistical significance was determined by log-rank (Mantel-Cox) test at 60 days post-implantation. [Figure 2C] FIG 2C. Mice were treated with CD8-depleting antibody (CD8 is depleted) or isotype control (no depletion). Mean tumor volume over time is shown with CD8 depletion (dotted line) and without depletion (solid line). ± SEM. Statistical significance was determined by two-way ANOVA and Tukey's multiple comparison test. [Figure 2D]Figure 2D. Second tumor implantation (rechallenge) of tumor-free mice engrafted with MC38 / CD86 and treated with PD1 mAb. For Figures 2A-2D, data from one experiment with 10 mice per group are shown. Data are representative of at least four separate experiments. Statistical significance is indicated (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). [Figure 3A] Figures 3A and 3B show the A431 human xenograft tumor model. Data corresponds to Figures 4 and 5. Figure 3A shows the engraftment of human CD45+ cells in peripheral blood before treatment. [Figure 3B] Figure 3B shows FACS analysis of A431. Markers of interest are indicated for each plot. [Figure 4]Figures 4 and 5 show that EGFRxCD28 (REGN7075) synergized with anti-PD1 (cemiplimab) treatment to induce antitumor immunity in VH mice engrafted with human fetal liver CD34+ cells. Specifically, in Figures 4 and 5, A431 epidermoid carcinoma tumor cells (obtained from ATCC) were subcutaneously implanted into SIRPAh / h TPOh / m Rag2- / - Il2rg- / - mice engrafted with fetal liver CD34+ cells. Mice were divided into treatment groups designated based on fetal liver donor, human immune cell engraftment frequency, and sex. Mice were treated intraperitoneally (IP) and dosed every 2-3 days for the duration of the experiment starting on the day of tumor challenge. Anti-PD-1 was dosed at 10 mg / kg and EGFRxCD28 or PSMAxCD28 at 5 mg / kg. Data show the mean A431 tumor volume (mm3 ± SEM) for each treatment group plotted against days post-challenge. Two-way ANONA, Tukey comparison: **P<0.01, ****P<0.001. (n=10-12 mice per group). Figure 4 shows the mean A431 tumor volume (mm3 ± SEM) for each treatment group (isotype, anti-PD1 monotherapy (cemiplimab), EGFRxCD28 (REGN7075) monotherapy, or EGFRxCD28 (REGN7075) + anti-PD1 combination therapy) plotted against days post-tumor challenge in VH mice engrafted with human CD34+ cells. Two-way ANONA, Tukey comparison: **P<0.01, ****P<0.0001. [Diagram 5] Figure 5 shows the mean A431 tumor volumes (mm3 ± SEM) of control groups (isotype, anti-PD1 monotherapy (cemiplimab), PSMAxCD28 monotherapy, or PSMAxCD28 + anti-PD1 combination therapy) plotted against days after tumor challenge in VH mice engrafted with human CD34+ cells. Two-way ANONA, Tukey's comparison: **P<0.01, ****P<0.0001. [Figure 6] Figure 6 shows validation of the lack of PSMA expression on A431 tumor cells. The MC38 mouse tumor cell line was engineered to express human PSMA and was used as a positive control. [Figure 7A]Figures 7A and 7B. Mean A431 tumor volume (mm3 ± SEM) for each treatment group administered EGFRxCD28 (REGN7075) plotted against days after tumor challenge in NSG mice receiving either prophylactic (Figure 7A) or therapeutic (Figure 7B) treatment. Two-way ANONA, Tukey's comparison: ****P<0.0001. [Figure 7B] Same as above. [Figure 8] Figure 8. Mean A549 tumor volume (mm3 ± SEM) for each treatment group (1 mg / kg or 10 mg / kg isotype or EGFRxCD28 (REGN7075)) plotted against days after tumor challenge in NSG mice. Two-way ANONA, Tukey comparison: *Isotype vs EGFRxCD28, 1 mg / kg (P<0.05), ****: P<0.0001; $Isotype vs EGFRxCD28, 10 mg / kg (P<0.05), $$$: P<0.001, $$$$: P<0.0001; @ EGFRxCD28 1 mg / kg vs 10 mg / kg (P<0.05). [Figure 9A] Figures 9A-9D. EGFRxCD28 (REGN7075) synergized with anti-PD1 (cemiplimab) treatment to induce anti-tumor immunity in VH mice engrafted with human fetal liver CD34+ cells. (Figure 9A) MFI of select tumor CD8+ T cell clusters identified by CITRUS (cluster identification, characterization and regression) analysis, (Figure 9B) cell frequency in each cluster from the indicated treatment group, (Figure 9C) MFI of select tumor CD4+ T cell clusters identified by CITRUS analysis, (Figure 9D) cell frequency in each cluster from the indicated treatment group. Figures 9B and 9D demonstrate that EGFRxCD28 synergized with anti-PD1 treatment to induce anti-tumor immunity in VH mice engrafted with human fetal liver CD34+ cells. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A]Figures 10A-10C. EGFRxCD28 (REGN7075) alone or in combination with anti-PD1 (cemiplimab) therapy does not induce systemic T cell activation compared to CD28 superagonist in cynomolgus monkeys. Cynomolgus monkeys were treated with a single dose of bispecific antibody at the indicated doses (mg / kg). Time indicates hours post-dose, serum cytokines (IFN gamma, IL-2, IL-6, IL-8, and IL-10) (Figure 10A), relative peripheral blood T cell counts (Figure 10B), and frequency of Ki67+ and ICOS+ T cells (% of CD3) (Figure 10C). Values ​​represent mean ± SEM. N=3 animals per group. P values ​​were calculated by two-way ANOVA in comparison with isotype control (**, p<0.01; ***, p<0.001, and ****, p<0.0001). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11] FIG. 11 shows the dose-dependent anti-tumor response over time mediated by an anti-EGFRX anti-CD28 antibody (REGN7075) when combined with a PD-1 antibody (REGN2810, described as H2M7798N or H4H7798N in US Patent Publication No. 20150203579). [Figure 12] FIG. 12 shows the dose-dependent anti-tumor response mediated by an anti-EGFRX anti-CD28 antibody (REGN7075) when combined with a PD-1 antibody (REGN2810). [Figure 13A] Figures 13A-13C describe the binding of various anti-EGFRx anti-CD28 antibodies to various cells including human and cynomolgus T cells, as well as Jurkat cells (CD28+ effector cells) (Figure 13A), and to various other tumor target cells including A375 melanoma cells, 22RV1 prostate cancer cells, PEO1 ovarian cells, CAPAN pancreatic cells, SW1990 pancreatic cells, and H292 lung cells (Figures 13B and 13C). Isotype controls I and II are antibodies that bind to Mers and EGFRvIII, respectively. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14A] Figures 14A and 14B show that anti-EGFRx anti-CD28 bispecific antibodies enhance the cytotoxicity of anti-tumor specific antigen (TSA)x anti-CD3 bispecific antibodies. The study included anti-EGFRx anti-CD28 antibodies combined with either anti-STEAP2x anti-CD3 bispecific antibodies (Figure 14A) or anti-PSMAX anti-CD3 bispecific antibodies (Figure 14B). Anti-STEAP2x anti-CD3 antibodies are described in WO2018 / 058001A. Anti-PSMAx anti-CD3 antibodies are described in WO2017 / 053856A1. [Figure 14B] Same as above. [Figure 15A] Figures 15A, 15B, 15C and 15D show that anti-EGFRX anti-CD28 bispecific antibody enhances the cytotoxicity of anti-tumor specific antigen (TSA)X anti-CD3 bispecific antibody (anti-MUC16X anti-CD3) across a variety of cell lines including PEO1 cells (Figure 15A), CAPAN2 cells (Figure 15B), SW1990 cells (Figure 15C), and H292 cells (Figure 15D). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Before describing the invention, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terms used in this specification are used for the purpose of describing specific embodiments only, and are not intended to be limiting, and the scope of the invention is limited only by the appended claims.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in connection with a specific recited numerical value means that the value can vary from the recited value by up to 1%.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0030] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described herein. All patents, patent applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0031] Checkpoint inhibition using PD-1 blocking mAbs is known to release a break in T cell activation, but its efficacy as a single agent has often been insufficient to consistently obtain tumor clearance or durable antitumor responses in many cancers. Several approaches to improve response rates to PD-1 inhibition are currently under evaluation. Indeed, identification of biomarkers predictive of responsiveness to PD-1 mAbs, agonistic antibodies that induce costimulatory receptors to improve T cell activation, or non-tumor targeted combination therapies using PD-1 inhibition along with chemotherapy or radiotherapy are all currently in preclinical and clinical trials. The challenge, however, is that many of these combinations are often based on the availability of existing agents and a posteriori rationale for combining therapies rather than a true hypothesis-driven approach, resulting in worse outcomes for patients in some cases. It is clear that checkpoint inhibition and reactivation of the immune system offers the potential for long-term remission in many patients, and methods to further improve or enhance T cell activity to promote even longer-lasting responses are warranted. Therefore, the concept of using EGFRxCD28 bispecific antibodies to enhance T cell signaling and activation was introduced to improve the antitumor effect of PD-1 mAb. Indeed, the novel combination immunotherapy of the present invention shows that CD28 costimulatory bispecific antibodies exert synergistic effects with PD-1 mAb to generate stable T cell activation as well as long-term antitumor responses without systemic toxicity. As a result, the tumor-targeted combination therapy of the present invention offers significant advantages over previously reported non-targeted methods. The use of CD28 bispecific antibodies offers the possibility of promoting costimulation only at the tumor site, avoiding the systemic toxicity of conventional CD28-activating antibodies (toxicity often observed with the combination of CLTA-4 and PD-1 blocking or other costimulatory agonist bivalent antibodies).Toxicity studies in genetically humanized immunocompetent mice and cynomolgus monkeys demonstrated that these bispecifics showed no toxicity as single agents or in combination with PD-1 mAb. Similar antitumor enhancement by EGFRxCD28 and anti-PD-1 mAb across syngeneic and xenogeneic models, along with a safety profile, suggested that this therapeutic modality is robust and not limited to tumor model specificity, and may have broad utility as a novel immunotherapy combination.

[0032] Tumor targeting methods have been developed to enhance T cell-mediated killing of tumor cells. Indeed, CD3-based bispecific antibodies have become emerging antibodies that can efficiently induce T cell activation by linking T cells to tumor cells and activating TCR / CD3, thereby mimicking normal "signal 1". However, despite promising clinical efficacy, CD3-bispecifics may be associated with cytokine release syndrome (CRS) due to the lack of tumor-only specificity in inducing T cell activation. Moreover, TCR / CD3 activation in the absence of costimulation (signal 2) may result in anergy or activation-induced cell death (AICD), which may limit or reduce the potential antitumor efficacy of these reagents. Herein, we first demonstrate that combination therapy of EGFRxCD28 bispecifics and anti-PD-1 mAb induced tumor-specific T cell activation. As shown herein, EGFRxCD28 bispecific antibodies have limited or no activity in the absence of "signal 1" and PD-1 blockade is dependent on endogenous antigen-specific T cell responses to tumor peptides. Thus, CD28-bispecifics, together with PD-1 blockade, can boost endogenous TCR / CD3-dependent T cell responses that induce long-term anti-tumor responses.

[0033] The antitumor activity of PD-1 mAbs is CD28-dependent, and PD-1 inhibition of T cell activation may decrease signaling through TCR / CD3 and / or CD28, affecting the spatial localization of these molecules. The data herein demonstrate that PD-1 accumulates at the immune synapse when PD-L1 is expressed by target cells, and this accumulation is associated with a decrease in CD28 at the synapse, suggesting that PD-1 may mediate T cell inhibition by disrupting the localization of CD28 at the synapse. Furthermore, we found that PD-1 blockade disrupted the synaptic localization of PD-1, which was accompanied by increased accumulation of CD28 at the synapse. This allows the EGFRxCD28 bispecific to significantly enhance the ability of PD-1 mAbs to promote T cell activation. This mechanism may be one of the mechanisms by which PD-1 blocking antibodies promote T cell activation. Overall, visualization of PD-1-PD-L1 interactions and / or PD-1 and CD28 localization at the immune synapse following PD-1 inhibition may advance our understanding of the effect of PD-1 blockade on T cell activation as well as the synergistic effect of EGFRxCD28 and PD-1 mAbs at the immune synapse level.

[0034] PD-1 mAbs are an important novel immunotherapy, but many cases will certainly require further optimization of their antitumor activity. Just as CAR-T approaches have employed chimeric receptors that artificially activate both "signal 1" and "signal 2" to improve antitumor activity, we show herein the potential benefit of combining PD-1 blockade with CD28-bispecifics (providing "signal 2") to enhance antitumor activity. This approach offers several practical benefits compared to CAR-T therapy in that it does not require laborious cell therapy preparations that must be individually customized for each patient, nor does it require preemptive "lymphodepletion" of patients with toxic chemotherapy (which is often associated with adverse effects and therefore renders patients unable to tolerate cell therapy). The bispecific approach offers the potential for increased efficacy as well as increased safety through specificity of action. Taken together, the data herein demonstrate that the combination of a CD28-based bispecific with a clinically proven PD-1 mAb, such as cemiplimab, may provide a well-tolerated biopharmaceutical solution with significantly enhanced and synergistic anti-tumor activity.

[0035] definition "EGFR" and "EGFR fragment" as used herein refer to known human EGFR proteins or fragments thereof, unless specified to be derived from a non-human species (e.g., "mouse EGFR", "mouse EGFR fragment", "monkey EGFR", "monkey EGFR fragment", etc.). In certain embodiments of the invention, the human EGFR comprises the amino acid sequence set forth in NCBI Accession No. NP_005219.2. In one embodiment, the human EGFR (amino acids L25-A647 of Accession No. 005228.4) is depicted with a C-terminal CPGG.myc epitope (E1-L10).GlyGly.myc epitope (E1-L10).SerGly.6XHis.SSG tag (SEQ ID NO: 69).

[0036] As used herein, "CD28" refers to the known human CD28 protein expressed on T cells as a costimulatory receptor, unless specified to be from a non-human species. In an embodiment of the present invention, human CD28 comprises the amino acid sequence set forth in NCBI accession number NP_006130.1.

[0037] "Isolated" antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides and vectors are at least partially free from other biological molecules derived from the cell or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments thereof, lipids, carbohydrates, or other substances, such as, for example, cell debris or growth medium. Isolated antigen binding proteins may further be at least partially free from components of the expression system, such as, for example, biological molecules derived from the host cell and its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers or salts, or the absence of components of a pharmaceutical formulation that includes the antigen binding protein (e.g., an antibody or antigen-binding fragment thereof).

[0038] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST Algorithm: Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul, SF, et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, JC, et al. al.,(1993) Comput. Chem. 17:149-163; Hancock, JMet al.,(1994) Comput. Appl. Biosci. 10:67-70; Alignment scoring systems: Dayhoff, MO,et al.,“A model of evolutionary change in proteins”in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.M Dayhoff(ed.),pp.345-352, Natl. Biomed. Res. Found.,Washington,DC; Schwartz, RM,et al.,“Matrices for detecting distant relationships”in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.''MODayhoff(ed.),pp.353-358,Natl.Biomed.Res.Found.,Washington,DC;Altschul,SF,(1991)J.Mol.Biol.219:555-565;States,DJ,et al.,(1991)Methods 3:66-70;Henikoff,S.,et al.,(1992)Proc.Natl.Acad.Sci.USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; Alignment statistics: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, NY. .

[0039] The present invention includes polypeptides comprising the amino acid sequence set forth in SEQ ID NOs: 6, 8, 10, 12, 14, 24, 34, 36, 38, 44, 46, 48, 54, 56 and / or 58.

[0040] An "antibody" is an immunoglobulin molecule that comprises four polypeptide chains, two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain (HC) contains a heavy chain variable region (HCVR or V H The heavy chain constant region is abbreviated as C H 1. C H 2, and C H Each light chain (LC) contains three domains: a light chain variable region (herein LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V LThe regions can be further divided into more conserved regions termed framework regions (FR) and intervening regions of hypervariability termed complementarity determining regions (CDRs). H and V L Each of the heavy chain CDRs contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain CDRs may also be referred to as HCDRs and the light chain CDRs may also be referred to as LCDRs. In different embodiments of the invention, the FRs of an antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified.

[0041] The antigen-binding arm of a Y-shaped IgG antibody (e.g., a CD28-binding arm or an EGFR-binding arm) refers to the structural portion of the antibody that confers binding specificity to an antigen. For example, the antigen-binding arm of an IgG antibody has a heavy chain (HC) associated with a light chain (LC).

[0042] For example, an antibody that is bispecific contains an arm that binds to a first antigen and another arm that binds to a second antigen, for example, an EGFRxCD28 bispecific antibody contains one arm that binds to EGFR and another arm that binds to CD28.

[0043] A bispecific antigen-binding molecule (e.g., a bispecific antibody) may have an effector arm that binds to a first antigen and a targeting arm that binds to a second antigen. The effector arm may be a first antigen-binding domain (e.g., anti-CD28) that binds to an antigen on an effector cell (e.g., a T cell). The targeting arm may be a second antigen-binding domain (e.g., an anti-EGFR antibody) that binds to an antigen on a target cell (e.g., a tumor cell). According to an exemplary embodiment, the effector arm binds to CD28 and the targeting arm binds to EGFR.

[0044] An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, etc., as used herein, includes any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. A multispecific antigen-binding fragment of an antibody binds to multiple antigens (e.g., binds to two different antigens if the fragment is bispecific). Antigen-binding fragments of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, and (vi) dAb fragments.

[0045] Antigen-binding fragments of antibodies, in certain embodiments of the invention, comprise at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, H Domain and V L The domains may be arranged relative to one another in any suitable configuration. For example, the variable region may be a dimer, and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H Domain or V L It may contain domains.

[0046] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting examples of variable and constant domain configurations that may be present in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the above configuration examples, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may be a region of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provides a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Additionally, antigen-binding fragments of antibodies of the invention may be non-covalently linked to each other (e.g., by disulfide bonds) and / or linked to one or more monomeric V domains. H Domain or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated.

[0047] The term "recombinant" antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, refers to a molecule that is produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, e.g., DNA splicing and transgenic expression. The term includes antibodies expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or a host cell (e.g., a Chinese Hamster Ovary (CHO) cell), or a cellular expression system, or antibodies isolated from a recombinant combinatorial human antibody library. The present invention includes recombinant antigen binding proteins as described herein.

[0048] The terms "specifically bind" or "specifically bind" refer to, for example, an antigen, such as an EGFR or CD28 protein, that binds to a specific antibody with a specific affinity of about 10, as measured, for example, at 25° C. or 37° C., by a real-time label-free biolayer interferometry assay, such as an Octet® HTX biosensor, or by surface plasmon resonance, such as a BIACORE® biosensor, or by a solution-affinity ELISA.-6 M (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M) less than K D "Anti-EGFR" refers to an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that specifically binds to EGFR. "Anti-CD28" refers to an antigen binding protein (or other molecule, e.g., an antigen-binding arm) such as an antibody or antigen-binding fragment thereof that specifically binds to CD28. "EGFRxCD28" refers to an antigen binding protein (or other molecule, e.g., an antibody or antigen-binding fragment thereof) that specifically binds to EGFR and CD28 (and optionally one or more other antigens).

[0049] The present invention includes antigen binding proteins, such as antibodies or antigen-binding fragments thereof, that bind to the same EGFR epitope and CD28 epitope as the antigen binding proteins of the invention (e.g., REGN7075 (also referred to herein as bsAb7075); REGN6321 (also referred to herein as bsAb6321); REGN6322 (also referred to herein as bsAb6322); REGN6323 (also referred to herein as bsAb6323). Other anti-EGFRX anti-CD28 antigen binding proteins can be found in Tables 9A, 9B and 9C. The amino acid sequences of the EGFR HCVR arms of the bispecific antibodies described herein are found in Table 1, and the CD28 epitopes of the bispecific antibodies described herein are also shown in Table 2. The amino acid sequences of the HCVR arms can be found in Table 3. Other EGFR parent antibodies for use in the present invention are described in WO2014 / 004427. The amino acid sequences of the EGFR HCVR arms and the CD28 HCVR arms of REGN7075, REGN6321, REGN6322, and REGN6323 are found in Table 6. The amino acid sequences of the common light chain variable regions described in the present invention are also found in Table 6.

[0050] The term "epitope" refers to an antigenic determinant (e.g., on EGFR or CD28) that interacts with a specific antigen-binding site of an antigen-binding protein, such as the variable region of an antibody molecule known as the paratope. An antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" may refer to a site on an antigen to which B cells and / or T cells respond and / or to a region of an antigen that is bound by an antibody. Epitopes may be structurally or functionally defined. Functional epitopes are generally a subset of structural epitopes, and have residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational; that is, composed of amino acids in a nonlinear fashion. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, epitopes may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0051] Methods for determining the epitope of an antigen-binding protein, such as an antibody or fragment or polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens may be employed (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids in a polypeptide that interact with an antigen-binding protein (such as an antibody or fragment or polypeptide) is hydrogen / deuterium exchange detected by mass spectrometry. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0052] The present invention includes antigen binding proteins that compete with the antigen binding proteins of the invention (e.g., REGN7075 (also referred to herein as bsAb7075); REGN6321 (also referred to herein as bsAb6321); REGN6322 (also referred to herein as bsAb6322); REGN6323 (also referred to herein as bsAb6323) and those set forth in Tables 9A, 9B and 9C) for binding to CD28 and EGFR. As used herein, the term "competes" refers to an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to an antigen and inhibits or prevents another antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) from binding to the antigen. Unless otherwise specified, the term also refers to bidirectional binding between two antigen binding proteins, e.g., antibodies. Competition is also included, i.e., a first antibody binds to an antigen and prevents binding by a second antibody, or vice versa. Thus, in certain embodiments of the invention, competition occurs in one of these directions. In certain embodiments, a first antigen binding protein (e.g., an antibody) and a second antigen binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, a first and a second antigen binding protein (e.g., an antibody) may bind to different, but overlapping or non-overlapping epitopes, in which case the binding of one inhibits or prevents the binding of the second antibody, e.g., via steric hindrance. Competition between antigen binding proteins (e.g., antibodies) can be measured by methods known in the art, e.g., real-time label-free biolayer interferometry assays. Alternatively, binding competition between antigen binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time label-free biolayer interferometry assays on an Octet RED384 biosensor (Pall ForteBio Corp.).

[0053] Typically, the antibodies or antigen-binding fragments of the invention, modified in some way, retain the ability to specifically bind to EGFR and CD28. For example, they retain at least 10% of the EGFR and CD28 binding activity (compared to the parent antibody) when activity is expressed on a molar basis. Preferably, the antibodies or antigen-binding fragments of the invention retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the EGFR and CD28 binding affinity of the parent antibody. It is also contemplated that the antibodies or antigen-binding fragments of the invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of the antibody) that do not substantially alter its biological activity.

[0054] For example, immunoglobulin chains (e.g., those comprising the amino acid sequences specifically described herein, such as the V of REGN7075 (also referred to herein as bsAb7075); REGN6321 (also referred to herein as bsAb6321); REGN6322 (also referred to herein as bsAb6322); REGN6323 (also referred to herein as bsAb6323) H , V LA "variant" of a polypeptide, such as a polypeptide sequence (e.g., HC or LC, or CDRs thereof) is a variant of a polypeptide that is a nucleotide sequence selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58-67) when compared to a reference amino acid sequence described herein (e.g., any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58-67) using the BLAST algorithm, with the parameters of the algorithm selected to give the largest match between the respective sequences over the entire length of each reference sequence. " refers to a polypeptide that contains an amino acid sequence that is at least about 70 to 99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) identical or similar to the "prediction threshold: 10, word size: 3, max matches in query scope: 0, BLOSUM62 matrix; gap cost: existence 11, extension 1, conditional composition score matrix adjusted)"

[0055] Additionally, a variant of a polypeptide can include an amino acid sequence of a reference polypeptide whose amino acid sequence is specifically described herein, but which is not described with respect to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as one or more missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions, in the V region of an immunoglobulin chain (e.g., REGN7075 (also referred to herein as bsAb7075); REGN6321 (also referred to herein as bsAb6321); REGN6322 (also referred to herein as bsAb6322); REGN6323 (also referred to herein as bsAb6323). H , V L For example, the present invention relates to an immunoglobulin light chain (or VHC or LC, or CDRs thereof) of an EGFR binding arm that comprises an amino acid sequence set forth in SEQ ID NO: 16, but that has one or more such mutations. L) variants, and / or immunoglobulin heavy chains (or V) comprising the amino acid sequence set forth in SEQ ID NO:2 but having one or more such mutations. H In one embodiment of the invention, the CD28xEGFR antigen binding protein comprises an immunoglobulin light chain variant comprising LCDR1, LCDR2 and LCDR3, in which one or more of the CDRs (e.g. one, or two or three) have one or more of such mutations (e.g. conservative substitutions), and / or an immunoglobulin heavy chain variant comprising HCDR1, HCDR2 and HCDR3, in which one or more of the CDRs (e.g. one, or two or three) have one or more of such mutations (e.g. conservative substitutions).

[0056] For example, "conservatively modified variants" or "conservative substitutions" of immunoglobulin chains described herein refer to variants in which there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure and rigidity, etc.). Such changes can often occur without significantly impairing the biological activity of the antibody or fragment. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to significantly impair biological activity. The present invention includes EGFRxCD28 and anti-EGFR antigen binding proteins and / or binding arms that include such conservatively modified variant immunoglobulin chains.

[0057] Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45.

[0058] antigen binding molecule The antibodies of the present invention may be monospecific, bispecific or multispecific. A multispecific antibody may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD28 antibodies of the present invention may be linked or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, or non-covalent or otherwise) to one or more other molecular entities, such as, for example, another antibody or antibody fragment, to generate a bispecific antibody having a second binding specificity, or a multispecific antibody.

[0059] The use of the phrase "anti-CD28 antibody" herein is intended to include both monospecific anti-CD28 antibodies as well as multispecific (e.g., bispecific) antibodies that comprise a CD28-binding arm and a second arm that binds to a target antigen. Thus, the present invention includes bispecific antibodies in which one arm of an immunoglobulin binds human CD28 and the other arm of the immunoglobulin is specific for a target antigen. The target antigen to which the other arm of the CD28 bispecific antibody binds can be any antigen expressed on or near a cell, tissue, organ, microorganism, or virus against which a targeted immune response is desired. The CD28-binding arm can comprise any of the amino acid sequences of HCVR / LCVR or CDRs described in Tables 3 and 8 herein. In certain embodiments, the CD28-binding arm binds to human CD28 and induces proliferation of human T cells.

[0060] In the case of a bispecific antibody of the invention, in which one arm of the antibody binds to CD28 and the other arm binds to a target antigen, the target antigen can be, for example, a tumor-associated antigen such as EGFR.

[0061] According to certain exemplary embodiments, the present invention comprises bispecific antigen binding molecules that specifically bind to CD28 and EGFR. Such molecules may be referred to herein as, for example, "anti-CD28 / anti-EGFR," or "anti-CD28xEGFR," or "CD28xEGFR," or "EGFRXCD28," or "anti-EGFR / anti-CD28," or "anti-EGFRxCD28," or "EGFRxCD28" bispecific molecules, or "anti-EGFRX anti-CD28," or "anti-CD28X anti-EGFR," or other similar terms.

[0062] According to an exemplary embodiment, a bispecific antigen-binding molecule (e.g., a bispecific antibody) may have an effector arm and a targeting arm. The effector arm may be a first antigen-binding domain (e.g., an anti-CD28 antibody) that binds to an antigen on an effector cell (e.g., a T cell). The targeting arm may be a second antigen-binding domain (e.g., an anti-EGFR antibody) that binds to an antigen on a target cell (e.g., a tumor cell). According to an exemplary embodiment, the effector arm binds to CD28 and the targeting arm binds to EGFR. The bispecific anti-CD28 / EGFR may send a costimulatory signal to an effector cell (e.g., a T cell).

[0063] As used herein, the term "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex that comprises or consists of at least one complementarity determining region (CDR) that, alone or in combination with one or more additional CDRs and / or framework regions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or an antibody fragment, as those terms are otherwise defined herein.

[0064] As used herein, the term "bispecific antigen-binding molecule" refers to a protein, polypeptide or molecular complex (e.g., an antibody or an antigen-binding fragment thereof) that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the present case, the first antigen-binding domain specifically binds to a first antigen (e.g., CD28) and the second antigen-binding domain specifically binds to a second distinct antigen (e.g., EGFR).

[0065] In certain exemplary embodiments of the invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR).

[0066] The first and second antigen-binding domains may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first and second antigen-binding domains may each be linked to a separate multimerization domain. Linking one multimerization domain to another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any polymer, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain may be linked to the immunoglobulin C H A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (C H 2-C H The Fc domain of an IgG antibody may comprise any of three domains, for example the Fc domain of an IgG antibody selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0067] A bispecific antigen-binding molecule of the present invention typically comprises two multimerization domains, e.g., two Fc domains, each of which is individually a separate antibody heavy chain portion. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0068] In one embodiment, the multimerization domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In another embodiment, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0069] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the invention. For example, an antibody or antigen-binding fragment thereof having a first antigen-binding specificity can be operatively linked (e.g., by chemical coupling, genetic fusion, or non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to form a bispecific antigen-binding molecule. Examples of specific bispecific formats that can be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (OVO)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEEO) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual acting Fab (OAF)-IgG, and Mab. 2 Bispecific formats are included (see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein for an overview of such formats).

[0070] In the case of bispecific antigen-binding molecules of the invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring Fc domain. For example, the invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain with an altered (e.g., enhanced or weakened) binding interaction between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises H 2nd area or C H The FcRn-binding domain includes modifications in three regions, where the modifications increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., an endosome with a pH in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), modifications at positions 250 and 428 (e.g., L or F), modifications at position 252 (e.g., LN / FIW or T), modifications at position 254 (e.g., S or T), and modifications at position 256 (e.g., S / R / Q / EID or T), or modifications at positions 428 and / or 433 (e.g., UR / S / P / Q or K) and / or 434 (e.g., H / F or V), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254 and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P).

[0071] The present invention also relates to a first C H 3 domain and second Ig C Hand a bispecific antigen-binding molecule comprising three domains, wherein the first and second IgC H The three domains differ from each other by at least one amino acid, and in this case, the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking that amino acid difference. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or eliminates Protein A binding, for example the H95R (according to IMGT exon numbering; H435R in EU numbering) modification. H 3 may further include a Y96F modification (Y436F according to IMGT, in EU). Additional modifications that may be present within the second CH3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I in EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU).

[0072] In certain embodiments, the Fc domain may be chimeric, comprising Fc sequences from multiple immunoglobulin isotypes. For example, the chimeric Fc domain may comprise the Fc sequences from human IgG1, human IgG2, or human IgG4. H C derived from two regions H 2 sequences and C derived from human IgG1, human IgG2, or human IgG4. HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region, combined with a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. Particular examples of chimeric Fc domains that may be contained in any of the antigen-binding molecules described herein include, from the N-terminus to the C-terminus, the following: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 C H Another example of a chimeric Fc domain that may be contained in any of the antigen-binding molecules described herein comprises, from the N-terminus to the C-terminus: [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 C H 2]-[IgG1 C H 3]. These and other examples of chimeric Fc domains that may be contained in any of the antigen-binding molecules of the present invention are described in WO2014 / 022540 A1. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding, which in turn affects Fc effector functions.

[0073] The antibodies and antigen-binding fragments of the invention include immunoglobulin chains comprising the amino acid sequences (and variants thereof) specifically described herein, as well as cellular or in vitro post-translational modifications to such antibodies or fragments. For example, the invention includes antibodies and antigen-binding fragments thereof that specifically bind to EGFR and CD28, comprising the heavy and / or light chain amino acid sequences described herein, as well as antibodies and fragments in which one or more asparagine, serine and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp and / or His) are oxidized, the N-terminal glutamine is pyroglutamate (pyroE), and / or the C-terminal lysine or other amino acid is missing.

[0074] Epidermal growth factor receptor (EGFR) binding molecules and anti-EGFR antigen-binding arms The present invention includes multispecific (eg, bispecific) antigen binding proteins, such as antibodies or antigen-binding fragments, that comprise one or more EGFR-binding arms and one or more CD28-binding arms.

[0075] An EGFR-binding arm is a portion of a multispecific antigen-binding protein that confers EGFR-binding ability to the protein. In one embodiment of the invention, the EGFR-binding arm of bispecific antibody REGN7075, REGN6321, REGN6322, or REGN6323 interferes with EGF binding to EGFR, and in another embodiment of the invention, the EGFR-binding arm does not interfere with EGF binding to EGFR. For example, the EGFR-binding arm of a Y-type IgG antibody refers to the structural portion of the antibody that confers binding specificity to EGFR. For example, in one embodiment of the invention, the EGFR-binding arm is a structural portion of the antibody that specifically binds to EGFR, comprising HCDR1, LCDR1, HCDR2, LCDR2, HCDR3, and LCDR3; HCVR(V H ) and LCVR(V L ) and / or HC and LC.

[0076] In one embodiment of the invention, the EGFR binding arm comprises a combination of heavy chain CDRs (HCDR1, HCDR2 and HCDR3). H and a V containing a combination of light chain CDRs (LCDR1, LCDR2 and LCDR3). L and corresponding light chain immunoglobulins comprising the same or similar amino acids as described herein or in International Patent Application Publication No. WO2014 / 004427.

[0077] "085N", "086N", "089N", "102N", "103N", "116N", "134P", "136P", "141P", "142P", "143P", "144P", "145P", "147P", "151P", "153P", "155P", "157P", "158P", "159P", "161P", "163P", "169P" and "171P" refer to the identification numbers of several anti-EGFR monospecific antibodies described in WO2014 / 004427. The HCVR arms of these antibodies may be used to generate bispecific antibodies that target tumor cells expressing EGFR and T cells expressing CD28, in combination with the HCVR amino acid sequences described in Table 3 and anti-CD28 arms with a common light chain having the amino acid sequence described in Table 6. "REGN7075", "REGN6321", "REGN6322" or "REGN6323" are also referred to as "bsAb7075", "bsAb6321", "bsAb6322" or "bsAb6323", respectively, and each is an immunoglobulin heavy chain variable region (V H ) or full-length heavy chains (or variants thereof), and the corresponding immunoglobulin light chain variable regions (V L or a V domain comprising a full-length light chain (or a variant thereof) and an EGFR-binding arm comprising the CDRs (HCDR1 (or a variant thereof), HCDR2 (or a variant thereof), and HCDR3 (or a variant thereof)). Hand the corresponding V including its CDRs (LCDR1 (or a variant thereof), LCDR2 (or a variant thereof) and LCDR3 (or a variant thereof)). L and wherein the variable region and / or CDRs comprise the specific amino acid sequences described herein and are not variants. H In some cases, the CDR-H may contain variant amino acid sequences that are not variants, and / or such V L In some embodiments of the invention, the VD-L may comprise a variant amino acid sequence, but the CDR-L is not variant. Such an EGFR-binding arm may be referred to in the context of an EGFRxCD28 multispecific antigen-binding protein, e.g., 085Nx14226P2. H is linked to an IgG heavy chain constant domain (e.g., IgG1 or IgG4 (e.g., containing an S228P mutation)), and / or L is linked to a lambda or kappa light chain constant domain.

[0078] The present invention also provides antigen binding proteins and antigen binding fragments thereof, such as, for example, antibodies (e.g., human antibodies, monoclonal antibodies and recombinant antibodies), that specifically bind to the EGFR protein or an antigenic fragment thereof (e.g., the extracellular domain of EGFR). Antigen binding proteins that bind to the same epitope on EGFR as any of the antigen binding proteins described herein, or that compete with them for binding to EGFR, are also part of the present invention.

[0079] Anti-EGFR antibodies and antigen-binding fragments thereof of the present invention include those described herein or in WO2014 / 004427, such as 085N, 086N, 089N, 102N, 103N, 116N, 134P, 136P, 141P, 142P, 143P, 144P, 145P, 147P, 151P, 153P, 155P, 157P, 158P, CDRs (HCDR1, HCDR2, and HCDR3) of the EGFR binding arms (or variants thereof) of 159P, 161P, 163P, 169P, 171P, mAb12999P2, mAb13008P2, mAb35193P2, mAb13006P2, REGN7075, REGN6321, REGN6322, or REGN6323, V H or full length immunoglobulin sequences, and / or 085N, 086N, 089N, 102N, 103N, 116N, 134P, 136P, 141P, 142P, 143P, 144P, 145P, 147P, 151P, 153P, 155P, 157P, 158P, 159 CDRs (LCDR1, LCDR2, and LCDR3) of the EGFR binding arm (or variants thereof) of P, 161P, 163P, 169P, 171P, mAb12999P2, mAb13008P2, mAb35193P2, mAb13006P2, REGN7075, REGN6321, REGN6322, or REGN6323, V L Alternatively, the sequences may include full-length immunoglobulin sequences.

[0080] "REGN7075," "REGN6321," "REGN6322," or "REGN6323" refers to any of the immunoglobulin heavy chain variable regions (V H ) or a full-length heavy chain (or a variant thereof), and the corresponding immunoglobulin light chain variable region (V L ) or a full-length light chain (or a variant thereof), or a V domain including its CDRs (HCDR1 (or a variant thereof), HCDR2 (or a variant thereof), and HCDR3 (or a variant thereof))H and the corresponding V including its CDRs (LCDR1 (or a variant thereof), LCDR2 (or a variant thereof) and LCDR3 (or a variant thereof)). L and the like, e.g., in which the variable regions and / or CDRs comprise the specific amino acid sequences described herein and are not variants. H In some cases, the CDR-H may contain variant amino acid sequences that are not variants, and / or such V L In some embodiments of the invention, the CDR-L may comprise a variant amino acid sequence that is not a variant. H is linked to an IgG heavy chain constant domain (e.g., IgG1 or IgG4), and / or L is linked to a lambda or kappa light chain constant domain.

[0081] The antigen binding proteins REGN7075, REGN6321, REGN6322, and REGN6323 are bispecific anti-EGFR antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) having one HCVR arm that binds to EGFR (and / or one or more different antigens or different EGFR epitopes) and one HCVR arm that binds to CD28 on T cells.

[0082] The amino acid sequences of the immunoglobulin chains of REGN7075, REGN6321, REGN6322, and REGN6323 are set forth below:

[0083] EGFR component of REGN7075 HCVR in the EGFR arm derived from parental EGFR mAb 12999P2): QVQLQESGPGLVKPSETLSLTCTVS GDSIITFY WSWIRQPPGRGLEWIGY IYYSGIT NYNPSLKSRVTISVDTSKNQVSLKLSSVTAADTAVYYC ARVSEDSYFHYGMDV WGQGTTVTVSS (SEQ ID NO: 2, underlined parts are CDRs) CDR-H1: GDSIITFY (SEQ ID NO: 4) CDR-H2: IYYSGIT (SEQ ID NO: 6) CDR-H3: ARVSEDSYFHYGMDV (SEQ ID NO: 8)

[0084] Full-length heavy chain of the EGFR arm of the bispecific antibody REGN7075 QVQLQESGPGLVKPSETLSLTTCTVSGDSIITFYWSWIRQPPGRGLEWIGYIYYSGITNYNPSLKSRVTISVDTSKNQVSLKLSSVTAADTAVYYCARVSEDSYFHYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:24)

[0085] LCVR of EGFR and CD28 parent antibodies and bispecific antibody REGN7075 EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO: 16, underlined CDRs) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0086] Full length light chains for both EGFR and CD28 parent antibodies and bispecifics EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28)

[0087] EGFR component of REGN6321 HCVR in the EGFR arm derived from parental EGFR mAb 13008P2): EVQLVESGGGLVRPGGSLRLSCTAS GFTFSTFI MFWVRQAPGKGLEYVSS ISSNGTI YYADSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYC TRGGDFWSGYYPFDY WGQGTLVTVSS (SEQ ID NO: 30, underlined CDRs) CDR-H1: GFTFSTFI (SEQ ID NO: 32) CDR-H2: ISSNGGTI (SEQ ID NO: 34) CDR-H3: TRGGDFWSGYYPFDY (SEQ ID NO: 36)

[0088] Full-length heavy chain of the EGFR arm of the bispecific antibody REGN6321 EVQLVESGGGLVRPGGSLRLSCTASGTFFSTFIMFWVRQAPGKGLEYVSSISSNGGTIYYADSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCTRGGDFWSGYYPFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:38)

[0089] LCVR of EGFR and CD28 parent antibodies and bispecific antibody REGN6321 EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO: 16, underlined CDRs) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0090] Full length light chains for both EGFR and CD28 parent antibodies and bispecifics EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28)

[0091] EGFR component of REGN6322 HCVR in the EGFR arm derived from the parental EGFR mAb 35193P2): EVQLVESGGGLVKPGGSLRLSCTAS GFSFRDAW MTWVRQVPGKGLEWVGR IRNKIDGGTT DYNTPVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYC TTDIWNYVLFYYYGLDV WGQGTTVTVSS (SEQ ID NO: 40, underlined CDRs) CDR-H1: GFSFRDAW (SEQ ID NO: 42) CDR-H2: IRNKIDGGTT (SEQ ID NO: 44) CDR-H3: TTDIWNYVLFYYYGLDV (SEQ ID NO: 46)

[0092] Full-length heavy chain of the EGFR arm of the bispecific antibody REGN6322 EVQLVESGGGLVKPGGSLRLSCTASGFSFRDAWMTWVRQVPGKGLEWVGRIRNKIDGGTTDYNTPVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDIWNYVLFYYYGL DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:48)

[0093] LCVR of EGFR and CD28 parent antibodies and bispecific antibody REGN6322 EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO: 16, underlined CDRs) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0094] Full length light chains for both EGFR and CD28 parent antibodies and bispecifics EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28)

[0095] EGFR component of REGN6323 HCVR in the EGFR arm derived from parental EGFR mAb 13006P2): QVQLQESGPGLVKPSETLSLTCTVS DDSIISYY WSWIRQPPGKGLEWIGY IYYSGRT NYNPSLKSRVTISVDTSKNQVSLKLNSVIAADTAVYYC ARVSEDSYYHYGMDV WGQGTTVTVSS (SEQ ID NO: 50, underlined CDRs) CDR-H1: DDSIISYY (SEQ ID NO: 52) CDR-H2: IYYSGRT (SEQ ID NO: 54) CDR-H3: ARVSEDSYYHYGMDV (SEQ ID NO: 56)

[0096] Full-length heavy chain of the EGFR arm of the bispecific antibody REGN6323 QVQLQESGPGLVKPSETLSLTTCTVSDDSIISYYWSWIRQPPGKGLEWIGYIYYSGRTNYNPSLKSRVTISVDTSKNQVSLKLNSVIAADTAVYYCARVSEDSYYHYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:58)

[0097] LCVR of EGFR and CD28 parental antibodies and the bispecific antibody REGN6323 EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO: 16, underlined CDRs) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0098] Full length light chains for both EGFR and CD28 parent antibodies and bispecifics EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28)

[0099] In certain embodiments of the invention, the heavy chain of mAb12999P2, mAb13008P2, mAb35193P2, or mAb13006P2 is paired with a light chain comprising an amino acid sequence selected from the following: EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:16); and DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPPIT FGQGTRLEIK (SEQ ID NO:67);

[0100] CD28 (Cluster of Differentiation 28) binding arm The present invention includes multispecific (eg, bispecific) antigen binding proteins, such as antibodies or antigen-binding fragments, that comprise one or more CD28-binding arms and one or more EGFR-binding arms.

[0101] A CD28-binding arm is a portion of a multispecific antigen-binding protein that confers CD28-binding ability to the protein. For example, the CD28-binding arm of a Y-shaped IgG antibody refers to the structural portion of the antibody that confers binding specificity to CD28. For example, in one embodiment of the invention, the CD28-binding arm is a structural portion of HCDR1, LCDR1, HCDR2, LCDR2, HCDR3 and LCDR3 that specifically bind to CD28; HVCR (V H ) and LCVR(V L ) and / or HC and LC.

[0102] In one embodiment of the invention, the CD28 binding arm comprises a V H and a V containing a combination of light chain CDRs (LCDR1, LCDR2 and LCDR3). L In one embodiment of the invention, the CD28-binding arm comprises a heavy chain variable region (V H ) and the corresponding light chain variable region (V L ).

[0103] The amino acid sequences of the immunoglobulin chains of the parent anti-CD28 antibodies 14226P2, 14193P2, and 14216P2 are set forth below:

[0104] Parent CD28 mAbs used in the preparation of bispecifics mAb14226P2 mAb14226P2 HCVR QVQLQESGPGLVKPSETLSLTCTVS GGSISSYY WSWIRQPPGKGLEWIGY IYYSGIT HYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYC ARWGVRRDYYYYGMDV WGQGTTVTVSS (SEQ ID NO:10) CDR-H1: GGSISSYY (SEQ ID NO: 12) CDR-H2: IYYSGIT (SEQ ID NO: 6) CDR-H3: ARWGVRRDYYYYGMDV (SEQ ID NO: 14)

[0105] mAb14226P2 Full length heavy chain of QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO:26)

[0106] mAb14226P2 LCVR EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:16) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0107] mAb14226P2 Full-length light chain of EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28)

[0108] mAb14193P2 mAb14193P2 HCVR QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV ISYAGNNK YYADSVKGRFTVSRDNSKKTLYLQMNSLRSEDTAVYYC AKDSYYDFLTDPDVLDI WGQGTMVTVSS (SEQ ID NO:59) CDR-H1: GFTFSSYG (SEQ ID NO: 60) CDR-H2: ISYAGNNK (SEQ ID NO: 61) CDR-H3: AKDSYYDFLTDPDVLDI (SEQ ID NO: 62)

[0109] mAb14193P2 LCVR EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:16) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0110] mAb14216P2 mAb14216P2HCVR EVQLVESGGGLVQPGGSLRLSCAAS GFTFSRNN MHWVRQAPGKGLEYVSG ISSNGGRT YYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFC TRDDELLSFDY WGQGTLVTVSS (SEQ ID NO:63) CDR-H1: GFTFSRNN (SEQ ID NO: 64) CDR-H2: ISSNGGRT (SEQ ID NO: 65) CDR-H3: TRDDELLSFDY (SEQ ID NO: 66)

[0111] mAb14216P2 LCVR EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:16) CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20) CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

[0112] In one embodiment of the invention, a heavy chain of 14226P2, 14193P2, or 14216P2 is paired with a light chain comprising an amino acid sequence selected from the following: EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK (SEQ ID NO:16); And DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPPIT FGQGTRLEIK (SEQ ID NO:67);

[0113] "14226P2", "14193P2" or "14216P2" or "mAb14226P2", "mAb14193P2" or "mAb14216P2" refers to the parent monospecific antibody from which the CD28 arm of the bispecific described herein is derived. H ) or a full-length heavy chain (or a variant thereof), and the corresponding immunoglobulin light chain variable region (V L ) or a CD28-binding arm comprising a full-length light chain, or a V comprising its CDRs (HCDR1 (or a variant thereof), HCDR2 (or a variant thereof), and HCDR3 (or a variant thereof) H and the corresponding V including its CDRs (LCDR1 (or a variant thereof), LCDR2 (or a variant thereof) and LCDR3 (or a variant thereof)). L and a CD28 binding arm comprising, for example, a V, in which the variable region and / or CDRs comprise the specific amino acid sequences described herein and are not variants. H In some cases, the CDR-H may contain variant amino acid sequences that are not variants, and / or such V L In some embodiments of the invention, the CD28 binding arm may be designated in the context of an EGFRxCD28 multispecific antigen binding protein, e.g., 085Nx14226P2. H is linked to an IgG heavy chain constant domain (e.g., IgG1 or IgG4), and / or L is linked to a lambda or kappa light chain constant domain.

[0114] The present invention also provides antigen binding proteins and antigen binding fragments thereof, such as, for example, antibodies (e.g., human antibodies, monoclonal antibodies and recombinant antibodies), that specifically bind to the CD28 protein or antigenic fragments thereof (e.g., the extracellular domain of CD28). Antigen binding proteins that bind to the same epitope on CD28 as any of the antigen binding proteins described herein, or that compete with them for binding to CD28, are also part of the present invention.

[0115] The multispecific EGFRxCD28 antigen binding proteins of the invention that bind to CD28 on the surface of T cells and agonize CD28 signaling to enhance T cell activation and / or proliferation may be referred to herein as "co-stimulatory" or "costimulatory". T cell activation is initiated by the binding of the T cell receptor (TCR) / CD3 complex to a peptide-MHC complex (signal 1), and subsequent activation is enhanced by engagement of a second "costimulatory" receptor (signal 2), e.g., the binding of the CD28 receptor on the T cell to its cognate ligand on a target cell. For example, activation of T cells by CD28 bispecific antibodies may result from amplification of a signal in response to recognition of an endogenous tumor antigen by the TCR / CD3 complex, or amplification of a signal in response to "signal 1" activation via a CD3-bispecific antibody.

[0116] Multispecific antigen-binding proteins The present invention provides antigen binding proteins that bind at least EGFR and CD28 in a multispecific (e.g., bispecific) manner. As used herein, such antibodies are designated in the format AxB, where A refers to the binding arm in the multispecific molecule that binds EGFR and B refers to the binding arm in the multispecific molecule that binds CD28. The reverse is also possible. EGFRxCD28 or CD28xEGFR refers to multispecific antigen binding proteins that bind EGFR and CD28. The specific EGFR and CD28 binding arms in the multispecific antigen binding proteins may be specified in the format AxB, where A refers to a specific arm and B refers to another specific arm. For example, 085Nx14226P2 refers to a multispecific antigen binding protein having an anti-EGFR binding arm of 085N as described herein and an anti-CD28 binding arm of 14426P2 as described herein. For example, 085N is a binding arm comprising the immunoglobulin heavy and light chains of 085N, or the variable regions thereof, or the CDRs thereof, the sequences of which are specifically described herein, or variants thereof.

[0117] In some embodiments, the multispecific antigen-binding protein comprises a bispecific antigen-binding protein. As used herein, the term "bispecific antigen-binding protein" refers to a protein, polypeptide or molecular complex (e.g., an antibody or an antigen-binding fragment thereof) that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the present case, the first antigen-binding domain specifically binds to a first antigen (e.g., CD28) and the second antigen-binding domain specifically binds to a second distinct antigen (e.g., EGFR).

[0118] Multispecific binding refers to binding to two or more different epitopes (EGFR and CD28, or more), which may be on the same antigen or on different antigens. Multispecificity includes bispecificity, trispecificity, and tetraspecificity.

[0119] The present invention includes any of the following multispecific antigen binding proteins (e.g., bispecific antibodies or antigen-binding fragments thereof): REGN7075, REGN6321, REGN6322, REGN6323. As well as bispecific antibodies prepared by combining any of the EGFR HCVR arms in Table 1 (e.g., the HCVR arms of parent monoclonal antibodies mAb12999P2, mAb13008P2, mAb35193P2 and mAb13006P2) with any of the CD28 HCVR arms in Table 3 (e.g., the HCVR arms of parent mAb14226, mAb14193 and mAb14216). The present invention also includes methods of their use as described herein.

[0120] Polynucleotides and methods of making Isolated polynucleotides encoding the immunoglobulin chains of any of the EGFRxCD28 multispecific antigen binding proteins or anti-EGFR antigen binding proteins described herein also form a part of the invention, as do vectors comprising the polynucleotides described herein, and / or host cells (e.g. Chinese Hamster Ovary (CHO) cells) comprising the polynucleotides, vectors or antigen binding proteins.

[0121] Polynucleotides include DNA and RNA. The present invention relates to, for example, immunoglobulin V of the EGFR binding arm and / or the CD28 binding arm. H , V LThe present invention includes polynucleotides of the present invention that encode a CDR-H, CDR-L, HC or LC, optionally operably linked to a promoter or other expression control sequence. For example, the present invention provides any polynucleotide (e.g., DNA) that comprises a nucleotide sequence set forth in Table 2, and a nucleotide sequence set forth in Table 4.

[0122] The present invention includes polynucleotides comprising the nucleotide sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 29, 31, 33, 35, 39, 41, 43, 45, 49, 51, 53 and / or 55, optionally operably linked to a promoter or other expression control sequence or other polynucleotide sequence.

[0123] Generally, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding (e.g., directly or through other promoter-binding proteins or agents) an RNA polymerase in a cell and initiating transcription of a coding sequence. A promoter may be operably linked to other expression control sequences, including enhancer and repressor sequences, and / or with a polynucleotide of the invention. Promoters that can be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (VIIIa-Komaroff, et al., (1983) Nature 297:111-112); al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25), see also "Useful proteins from recombinant bacteria" in Scientific American (1980) 242:74-94, and promoter elements derived from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0124] When a polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression control sequence, in a cell or other expression system, the coding sequence is transcribed by that sequence via RNA polymerase into RNA, preferably mRNA, which is then subjected to RNA splicing (if it contains introns), and optionally translated into the protein encoded by the coding sequence.

[0125] The present invention includes polynucleotides encoding immunoglobulin polypeptide chains whose nucleotide sequences are variants of the immunoglobulin polypeptide chains specifically described herein. A "variant" of a polynucleotide refers to a polynucleotide that comprises a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to a reference nucleotide sequence described herein when the comparison is performed using the BLAST algorithm and the parameters of the algorithm are selected to give the greatest match between each sequence over the entire length of each reference sequence (e.g., expectation threshold: 10, word size: 28, maximum match in query range: 0, match / non-match score: 1, -2; gap cost: linear). In certain embodiments of the invention, variants of the nucleotide sequences embodied herein contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) point mutations, insertions (e.g., in-frame insertions), or deletions (e.g., in-frame deletions) of one or more nucleotides. Such mutations may be missense or nonsense mutations in certain embodiments of the invention. In certain embodiments of the invention, such variant polynucleotides encode immunoglobulin polypeptide chains that can incorporate within an EGFR binding arm and / or a CD28 binding arm, i.e., the protein retains specific binding to EGFR and / or CD28.

[0126] Eukaryotic and prokaryotic host cells, including mammalian cells, may be used as hosts for the expression of anti-EGFR and EGFRxCD28 antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) or antigen-binding arms thereof. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeasts and filamentous fungi, such as Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp.), Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. The invention includes an isolated host cell (e.g., a CHO cell or any type of host cell set forth above) comprising an anti-EGFR antibody (e.g., an EGFR antibody found in WO2014 / 004427), or an anti-EGFRx anti-CD28 antigen binding protein of the invention, such as REGN7075, REGN6321, REGN6322, REGN6323, and an anti-EGFRx anti-CD28 antigen binding protein set forth in Tables 9A, 9B or 9C, or a polynucleotide encoding an immunoglobulin (Ig) heavy and / or light chain thereof, and / or one or more polynucleotides encoding the EGFR binding arm and the CD28 binding arm of a multispecific antigen binding protein of the invention.

[0127] The present invention also relates to a combination of EGFRxCD28 and / or anti-EGFR antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) of the invention, such as REGN7075, REGN6321, REGN6322, REGN6323, and any of the CD28 HCVR arms of Table 3 (e.g., the HCVR arms of parent mAb14226, mAb14193, and mAb14216) with an EGFRxCD28 or ... Also included are bispecific antibodies prepared by combining HCVR arms (e.g., the HCVR arms of parent monoclonal antibodies mAb12999P2, mAb13008P2, mAb35193P2, and mAb13006P2), or cells expressing EGFR and / or CD28, or antigenic fragments thereof, or fusions (e.g., His6 (SEQ ID NO: 68), Fc and / or myc) bound to any of the bispecific antibodies shown in Tables 9A, 9B and 9C, e.g., in which case the cells are within the body of a subject or in vitro.

[0128] Furthermore, the present invention also provides complexes comprising the EGFRxCD28 and / or EGFR antigen binding proteins of the present invention, such as complexes comprising an antibody or antigen-binding fragment thereof as discussed herein complexed with an EGFR and / or CD28 polypeptide, or an antigenic fragment thereof or a fusion thereof, and / or a secondary antibody or antigen-binding fragment thereof (e.g., a detectably labeled secondary antibody) that specifically binds to an anti-EGFR antibody or an EGFRxCD28 antibody or a fragment thereof. In certain embodiments of the present invention, the complex is in vitro (e.g., fixed to a solid substrate) or in the body of a subject. In certain embodiments of the present invention, the EGFR is on the surface of a tumor cell and the CD28 is on the surface of an immune cell, such as a T cell. In certain embodiments of the present invention, the T cell is activated.

[0129] There are several methods for producing recombinant antibodies, which are known in the art. One example of a method for recombinantly producing antibodies is disclosed in U.S. Pat. No. 4,816,567. Transformation may be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, biolistic injection, and direct DNA microinjection into nuclei. Additionally, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.

[0130] The invention includes recombinant methods for making an anti-EGFRxanti-CD28 (e.g., REGN7075, REGN6321, REGN6322, or REGN6323) antigen binding protein of the invention, such as an antibody or antigen-binding fragment thereof, or an immunoglobulin chain thereof of the invention, the method comprising: (i) introducing into a host cell one or more polynucleotides encoding EGFRxCD28, or light and heavy chain immunoglobulins encoding the antigen binding arms of an anti-EGFR antigen binding protein, e.g., where the polynucleotides are in a vector and / or integrated into a chromosome of the host cell and / or operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions suitable for expression of the polynucleotide; and (iii) optionally, isolating the antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) or chain from the host cell and / or from the medium in which the host cell was grown. The present invention also includes anti-EGFR or EGFRxCD28 antigen binding proteins, such as antibodies or antigen-binding fragments thereof, that are the product of the production methods described herein, and optionally the purification methods described herein.

[0131] In certain embodiments of the invention, methods of making EGFRxCD28 (e.g., REGN7075, REGN6321, REGN6322, or REGN6323) antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, include methods of purifying the antigen binding protein, e.g., by column chromatography, precipitation, and / or filtration. As discussed, the products of the methods also form part of the invention.

[0132] Sequence variants The antibodies and bispecific antigen-binding molecules of the present invention may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may include antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In one embodiment, the V H and / or V LAll of the framework and / or CDR residues in the domain are mutated back to the residues present in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence. For example, only mutated residues present within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only mutated residues present within CDR1, CDR2, or CDR3 are mutated back. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen binding domain was originally derived). Furthermore, the antigen binding domain may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antigen binding domains containing one or more germline mutations can be readily tested for one or more desired properties. Desired properties include, for example, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained by this general method are encompassed within the present invention.

[0133] The present invention also includes antigen-binding molecules in which one or both antigen-binding domains contain variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes antigen-binding molecules comprising an antigen-binding domain having an HCVR amino acid sequence, an LCVR amino acid sequence, and / or a CDR amino acid sequence with, for example, 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution does not substantially change the functional properties of a protein. Examples of amino acid groups with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0134] The present invention also includes antigen-binding molecules comprising an antigen-binding domain having an amino acid sequence of HCVR, LCVR and / or CDR that is substantially identical to any of the amino acid sequences of HCVR, LCVR and / or CDR disclosed herein. When referring to an amino acid sequence, the term "substantial identity" or "substantially identical" means that two amino acid sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, with a predefined gap weighting. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331.

[0135] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, using the program GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the region of best overlap between the query and search sequences and the percentage of sequence identity (Pearson (2000), supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0136] Antibodies containing Fc variants According to one embodiment of the invention, there is provided an anti-EGFRX, anti-CD28 bispecific antigen binding molecule comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides an anti-EGFRX, anti-CD28 bispecific antigen binding molecule comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThese include antibodies and antigen-binding molecules that contain mutations in three regions, where the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes at a pH range of about 5.5 to about 6.0). Such mutations can extend the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example: 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and / or 256 (e.g., S / R / Q / E / D or T); or 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or 434 (e.g., H / F or Y); or 250 and / or 428; or 307 or 308 (e.g., 308F, V308F), and / or An example of the modification is at position 434.

[0137] In one embodiment, the modification is 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E); 250Q and 428L modifications (e.g., T250Q and M428L); and / or Contains modifications of 307 and / or 308 (e.g., 308F or 308P).

[0138] For example, the present invention includes EGFRxCD28 bispecific antigen binding molecules comprising an Fc domain that comprises one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F).

[0139] All possible combinations of the aforementioned Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

[0140] Biological characteristics of antibodies and antigen-binding molecules The present invention includes antibodies and antigen-binding fragments thereof that bind to CD28 and EGFR with high affinity. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD28 and / or EGFR with moderate or low affinity, depending on the therapeutic context and the specific targeting properties desired. For example, in the case of a bispecific antigen-binding molecule in which one arm binds to CD28 and another arm binds to a target antigen (e.g., EGFR), it may be desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-CD28 arm binds to CD28 with only moderate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding general / non-target CD28 binding and the associated adverse side effects.

[0141] According to one embodiment, the present invention provides a method for the production of antibodies that bind to human CD28 (e.g., at 25° C.) with a K of less than about 200 nM as measured by surface plasmon resonance using an assay format, e.g., as defined in Examples 1 and 13 herein. DIn certain embodiments, the antibodies or antigen-binding fragments thereof of the invention have a K of less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 60 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by surface plasmon resonance, e.g., using the assay formats set forth in Examples 1 and 13 herein, or closely similar assays. D In one embodiment, the antibody or antigen-binding fragment of the invention binds to CD28 with a K of about 5 nM to about 20 nM. D and binds to CD28.

[0142] The present invention further provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to the patient a medicament for which the medicament has a dissociation half-life (t ) of greater than about 3 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., for example using the assay formats set forth in Examples 1 and 13 herein, or closely similar assays. 1 / 2 In certain embodiments, the antibodies or antigen-binding fragments of the invention have a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay formats set forth in Examples 1 and 13 herein, or closely similar assays. 1 / 2 and binds to CD28.

[0143] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) capable of simultaneously binding to human CD28 and human EGFR. According to certain embodiments, the bispecific antigen-binding molecules of the present invention specifically interact with cells expressing CD28 and / or EGFR. The extent to which the bispecific antigen-binding molecules bind to cells expressing CD28 and / or EGFR can be assessed by fluorescence-activated cell sorting (FACS) as described in Examples 10A and 10B herein. For example, the present invention includes bispecific antigen-binding molecules that specifically bind to a human cell line that expresses CD28 but not EGFR (e.g., Jurkat cells), and a human ovarian cancer cell line that expresses EGFR but not CD28 (e.g., PEO1). In some embodiments, the bispecific antigen-binding molecules are delivered to human or cynomolgus T cells expressing CD28 at 1x10 -5 In some embodiments, the bispecific antigen-binding molecule binds to human or cynomolgus T cells expressing CD28 at a concentration of less than 1x10 -12 M~1x10 -5 In one embodiment, the bispecific antigen-binding molecule binds to human or cynomolgus T cells expressing CD28 with an EC50 value of 1x10 -9 M~1x10 -5 In one embodiment, the bispecific antigen-binding molecule binds to the surface of an EGFR-expressing cell line with an EC50 value of about 2.5×10 -8 EC below M 50 Binding of the bispecific antigen-binding molecules to the surface of cells or cell lines can be measured by an in vitro FACS binding assay as described in Examples 10A and 10B.

[0144] The EGFRxCD28 antigen binding proteins described herein, including, for example, variant immunoglobulin chains, may exhibit one or more of the following properties: - Mice engrafted with CD34+ cells (e.g., fetal liver CD34+ cells) (e.g., VH mice (Rag2 null / γ c null / huSirp-a / huTPO) to reduce the proliferation and / or survival of human tumor cells (e.g., A431 tumor cells). Optionally, in this case, EGFRxCD28 is administered in association with a PD1 antagonist (e.g., anti-PD1 such as cemiplimab). - Mice containing human PBMCs (e.g., NOD / SCID / γ c null )) to reduce the proliferation and / or survival of human tumor cells (e.g., A431 tumor cells) (e.g., in this case human PBMCs mixed with tumor cells are subcutaneously implanted into mice). Optionally, in this case EGFRxCD28 is administered in association with a PD1 antagonist (e.g., anti-PD1 such as cemiplimab). - Mice containing human PBMCs (e.g. NOD / SCID / γ c null ) to reduce the proliferation and / or survival of human tumor cells (e.g., A549 tumor cells) (e.g., in this case human PBMCs are implanted intraperitoneally into mice and tumor cells are implanted subcutaneously into mice). Optionally, in this case EGFRxCD28 is administered in association with a PD1 antagonist (e.g., anti-PD1 such as cemiplimab). - Binds to Jurkat CD28+ cells. - PE01 binds to EGFR+ cells; and / or - does not result in significant cytokine release (e.g., interferon gamma, IL-2, IL-6, IL-8 and / or IL-10) when administered to cynomolgus monkeys, e.g., at 10 mg / kg.

[0145] The present invention includes anti-EGFR and EGFRxCD28 bispecific antigen-binding molecules capable of depleting tumor cells in a subject (see, e.g., Examples 3-5). For example, according to one embodiment, anti-EGFR and EGFRxCD28 bispecific antigen-binding molecules are provided, where a single administration of the antigen-binding molecule to a subject in a therapeutically effective dose results in a reduction in the number of tumor cells in the subject.

[0146] The present invention comprises an anti-EGFRX-anti-CD28 bispecific antigen binding molecule capable of binding to a variety of tumor cells, including A375 melanoma cells, 22RV1 prostate cells, PEO1 ovarian cells, CAPAN2 pancreatic cells, SW1990 pancreatic cells, and H292 embryonic cells (see Examples 10A and 10B). Thus, the bispecific antibodies of the present invention may prove useful in the treatment of a number of cancer conditions.

[0147] The present invention includes anti-EGFRX-anti-CD28 bispecific antigen binding molecules capable of enhancing the cytotoxic efficacy of anti-tumor specific antigen (TSA)X-anti-CD3 bispecific antibodies in various cell lines (see Example 11). When using this method, the TSAXCD3 bispecific antibody may include anti-STEAP2X-anti-CD3, anti-PSMAX-anti-CD3, or anti-MUC16X-anti-CD3 bispecifics. Anti-EGFRX-anti-CD28 antibodies may also prove useful when combined with checkpoint inhibitors, such as antibodies against PD-1 or PD-L1, or any other checkpoint inhibitors (see Example 9). In certain embodiments, it may be beneficial to combine anti-EGFRX-anti-CD28 with both anti-TSAX-anti-CD3 bispecifics and checkpoint inhibitors.

[0148] The antibodies of the invention have also been demonstrated to bind to specific epitopes on human EGFR using hydrogen-deuterium exchange (see Example 12). In particular, certain antibodies of the invention have been found to bind / interact with amino acid residues 345-368 of human EGFR (SEQ ID NO:70), amino acid residues 399-416 of human EGFR (SEQ ID NO:71), and amino acid residues 133-154 of human EGFR (SEQ ID NO:72).

[0149] Epitope mapping and related techniques The epitope on CD28 or EGFR to which the antigen-binding molecule of the present invention binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD28 or EGFR protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD28 or EGFR. The antibodies of the present invention may interact with amino acids contained within a CD28 monomer, or with amino acids on two different CD28 chains of a CD28 dimer. As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. An antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. An epitope may be either conformational or linear. A conformational epitope is generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on an antigen.

[0150] Various techniques known to those skilled in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Examples of techniques that can be used to determine the epitope or binding domain of a particular antibody or antigen-binding domain include, for example, AntibodiesThese include the routine cross-blocking assay described in Reineke, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide that interact with an antibody is the hydrogen / deuterium exchange method detected by mass spectrometry. As a general term, the hydrogen / deuterium exchange method involves deuterium labeling of a protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry to reveal deuterium-labeled residues, which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. Alternatively, in one embodiment, the protein of interest is bound to the antibody, and then hydrogen-deuterium exchange occurs. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry to reveal non-deuterium-labeled residues, which correspond to the specific amino acids with which the antibody interacts. X-ray crystallography can also be used to identify amino acids within a polypeptide with which the antibody interacts.

[0151] The present invention further includes anti-CD28 and anti-EGFR antibodies that bind to the same epitope as certain exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Tables 1, 3, 6, 9A, 9B, and 9C). The present invention also includes anti-CD28 and / or anti-EGFR antibodies that compete for binding to CD28 and / or EGFR with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Tables 1, 3, 6, 9A, 9B, and 9C).

[0152] The present invention also includes bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds human CD28 and a second antigen binding fragment that specifically binds human EGFR, where the first antigen binding domain binds to the same epitope on CD28 as any of the specific exemplary CD28-specific antigen binding domains described herein, and / or the second antigen binding domain binds to the same epitope on EGFR as any of the specific exemplary EGFR-specific antigen binding domains described herein.

[0153] In certain embodiments, the invention comprises bispecific antigen binding molecules that interact with one or more of amino acid residues 133-154 of EGFR set forth in SEQ ID NO:72, amino acid residues 345-368 of EGFR set forth in SEQ ID NO:70, or amino acid residues 399-416 of EGFR set forth in SEQ ID NO:71.

[0154] Similarly, the present invention also includes bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds human CD28 and a second antigen binding fragment that specifically binds human EGFR, where the first antigen binding domain competes for binding to CD28 with any of the specific exemplary CD28-specific antigen binding domains described herein and / or the second antigen binding domain competes for binding to EGFR with any of the specific exemplary EGFR-specific antigen binding domains described herein.

[0155] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to or competes with the same epitope as a reference antigen-binding molecule of the present invention can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CD28 (or EGFR) as a reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first bound to CD28 protein (or EGFR protein). The ability of the test antibody to bind to CD28 (or EGFR) molecule is then evaluated. If the test antibody can bind to CD28 (or EGFR) after saturating the binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody does not compete with the reference bispecific antigen-binding molecule for binding to CD28 (or EGFR) and / or there is steric interference between antibodies that bind to different sites on the antigen. On the other hand, if the test antibody cannot bind to the CD28 (or EGFR) molecule after saturating the binding with the reference bispecific antigen-binding molecule, the test antibody competes with the reference bispecific antigen-binding molecule of the present invention for binding to CD28 (or EGFR). Additional routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the invention, two antigen binding proteins compete for binding to an antigen if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antigen binding protein inhibits binding of the other antibody by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antigen binding proteins may bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen binding protein also reduce or eliminate binding of the other. Two antigen binding proteins may have "overlapping epitopes" if only a subset of amino acid mutations that reduce binding of one antigen binding protein also reduce or eliminate binding of the other.

[0156] To determine whether an antibody or its antigen-binding domain competes with a reference antigen-binding molecule for binding, the above-mentioned binding method is carried out in two directions: in the first direction, the reference antigen-binding molecule is bound to the CD28 protein (or EGFR protein) under saturating conditions, and then the binding of the test antibody to the CD28 (or EGFR) molecule is evaluated. In the second direction, the test antibody is bound to the CD28 (or EGFR) molecule under saturating conditions, and then the binding of the reference antigen-binding molecule to the CD28 (or EGFR) molecule is evaluated. If only the first (saturating) antigen-binding molecule can bind to the CD28 (or EGFR) molecule in both directions, it is concluded that the test antibody and the reference antibody compete for binding to CD28 (or EGFR). As will be recognized by those skilled in the art, an antibody that competes with a reference antigen-binding molecule for binding does not necessarily bind to the same epitope as the reference antibody, and the binding of the reference antibody may be sterically hindered by overlapping or adjacent epitopes.

[0157] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific to a particular antigen can be prepared by any antibody production method known in the art. Once two different antigen-binding domains specific to two different antigens (e.g., CD28 and EGFR) are obtained, they can be appropriately positioned relative to each other using routine methods to generate the bispecific antigen-binding molecules of the invention. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the invention is presented elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized, or fully human antibodies. Methods for generating such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), a high affinity chimeric antibody with human variable regions and mouse constant regions against a particular antigen (e.g., CD28 or EGFR) is first isolated. Antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant regions may be replaced with the desired human constant regions to generate fully human heavy and / or light chains which are incorporated into the bispecific antigen-binding molecules of the invention.

[0158] Genetically engineered animals may be used to generate human bispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences may be used, in which the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically modified mice can be used to generate fully human bispecific antigen-binding molecules that contain two different heavy chains associated with the same light chain, the heavy chains containing variable domains derived from one of two different human light chain variable region gene segments. (See, for example, US Patent Publication No. 2011 / 0195454 for a detailed discussion of such modified mice and their use to generate bispecific antigen-binding molecules).

[0159] bioequivalent The present invention encompasses antigen-binding molecules having amino acid sequences that differ from the amino acid sequences of the described antibodies, but that retain the ability to bind to CD28 and EGFR. Such variant molecules contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit essentially equivalent biological activity to the described antigen-binding molecules. Similarly, DNA sequences encoding the antigen-binding molecules of the present invention encompass sequences that contain one or more nucleotide additions, deletions, or substitutions when compared to the disclosed sequences, but that encode antigen-binding molecules that are essentially biologically equivalent to the described antigen-binding molecules of the present invention. Examples of such variant amino acid sequences and DNA sequences are discussed above.

[0160] The present invention includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered biologically equivalent if they are pharmaceutical equivalents or pharmaceutical substitutes and do not show significant differences in their rate and extent of absorption when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered equivalents, or pharmaceutical substitutes, if the extent of absorption is equivalent but the rate of absorption is not, but the antibodies can be considered biologically equivalent because the difference in absorption rate is intentional, reflected in the labeling, is not essential for achieving effective body drug concentrations in chronic use, and is not considered medically significant for the particular pharmaceutical product tested.

[0161] In one embodiment, two antigen binding proteins are bioequivalent if they have no clinically meaningful differences in their safety, purity, and efficacy.

[0162] In one embodiment, two antigen binding proteins are biologically equivalent if one or more exchanges between a reference product and a biological product can be made without a predicted increase in the risk of adverse effects, including a clinically meaningful change in immunogenicity, or loss of efficacy, compared to continued treatment if the exchange were not made.

[0163] In one embodiment, two antigen binding proteins are biologically equivalent if they both act through a common mechanism of action to the extent that mechanism is known for the disease in which they are used.

[0164] Bioequivalence may be demonstrated by in vivo and in vitro methods, including, for example, (a) in vivo tests in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum or other bodily fluids as a function of time, (b) in vitro tests that correlate and are a reasonable indicator of in vivo bioavailability in humans, (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time, and (d) appropriately controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0165] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein may be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unnecessary or inappropriate intramolecular disulfide bridges upon renaturation. In other circumstances, biologically equivalent antibodies may include the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that abolish or eliminate glycosylation.

[0166] Species selectivity and species cross-reactivity According to one embodiment, the present invention provides an antigen-binding molecule that binds to human CD28 but not to CD28 from other species. The present invention also provides an antigen-binding molecule that binds to human EGFR but not to EGFR from other species. The present invention further includes antigen-binding molecules that bind to human CD28 and CD28 from one or more non-human species, and / or antigen-binding molecules that bind to human EGFR and EGFR from one or more non-human species.

[0167] According to certain exemplary embodiments of the present invention, there is provided an antigen-binding molecule that binds to human CD28 and / or human EGFR, and may or may not also bind to one or more of CD28 and / or EGFR of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. For example, in certain exemplary embodiments of the present invention, there is provided a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human CD28 and cynomolgus monkey CD28, and a second antigen-binding domain that specifically binds to human EGFR.

[0168] immune complex The present invention encompasses EGFRxCD28 antigen binding proteins, e.g., antibodies or antigen-binding fragments, conjugated with another moiety, e.g., a therapeutic moiety ("immunoconjugate"), e.g., any combination of anti-EGFR HCVRs paired with HCVRs from REGN7075, REGN6321, REGN6322, REGN6323, or any of the CD28 antibodies described herein. In certain embodiments of the present invention, anti-EGFR, or EGFRxCD28 antigen binding proteins, e.g., antibodies or antigen-binding fragments, are conjugated with any of the additional therapeutic agents described herein. As used herein, the term "immunoconjugate" refers to an antigen binding protein, e.g., an antibody or antigen-binding fragment, chemically or biologically linked to another antigen binding protein, drug, radiopharmaceutical, receptor moiety, enzyme, peptide, protein, or therapeutic agent.

[0169] In some embodiments, the therapeutic moiety may be a cytokine, a chemotherapeutic agent, an immunosuppressant, or a radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents and chemotherapeutic agents suitable for forming immunoconjugates are known in the art (see, for example, WO 05 / 103081).

[0170] Administration and Treatment The invention provides methods for administering an EGFRxCD28 multispecific antigen binding protein, such as, for example, REGN7075, REGN6321, REGN6322, REGN6323, or any combination of anti-EGFR HCVRs paired with HCVRs from any of the CD28 antibodies described herein, or a pharmaceutical composition thereof, to a subject (e.g., a human suffering from a hyperproliferative disorder), which comprises introducing the antigen binding protein or pharmaceutical composition thereof into the body of the subject (e.g., human), e.g., parenterally. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen binding protein or pharmaceutical composition into the body of the subject, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0171] The manner in which EGFRxCD28, or anti-EGFR antigen binding protein, or pharmaceutical composition thereof, is administered can vary, including parenteral, oral, buccal, rectal, transmucosal, intestinal, oral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, intraocular, intravitreal, transdermal, or intraarterial.

[0172] The invention also provides a container (e.g., a capped plastic or glass vial or ampoule, a chromatography column, a hollow bore needle or syringe cylinder) comprising an EGFRxCD28 or anti-EGFR antigen binding protein of the invention, or a pharmaceutical composition thereof.

[0173] The present invention also provides an injection device comprising one or more antigen binding proteins (e.g., antibodies or antigen binding fragments) that specifically bind to EGFR, or EGFR and CD28 (EGFRxCD28), or a pharmaceutical formulation thereof. The injection device may be packaged in a kit. An injection device is a device that introduces a substance into a subject's body via a parenteral route, such as intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe or an autoinjector (e.g., pre-filled with a pharmaceutical formulation), which includes, for example, a cylinder or barrel for holding a liquid to be injected (e.g., comprising an antibody or fragment or a pharmaceutical formulation thereof), a needle for piercing the skin, blood vessel, or other tissue for injection of the liquid, and a plunger for forcing the liquid from the cylinder through the inner bore of the needle and into the subject's body.

[0174] A pre-filled syringe is a syringe that is filled with a composition (e.g., a pharmaceutical composition comprising a multispecific antigen binding protein and a pharma- ceutically acceptable carrier) and then sold or shipped to an end user, such as a physician or caregiver, who will administer the composition to a patient / subject.

[0175] Therapeutic uses of antigen-binding molecules The present invention provides a method of treating or preventing a hyperproliferative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an EGFRxCD28 antigen binding protein (such as REGN7075, REGN6321, REGN6322, REGN6323, or any combination of anti-EGFR HCVRs paired with HCVRs from any of the CD28 antibodies described herein), optionally in association with a PD-1 and / or PD-L1 inhibitor, such as, for example, an antibody (e.g., pembrolizumab, nivolumab, and / or cemiplimab) or an antigen-binding fragment thereof. In an embodiment of the invention, the method comprises determining whether the subject's cancer expresses EGFR. If such expression is observed, EGFRxCD28 and / or anti-EGFR antigen binding protein is administered. For example, in one embodiment of the invention, the method comprises taking a biopsy of the cancer (e.g., performed by the treating physician) and determining, or instructing another individual or entity (e.g., on behalf of the patient or subject) to do so, whether cells of the cancer express EGFR, and administering an anti-EGFR or EGFRxCD28 antigen binding protein to the subject if EGFR expression is present. In one embodiment of the invention, the physician instructs (on behalf of the patient or subject) another individual or entity, such as, for example, a pathologist, to perform the biopsy. In one embodiment of the invention, EGFR expression is verified by immunohistochemistry (IHC) or by ELISA (enzyme-linked immunosorbent assay).

[0176] The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising EGFR, or a bispecific antigen-binding molecule that specifically binds CD28 and EGFR. The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules disclosed herein and a pharma- ceutically acceptable carrier or diluent.

[0177] The antibodies and bispecific antigen-binding molecules of the present invention (and therapeutic compositions comprising them) are particularly useful for the treatment of any disease or disorder in which stimulating, activating and / or targeting the immune response is beneficial. In particular, the anti-EGFR or EGFRxCD28 bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by EGFR expression or activity, or the proliferation of EGFR+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved includes killing cells expressing EGFR in the presence of effector cells, such as T cells. EGFR-expressing cells that can be inhibited or killed using the bispecific antigen-binding molecules of the present invention include, for example, lung cancer cells.

[0178] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, in need of prevention and / or treatment of EGFR-expressing cancer. The subject may have an EGFR-expressing cancer, may be susceptible to developing such a condition, and / or may benefit from inhibition or reduction of EGFR activity, or depletion of EGFR+ cells. In one embodiment, the subject may have or be at risk of developing a hyperproliferative disease.

[0179] For the purposes of this specification, hyperproliferative disease refers to a disease characterized by abnormal, excessive, and / or uncontrolled cell proliferation, for example, where the cells express EGFR. For example, hyperproliferative disease includes EGFR-expressing cancer. Various cancers express EGFR. Examples of EGFR-expressing cancer include, but are not limited to, esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, glioma, thyroid cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, colon cancer, bladder cancer, rectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, gastroesophageal cancer (e.g., gastroesophageal adenocarcinoma), and melanoma. Thus, the antibodies and bispecific antigen-binding molecules of the present invention can be used to treat various cancers.

[0180] For example, in this case, cancers characterized by solid tumor cells or cancerous blood cells, which may be EGFR-expressing cancers, where EGFR expression has been confirmed in the cells of the particular subject being treated, include esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer (e.g., non-small cell lung carcinoma), colorectal cancer, rectal cancer, endometrial cancer, skin cancer (e.g., head and neck squamous cell carcinoma), brain cancer (e.g., glioblastoma multiforme), breast cancer, gastroesophageal cancer (e.g., gastroesophageal adenocarcinoma), prostate cancer, and / or ovarian cancer.

[0181] The antigen-binding molecules of the present invention may also be used to treat, for example, primary and / or metastatic tumors arising in the colon, lung, brain, kidney and bladder (or derived from any of the cancers discussed herein). According to an exemplary embodiment, the bispecific antigen-binding molecules of the present invention are used to treat ovarian cancer.

[0182] The present invention also includes methods of treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject following anti-cancer treatment.

[0183] According to one embodiment, the invention provides a method for treating a hyperproliferative disease, e.g., associated with EGFR expression (e.g., lung cancer), comprising administering to a subject one or more of the anti-EGFR or EGFRxCD28 bispecific antigen binding molecules described herein, e.g., after the subject has been shown to be non-responsive to other types of anti-cancer treatment. For example, the invention includes a method for treating a hyperproliferative disease, e.g., lung cancer, comprising administering to a patient suffering from cancer, e.g., lung cancer, an anti-EGFR or EGFRxCD28 bispecific antigen binding molecule to the patient or subject 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received a standard of care treatment. In other embodiments, an anti-EGFR or EGFRxCD28 bispecific antigen-binding molecule of the invention comprising an IgG4 Fc domain is first administered to a subject at one or more time points (e.g., to provide an initial stable depletion of ovarian cancer cells), followed by administration of an equivalent bispecific antigen-binding molecule comprising a different IgG domain, e.g., an IgG1 Fc domain, at a subsequent time point. It is anticipated that the anti-EGFR or EGFRxCD28 antibody of the invention may be used in conjunction with other bispecific antigen-binding molecules, e.g., anti-EGFR / anti-CD3 bispecific antibodies. It is also anticipated that the bispecific antibodies of the invention may be used in conjunction with additional therapeutic agents, e.g., targeting PD-1 and targeting others. It may be beneficial to combine two bispecific antibodies that target the same tumor antigen (e.g., EGFR), but it may also be beneficial to combine a bispecific that targets CD3 on T cells and another that targets a costimulatory molecule such as CD28. This combination may be used alone to enhance tumor cell killing, or may be used in combination with a checkpoint inhibitor.

[0184] An "effective" or "therapeutically effective" dose of EGFRxCD28, or an anti-EGFR antigen binding protein, e.g., an antibody or antigen binding fragment, for the treatment or prevention of a hyperproliferative disease, e.g., an EGFR-expressing cancer, is an amount of antigen binding protein sufficient to ameliorate one or more signs and / or symptoms of the disease in a treated subject, by inducing regression or elimination of said signs and / or symptoms, or by inhibiting the progression of said signs and / or symptoms. In one embodiment of the invention, a therapeutically effective dose of anti-EGFR, or an EGFRxCD28 antigen binding protein is 0.1-2000 mg, e.g., 0.1 mg IV (intravenous) Q2W (every two weeks) to 2000 mg IV Q2W. Dosage may vary depending on the age and size of the subject to which it is administered, the target disease, condition, route of administration, etc. In certain embodiments, a second or subsequent multiple administrations of the antigen binding protein following an initial dose may be in an amount that is about the same as the initial dose, or less than the initial dose, or may be more than the initial dose, where the subsequent administrations are at two week intervals.

[0185] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, etc. The preferred dose is typically calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. Effective doses and schedules for administering bispecific antigen-binding molecules may be empirically determined, for example, the progress of the patient may be monitored by periodic evaluation, and the dose adjusted accordingly. Furthermore, inter-species expansion of doses may be performed using methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0186] According to an embodiment of the invention, multiple doses of an antigen-binding molecule (e.g., an anti-CD28 antibody, or a bispecific antigen-binding molecule that specifically binds to EGFR and CD28) may be administered to a subject over a predetermined time course. The method according to this aspect of the invention includes sequentially administering multiple doses of an antigen-binding molecule of the invention to a subject. As used herein, "sequentially administering" means that each dose of an antigen-binding molecule is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes a method comprising sequentially administering an initial dose of an antigen-binding molecule, followed by one or more second doses of the antigen-binding molecule, and optionally then one or more third doses of the antigen-binding molecule.

[0187] The terms "initial dose", "second dose", and "third dose" refer to the temporal order of administration of the antigen-binding molecule of the present invention. Thus, the "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "reference dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial dose, second dose, and third dose may all contain the same amount of antigen-binding molecule, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecule contained in the initial dose, second dose, and / or third dose differs from each other during the course of treatment (e.g., adjusted up or down). In certain embodiments, two or more doses are administered as a "loading dose" at the beginning of a treatment regimen, and subsequent doses are administered on a less frequent basis (e.g., "maintenance doses").

[0188] In one exemplary embodiment of the present invention, each of the second and / or third doses is administered one to several weeks after the immediately preceding dose. The term "immediately preceding dose" as used herein refers to a sequence of multiple doses, and refers to an administration of an antigen-binding molecule administered to a patient before the administration of the very next dose in the sequence is administered without any intervening administrations.

[0189] The method according to this aspect of the invention may include administering to the patient a second and / or third dose of an antigen binding molecule (e.g., an anti-CD28 antibody, or a bispecific antigen binding molecule that specifically binds EGFR and CD28) any number of times. For example, in some embodiments, only one second dose is administered to the patient. In other embodiments, two or more second doses are administered to the patient. Similarly, in some embodiments, only one third dose is administered to the patient. In other embodiments, two or more third doses are administered to the patient.

[0190] In embodiments that include multiple second doses, each second dose may be administered at the same frequency as the other second doses. Similarly, in embodiments that include multiple third doses, each third dose may be administered at the same frequency as the other third doses. Alternatively, the frequency with which the second and / or third doses are administered to a patient may vary over the course of a treatment regimen. The frequency of administration may also be adjusted by a physician over the course of treatment depending on the needs of an individual patient after clinical testing.

[0191] Diagnostic Uses Bispecific antibodies of the invention may also be used to detect and / or measure CD28 or EGFR, or CD28- or EGFR-expressing cells in a sample, e.g., for diagnostic purposes. For example, anti-EGFR, or EGFRxCD28 antibodies, or antigen-binding fragments thereof, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of CD28 or EGFR. An exemplary diagnostic assay for CD28 or EGFR may include, e.g., contacting a sample obtained from a patient with an antibody of the invention, where the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled antibody may be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be, e.g., 3 H, 14 C. 32 p, 35 S, or125 The radioisotope may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure CD28 or EGFR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Samples that can be used in the CD28 or EGFR diagnostic assay according to the present invention include any tissue or liquid sample obtainable from a patient that contains a detectable amount of CD28 protein or EGFR protein or fragments thereof under normal or pathological conditions. In general, the level of CD28 or EGFR in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal CD28 or EGFR level or activity) is measured to first establish a reference or standard CD28 or EGFR level. The baseline level of CD28 or EGFR may then be compared to the level of CD28 or EGFR measured in a sample obtained from an individual suspected of having a disease or condition associated with CD28 or EGFR.

[0192] Combinations and Pharmaceutical Formulations The present invention provides compositions comprising EGFRxCD28, and / or anti-EGFR antigen binding proteins, and one or more components, as well as methods of using them, and methods of making such compositions. Pharmaceutical formulations comprising EGFRxCD28, or anti-EGFR antigen binding proteins of the invention, and a pharma- ceutically acceptable carrier or excipient (e.g., aqueous pharmaceutical formulations comprising water) are part of the invention.

[0193] The present invention provides pharmaceutical compositions containing the antigen-binding molecules of the present invention. The pharmaceutical compositions of the present invention may be formulated with suitable carriers, excipients, and other agents that improve transport, delivery, tolerability, and the like. Numerous suitable formulations can be found in the following formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0194] To prepare a pharmaceutical formulation of an EGFRxCD28 antigen binding protein, e.g., an antibody and antigen binding fragment thereof (e.g., REGN7075; REGN6321; REGN6322; REGN6323, or any combination of anti-EGFR HCVRs paired with a HCVR derived from any of the CD28 antibodies described herein), the antigen binding protein is mixed with a pharma- ceutically acceptable carrier or excipient. For example, Remington's Pharmaceutical Sciences and USPharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Disperse See, for example, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY; Weiner and Kotkoskie (2000) Excipient Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In certain embodiments of the invention, the pharmaceutical formulations are sterile. Such compositions are part of the invention.

[0195] Pharmaceutical formulations of the invention comprise EGFRxCD28, or an anti-EGFR antigen binding protein, and a pharma- ceutically acceptable carrier, including, for example, water, a buffer, a preservative, and / or a detergent.

[0196] The scope of the invention includes a dried, e.g., lyophilized, composition comprising EGFRxCD28, or an anti-EGFR antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, or a pharmaceutical formulation thereof that comprises a pharma- ceutically acceptable carrier but is substantially free of water.

[0197] A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. BioI. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, such as, for example, drip or bolus injection, absorption through epithelial or mucocutaneous layers (such as oral mucosa, rectal mucosa, and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or local.

[0198] As discussed herein, the invention provides a container (e.g., a plastic or glass vial) or an injection device (e.g., a syringe, pre-filled syringe, or autoinjector) containing any of the EGFRxCD28 or anti-EGFR antigen binding proteins herein, such as antibodies or antigen-binding fragments thereof, or pharmaceutical formulations thereof comprising a pharma- ceutically acceptable carrier or excipient.

[0199] The pharmaceutical composition of the present invention may be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In the case of disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold pre-filled with the pharmaceutical composition, which is held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0200] Many reusable and disposable pen and autoinjector delivery devices are adapted for subcutaneous delivery of the pharmaceutical compositions of the present invention, see, for example, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK) or HUMIRA™ pen (Abbott Labs, Abbott Park, Ill.).

[0201] In some circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be placed in the vicinity of the target of the composition, such that only a fraction of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0202] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injection. Aqueous media for injection include, for example, saline, and other isotonic solutions that may be used in combination with a suitable solubilizer. Oily media for injection are also part of the present invention. Such oily media may be mixed with a solubilizer.

[0203] The above-mentioned pharmaceutical composition for oral or parenteral use is advantageously prepared in a dosage form of a unit dose suitable for the dosage of the active ingredient. Examples of such dosage forms of unit doses include tablets, pills, capsules, injection solutions (ampoules), suppositories, etc. The amount of the antibody contained in the above-mentioned dosage form of a unit dose, particularly in the form of an injection solution, is generally about 0.1 to about 2000 mg.

[0204] The present invention includes compositions and therapeutic formulations comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutically active ingredients, and includes methods of treatment comprising administering the combination to a subject in need thereof.

[0205] Exemplary additional therapeutic agents that may be mixed with or administered in association with the antigen-binding molecules of the invention include, for example, chemotherapeutic agents (e.g., anti-cancer chemotherapeutic agents such as paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, or oxaliplatin), radiation therapy, checkpoint inhibitors targeting PD-1 (e.g., anti-PD-1 antibodies such as pembrolizumab, nivolumab, or cemiplimab (see U.S. Pat. No. 9,987,500)), CTLA-4, LAG3, TIM3, and others, costimulatory agonist bivalent antibodies targeting molecules such as GITR, OX40, 4-1BB, other costimulatory CD28 bispecific antibodies, and multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that bind tumor-associated antigens (e.g., MUC16 (mucin 16), PSMA, or STEAP2) and, for example, CD3 (MUC16xCD3, PSMAxCD3, or STEAP2xCD3). Exemplary bispecific antibodies that contain an antigen-binding domain that binds to CD3 include, but are not limited to, those described in WO2017 / 053856A1, WO2014 / 047231A1, WO2018 / 067331A1, and WO2018 / 058001A1. EGFR is expressed in a variety of cancers. Thus, the bispecific anti-EGFRxCD28 antibodies of the present invention can be used in combination with various bispecific antibodies that contain an antigen-binding domain that binds to CD3 in the treatment of various cancers.

[0206] In further embodiments of the invention, the additional therapeutic agent administered to the subject in association with EGFRxCD28, and / or an EGFR antigen binding protein, such as an antibody or antigen-binding fragment thereof, is selected from the group consisting of EGFRxCD28, ... th The drug is administered to the subject in accordance with the Prescribing Information for Drugs and Drug Administration (PHI) (Nov. 1, 2002) or in accordance with the approved prescribing information typically provided with the particular drug.

[0207] Methods for treating or preventing EGFR-expressing cancer in a subject in need thereof by administering a therapeutically effective dose of EGFRxCD28 and / or an anti-EGFR antigen binding protein in association with an additional therapeutic agent are part of the present invention.

[0208] The term "associated with" indicates that the components of the present invention, EGFRxCD28, or anti-EGFR antigen binding protein, e.g., an antibody or antigen binding fragment thereof, can be formulated in one composition, e.g., for simultaneous delivery, or can be formulated separately in two or more compositions (e.g., a kit containing the components), with an additional therapeutic agent, e.g., cemiplimab. Components administered in association with each other can be administered to a subject at a time different from when the other components are administered. For example, each administration may be administered non-simultaneously (e.g., separately or sequentially), with an interval of more than a given time period as part of a treatment regimen. Separate components administered in association with each other may be administered sequentially, albeit essentially simultaneously, during the same administration session. Furthermore, separate components administered in association with each other may be administered to a subject by the same route or by different routes. EXAMPLES

[0209] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention.

[0210] Example 1: Construction of anti-EGFRxCD28 antibodies Generation of anti-EGFR antibodies Anti-EGFR antibodies were obtained by stimulating an immune response by administering an EGFR-expressing cell line (A431) together with an adjuvant directly to VELOCIMMUNE® mice, which contain DNA encoding a human immunoglobulin heavy chain and a universal light chain variable region, such that the antibodies generated in the mice have different heavy chain variable regions but essentially identical light chain variable domains.

[0211] The antibody immune response was monitored by EGFR-specific immunoassay. When a desired immune response was obtained, anti-EGFR antibodies were directly isolated from antigen-positive B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298.

[0212] Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-EGFR antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. Table 1: Amino acid sequence identifiers of parent EGFR monoclonal antibodies (mAbs) [Table 1] Table 2: Nucleic acid sequence identifiers of parent EGFR monoclonal antibodies (mAbs) [Table 2]

[0213] Preparation of anti-CD28 antibody Anti-CD28 antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding a human immunoglobulin heavy chain and a universal light chain variable region) with human CD28 protein fused to the Fc portion of mouse IgG2a, or with cells expressing CD28, or with DNA encoding CD28.

[0214] The antibody immune response was monitored by CD28-specific immunoassay. When a desired immune response was obtained, anti-CD28 antibodies were isolated directly from antigen-positive B cells as described in U.S. Patent No. 7,582,298.

[0215] Table 3 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-CD28 antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 4.

[0216] The particular biological properties of the exemplary anti-CD28 antibodies generated according to the methods of this example are described in detail in the Examples set forth below. Table 3: Amino acid sequence identifiers of parent CD28 monoclonal antibodies (mAbs) [Table 3] Table 4: Nucleic acid sequence identifiers of parent CD28 antibodies [Table 4]

[0217] Generation of bispecific antibodies (bsAb) that bind to CD28 and EGFR Bispecific antibodies comprising an anti-EGFR specific binding domain and an anti-CD28 specific binding domain were constructed using standard methods, where the anti-EGFR antigen binding domain and the anti-CD28 antigen binding domain each comprise a different distinct HCVR paired with a common LCVR. In some examples, bispecific antibodies are constructed utilizing an anti-CD28 antibody heavy chain, an anti-EGFR antibody heavy chain, and a common light chain, and contain components such as the amino acid and nucleic acid sequences encoding the antibodies shown in Tables 5, 6, 7, and 8 below. Additional bispecific antibodies that bind to EGFR and CD28 may be prepared using parent monoclonal antibodies having the names shown in Tables 9A, 9B, and 9C. Table 5: Summary of antibody designations for the HCVR arms of anti-EGFRxanti-CD28 bispecific antibodies [Table 5] Table 6: Amino acid sequences of anti-EGFRxanti-CD28 bispecific antibodies [Table 6] Table 7: Nucleic acid sequences encoding anti-EGFRxanti-CD28 bispecific antibodies [Table 7] Table 8. Amino acid and nucleotide sequences of full-length immunoglobulin chains of bispecific antibodies bsAb7075, bsAb6321, bsAb6322, and bsAb6323 [Table 8] D = the nucleotide sequence of the DNA encoding the specified sequence P = amino acid of a polypeptide of the specified sequence The numbers refer to the sequence number of the specified sequence. HC is the full length heavy chain of the designated antibody. LC is the full length light chain of the indicated antibody.

[0218] Additional bispecific antibodies comprising one HCVR arm from a parent EGFR antibody and the other HCVR arm from a parent CD28 antibody may be generated using the techniques described herein. The parent EGFR antibodies used to generate these additional anti-EGFR x anti-CD28 bispecific antibodies have the HCVR sequences described in WO2014 / 004427. The parent CD28 antibodies used to generate these additional anti-EGFR x anti-CD28 bispecific antibodies have the amino acid sequences set forth above in Table 3. These anti-EGFR and anti-CD28 binding domains (pairings) are shown below in Tables 9A, 9B and 9C. Table 9A: Summary of parent antibody names for the HCVR arms of additional anti-EGFR x anti-CD28 bispecific antibodies [Table 9A-1] [Table 9A-2] Table 9B: Summary of parent antibody names for the HCVR arms of additional anti-EGFR x anti-CD28 bispecific antibodies [Table 9B] Table 9C: Summary of parent antibody names for the HCVR arms of additional anti-EGFR x anti-CD28 bispecific antibodies [Table 9C]

[0219] The bispecific antibody described in the Examples below consists of a human soluble heterodimeric hCD28 protein and a binding arm that has been shown to bind to human EGFR (see Biacore binding data below). The exemplified bispecific antibody was engineered to have an engineered (chimeric) IgG4 Fc domain as described in U.S. Patent Application Publication No. 20140243504A1, published August 28, 2014.

[0220] The bispecific antibody generated according to this example comprises two distinct antigen-binding domains (i.e., binding arms). The first antigen-binding domain comprises a heavy chain variable region derived from an anti-CD28 antibody (CD28-VH) and the second antigen-binding domain comprises a heavy chain variable region derived from an anti-EGFR antibody (EGFR-VH). Both anti-EGFR and anti-CD28 share a common light chain. The pairing of CD28-VH / EGFR-VH generates an antigen-binding domain that specifically recognizes CD28 on T cells and EGFR on tumor cells.

[0221] Characterization of a bispecific antibody that binds CD28 and EGFR SPR kinetics: Kinetics binding parameters were determined by surface plasmon resonance (SPR) using a T-200 instrument and a CM5 sensor (GE Healthcare). Double referenced sensorgrams were extensively fitted to a 1:1 binding model using Scrubber (BioLogics Software). The dissociation rate constant (kd) was determined by fitting the change in the binding reaction during the dissociation phase, and the association rate constant (ka) was determined by fitting the binding of the analyte at different concentrations. K D was calculated from the ratio of kd and ka. The dissociation half-life (t1 / 2) was calculated as ln2 / kd and converted to minutes.

[0222] EGFR specific method: Anti-hFc monoclonal Ab (REGN2567) was coupled to a CM5 sensor surface using conventional EDC / NHS coupling chemistry. 200-250 RU of EGFRxCD28 Ab was captured on the surface. Six 3-fold dilutions of hEGFR.mmH starting at 30 nM were injected over the surface for 5 min at a flow rate of 50 uL / min. The surface was regenerated with a 10 second pulse of 20 mM H3PO4. Dissociation was measured for 10 min. Sensorgrams were processed and fitted as described above.

[0223] CD28 capture method details: Anti-mFc polyclonal Ab (GE) was coupled to a CM5 sensor surface using conventional EDC / NHS coupling chemistry. Approximately 30 RU of hCD28.mFc was captured on the surface. Six 3-fold dilutions of EGFRxCD28 Ab starting at 90 nM were injected over the surface for 4 min at a flow rate of 50 uL / min. Dissociation was measured for 5 min. The surface was regenerated by injecting 10 mM glycine, pH 1.5 for 40 s. Sensorgrams were processed and fitted as described above.

[0224] Biacore analysis showed that EGFRxCD28 had a K of approximately 9.3E-10. D and bound to hEGFR with a K of approximately 5.2E-08. D It was shown that the antibody bound to hCD28 at 100 ng / mL (Tables 10 and 11). Table 10: Biacore kinetics for binding to EGFR [Table 10] Table 11: Biacore kinetics for binding to CD28 [Table 11]

[0225] We next verified the ability of EGFRxCD28 to induce cytotoxic activity and T cell activation using co-cultures of human T cell-containing peripheral blood mononuclear cells (PBMCs) and PEO-1. Human peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte packs of healthy donors. PBMC isolation was performed by density gradient centrifugation using 50 mL SepMate™ tubes according to the manufacturer's recommended protocol. Briefly, 15 mL of FicollPaque PLUS was layered into a 50 mL SepMate tube, followed by the addition of 30 mL of leukocytes diluted 1:2 in D-PBS. Subsequent steps followed the SepMate manufacturer's protocol. CD3+ T cells were then isolated from PBMCs using StemCell Technologies' EasySep Human T Cell Isolation Kit according to the manufacturer's recommended instructions. Isolated CD3+ T cells were 50 × 10 per vial. 6 Cells were frozen in FBS containing 10% DMSO at a concentration of 100 mM.

[0226] EGFRxCD28 significantly increased the ability of MUC16xCD3-mediated T cell induction of tumor cell killing (data not shown) from 18.8% to 71.5% (Figure 1C). Consistent with this enhanced T cell cell killing, EGFRxCD28 also enhanced T cell activation as assessed by CD25 expression and IFNγ cytokine release (Figures 1D and 1E). Of note, EGFRxCD28 in the presence of a non-targeting CD3 binding control (absence of "signal 1") had no effect on T cell cytotoxicity or activation (Figures 1C-1E).

[0227] Example 2: Overexpression of the natural CD28 ligand on tumor cells synergizes with PD-1 mAb therapy to induce CD8 T cell-dependent long-term antitumor activity in vivo. To determine whether binding of CD28 to its natural ligand could enhance the antitumor effect of PD-1 mAb in vivo, MC38 tumor cells were engineered to overexpress CD86, one of the costimulatory ligands for CD28. Specifically, to generate tumor cell lines engineered to express costimulatory ligands, pLVX lentiviral plasmids carrying the EF1a promoter encoding mouse CD86 or an empty vector and a puromycin resistance gene (pLVX.EF1a.CD86-puro and pLVX.EF1a.EV-puro, respectively) were used to transfect HEK293T cells to drive production of viral particles, which were then used to infect MC38 (National Cancer Institute, Laboratory of Tumor Immunology & Biology). Engineered cell lines expressing CD86 were isolated by fluorescence-activated cell sorting (FACS). Cells were maintained in the presence of 0.5 μg / ml puromycin under ATCC recommended conditions. The resulting cell lines were designated MC38 / CD86 and MC38 / EV.

[0228] The combination of MC38 / CD86 cells and anti-PD-1 mAb treatment significantly inhibited tumor growth (Figure 2A) and led to complete tumor regression associated with a stable survival benefit compared to negative control MC38 transfected with an empty vector control (MC38-EV) (Figure 2B). + T cell depletion completely abolished the antitumor effect induced by the combination of MC38 / CD86 and anti-PD-1 mAb treatment, suggesting that CD8 +The results demonstrated a dependency on T cells (Figure 2C). Of note, tumor-free mice initially implanted with MC38 / CD86 and treated with anti-PD-1 mAb rejected a second MC38 parental tumor implanted >60 days after the first tumor implantation, suggesting the presence of a T cell memory response (Figure 2D). In conclusion, these data indicate that constitutive expression of the CD28 ligand and synergistic anti-PD-1 treatment can confer long-term CD8-dependent antitumor immunity in vivo.

[0229] Example 3: Administration of EGFRxCD28 antibody in combination with anti-PD-1 antibody controlled and eliminated mouse-human tumor xenografts. H is human CD34 + The stem cells engrafted. The efficacy of EGFRxCD28 bispecific antibody in combination with PD-1 blocking Ab (REGN2810 (cemiplimab), E. Burova et al., Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice. Mol Cancer Ther 16, 861-870 (2017)) was validated using a human tumor xenograft model. Engraftment of human immune cell populations was demonstrated by FACS (Figure 3A). A431 epidermoid carcinoma tumor cells (obtained from ATCC) were cultured in a SIRPA-treated control group with fetal liver CD34+ cells engrafted. h / h TPO h / m Rag2 - / - Il2rg - / - Mice were subcutaneously implanted in the foetal liver. Mice were divided into four groups based on fetal liver donor, human immune cell engraftment frequency, and sex. Mice received intraperitoneal injections of isotype control, 5 mg / kg EGFRxCD28, 10 mg / kg PD-1, or the combination twice per week, starting on the day of implantation (day 0). Antibody injections were then administered every 2-3 days for the duration of the experiment. Tumors were measured in two dimensions (length x width) and tumor volumes were calculated (length x width). 2x0.5). Tumors were assigned a tumor endpoint (tumor volume >2000 mm 3 Mice were euthanized when the tumor reached atrophy or tumor ulceration.

[0230] Expression of EGFR and PD-L1 on A431 tumor cells was demonstrated by FACS (Figure 3B). Starting on the day of tumor challenge, each group was treated intraperitoneally (IP) with: (1) 10 mg / kg human isotype control No. 1 + 5 mg / kg human isotype control No. 2 (2) 10 mg / kg anti-human PD-1 (REGN2810, cemiplimab) + 5 mg / kg human isotype control No. 2 (3) 5 mg / kg EGFRxCD28 (REGN7075) + 10 mg / kg human isotype control No. 1, or (4) 5 mg / kg EGFRxCD28 (REGN7075) + 10 mg / kg anti-human PD-1 (REGN2810). Antibody injections were then administered twice per week for the duration of the experiment. Tumors were measured in two dimensions (length x width) and tumor volumes were calculated (length x width). 2 x0.5). Tumors were assigned a tumor endpoint (tumor volume >2000 mm 3 Mice were euthanized when the tumor reached atrophy or tumor ulceration. Isotype control No. 1 and Isotype control No. 2 are negative control antibodies that bind to Fel d1.

[0231] The size of A431 tumors over time in each treatment group is described in Figure 4. EGFRxCD28 in combination with anti-PD-1 synergistically controlled and eliminated human tumor xenografts in humanized mice. Monotherapy of EGFRxCD28 could significantly delay tumor growth, and combination with anti-PD-1 Ab further inhibited tumor progression. When control PSMAxCD28 Ab was used in the same treatment setting, with or without anti-PD1 treatment, no tumor growth inhibition was observed (Figure 5). No expression of PSMA on A431 tumor cells was demonstrated by FACS (Figure 6).

[0232] These data demonstrated that anti-PD1 monotherapy induced a limited anti-tumor response, anti-EGFR monotherapy induced a partial anti-tumor response, whereas the combination therapy induced a robust and stable anti-tumor immune response.

[0233] Example 4: Administration of EGFRxCD28 antibody as monotherapy controlled and eliminated human A431 tumor xenografts in NSG mice engrafted with human PBMC cells. A431 epidermoid carcinoma tumor cells (obtained from ATCC) were mixed with human PBMCs and cultured in NSG mice (NOD / SCID / γ c null ) were implanted subcutaneously in mice. Mice were treated with either a prophylactic (treatment on days 0, 3, 7) or therapeutic (days 3, 7, 10) protocol.

[0234] For the prophylactic protocol, mice were divided into four treatment groups. Each group was treated intraperitoneally (IP) with: (1) 5 mg / kg control Ab EGRvIIIxCD3 (bs17664D) (2) 5mg / kg EGFRxCD28 (REGN7075) (3)0.5mg / kg EGFRxCD28(REGN7075) (4)0.05mg / kg EGFRxCD28(REGN7075)

[0235] For the therapeutic protocol, mice were divided into three treatment groups. Each group was treated with: (1) 5 mg / kg control Ab EGRvIIIxCD3 (bs17664D) (2) 5mg / kg EGFRxCD28 (REGN7075) (3)0.05mg / kg EGFRxCD28(REGN7075)

[0236] Tumors were measured in two dimensions (length x width) and tumor volumes were calculated (length x width). 2x0.5). Tumors were assigned a tumor endpoint (tumor volume >2000 mm 3 Mice were euthanized when the tumor reached atrophy or tumor ulceration.

[0237] The mean tumor sizes of mice treated with each treatment group are summarized in Figures 7A and 7B. These data show that with prophylactic treatment, strong antitumor responses were observed at all three doses tested, although responses were less complete than with therapeutic treatment.

[0238] Example 5: Administration of EGFRxCD28 antibody as monotherapy controlled and eliminated human A549 tumor xenografts in NSG mice engrafted with human PBMC cells. A549 lung adenocarcinoma tumor cells (obtained from ATCC) were cultured in NSG mice (NOD / SCID / γ c null ) were subcutaneously implanted on day 0. Human PBMCs were engrafted intraperitoneally on day 3. Mice were divided into four treatment groups. Each group was treated intraperitoneally (IP) with: (1) 10 mg / kg control Ab EGRvIIIxCD3 (bs17664D) (2) 1mg / kg EGFRxCD28 (REGN7075) (3)10mg / kg EGFRxCD28(REGN7075)

[0239] Ab treatments were administered on days 15, 22 and 29. Tumors were measured in two dimensions (length x width) and tumor volumes were calculated (length x width). 2 x0.5). Tumors were assigned a tumor endpoint (tumor volume >2000 mm 3 Mice were euthanized when the tumor reached atrophy or tumor ulceration.

[0240] The mean tumor size of mice treated with each treatment group is summarized in Figure 8. In the setting of therapeutic treatment with engrafted tumors, a significant dose-dependent anti-tumor response was observed.

[0241] Example 6: EGFRxCD28 costimulatory bispecific antibody binds to CD28+ and EGFR+ cells. Flow cytometry analysis was used to determine the binding of EGFRxCD28 (REGN6323) to Jurkat cells (CD28+ cells) and PEO1 cells (EGFR+ cells). Binding of the antibody to each cell type was observed. See Figures 1A and 1B.

[0242] Cell lines that endogenously express EGFR (PEO1, EGFR + ) were labeled with 1 μM Violet Cell Tracker and incubated overnight at 37° C. Separately, human PBMCs (New York Blood Center) or cynomolgus monkey PBMCs (Covance, Cranford, NJ) were incubated at 1×10 6 Cells were seeded at 2000 cells / mL in supplemented RPMI medium and incubated overnight at 37° C. to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, target cells were co-incubated for 96 h at 37° C. with adherent cell-depleted naïve human PBMCs (effector / target cell ratio of 4:1) and serial dilutions of either TSAxCD3 or non-targeted CD3-based bispecifics alone or in combination with a fixed concentration (2.5 μg / ml) of TSAxCD28 bispecifics. Cells were removed from cell culture plates using enzyme-free cell dissociation buffer and analyzed by flow cytometry (FACS).

[0243] For FACS analysis, cells were stained with viability far red cell tracker (Invitrogen) and directly conjugated antibodies against CD2, CD4, CD8 and CD25 (BD). For cell counting, samples were analyzed with calibration beads. To assess the specificity of killing, target cells were gated as the Violet cell tracker positive population. The percentage of live target cells was calculated as follows: % live cells = (R1 / R2) * 100, where R1 = % live target cells in the presence of antibody, and R2 = % live target cells in the absence of the test antibody. T cell activation was determined by CD2 + / CD4 + T cells or CD2 + / CD8 + Activation of T cells (CD25 + ) T cell counts were measured by the percentage of live CD4 + Cells or CD8 + The number of cells was determined by counting.

[0244] The levels of cytokines accumulated in the medium were analyzed using the BD cytometric Bead Array (CBA) human Th1 / Th2 / Th17 cytokine kit according to the manufacturer's protocol.

[0245] Example 7: Analysis of intratumoral T cell clusters. To examine whether treatments had a significant effect on human T cell activation in the A431 tumor xenograft model, intratumoral T cells were profiled. CITRUS (cluster identification, characterization, and regression), a method for independently stratifying statistically significantly distinct T cell clusters, was used to identify responsive human CD8+ / CD4+ T cell clusters upon single or combination treatments.

[0246] For flow cytometric analysis of in vivo experiments, tumors were harvested, single cell suspensions were prepared, and red blood cells were lysed using ACK Lysis buffer (ThermoFisher Scientific). Live / dead cell discrimination was performed using the Live / dead fixable blue dead cell staining kit (ThermoFisher Scientific). Samples were acquired on a Symphony (BD Bioscience) and analyzed using Cytobank software (Cytobank, Santa Clara, CA). Analysis was performed with equal numbers of events per sample. Event range was determined by the sample with the fewest events acquired. CITRUS analysis from Cytobank was used to automatically cluster T cells based on specific markers.

[0247] Both single-agent treatment with EGFRxCD28 (REGN7075) and anti-PD-1 Ab (cemiplimab) reduced the CD8+ T cell cluster C1, which expresses high levels of PD-L1 and ICOS (see Figures 9A and 9B). Blockade of PD-1 alone stably induced the expansion of CD8+ T cell cluster C2 with a highly activated phenotype (high levels of CD2, CD86, GITR, TIGIT and ICOS). Surprisingly, more diverse CD4+ T cell clusters were observed to respond to single-agent or combination treatment (Figures 9C and 9D). Only combination treatment significantly expanded cluster C2, which had expanded effector memory-like phenotype (high Ki67, low CD45RA and CCR7), and less expanded exhausted effector memory-like cluster C3 (low CD38, CD45RA and CCR7). Similar to CD8+ T cells, combination treatment significantly reduced cluster C1 with high levels of PDL1 expression. PD-1 blockade monotherapy induced expansion of effector memory cells with high levels of CD38 expression (C4), representing a more activated phenotype. EGFRxCD28 monotherapy maintained a small population of naive-like CD4+ T cells (C5) in the tumor, which could be further primed / activated by anti-PD1 Ab treatment.

[0248] These data indicate that human xenograft tumor models in humanized mice provided a powerful platform to validate EGFRxCD28 bispecific antibodies as monotherapy and in combination with PD-1 blockade. Advanced FACS analysis and fully automated clustering identification methods allow detailed analysis of underlying immune mechanisms in human immune-reconstituted mice. PD1 blockade alone was capable of inducing robust expansion of CD8+ and CD4+ T cells with an activated phenotype, but was insufficient to promote memory development and induce significant tumor inhibition. In combination with EGFRxCD28 treatment, a balanced T cell activation state could be achieved to induce robust antitumor responses.

[0249] Example 8: EGFRxCD28 alone or in combination with anti-PD1 therapy does not induce systemic T cell activation compared to CD28 superagonists in cynomolgus monkeys. A study in cynomolgus monkeys was conducted to evaluate the tolerability of the EGFRxCD28 bispecific (REGN7075) alone or for potential synergistic pharmacology in combination with an anti-PD1 antibody. Three monkeys per treatment group received a single dose (10 mg / kg) of EGFRxCD28 alone or in combination with REGN2810 (cemiplimab) (10 mg / kg) via intravenous infusion (continuous infusion was given in the combination group).

[0250] Cynomolgus monkey studies were performed in accordance with IACUC guidelines. For studies with EGFRxCD28, female cynomolgus monkeys (Macaca fascicularis) were performed at SNBL USA (Accredited by the Assessment and Acredibility of Laboratory Animal Care (AAALAC); Animal Welfare Assurance published by the Office of Laboratory Animal Welfare (OLAW); Registered with the United States Department of Agriculture (USDA) and the institutional animal care and use committee (IACUC)). For flow cytometry, blood was collected from a peripheral vein in restrained, conscious animals into potassium EDTA tubes. 100 μl of whole blood was stained with the indicated antibodies. Flow cytometry data acquisition was performed using a FACS Canto II. The absolute numbers of individual groups were calculated from the relative percentages derived from the gating of the parent / grandparent groups and the total number of parent / grandparent groups from the validated hematology analyzer according to the following formula: absolute number of groups (x10 3 / μL) = (relative percentage of group % x total number of parent / grandparent groups) / 100. For serum cytokines, blood was collected into serum separators containing anticoagulant. Serum was separated by centrifugation in a centrifuge set at 4°C. Analyzed using the MSD U-Plex platform (IL-2, 4, 6, 8, 10, TNFα and IFNγ).

[0251] Toxicity assessment was based on clinical observations, qualitative food intake, body weight and vital signs (temperature, heart rate, pulse oximetry, and respiratory rate), as well as clinical and anatomical pathology findings at the completion of the study. Blood samples were taken for cytokine analysis and FACS immunophenotyping. EGFRxCD28 alone or in combination with cemiplimab was well tolerated, and all animals survived until scheduled necropsy. No test article-related clinical observations were noted (data not shown). No changes in organ weights were observed, and no gross changes were noted at terminal necropsy (data not shown). Furthermore, no significant cytokine release, T cell margination, or activation was observed (Figures 10A-10C). In contrast, significant cytokine release, lymphocyte margination, and T cell activation were observed in monkeys receiving the CD28 "superagonist" (data not shown). No significant treatment-related histological changes were observed in monkeys receiving EGFRxCD28 alone or in combination with cemiplimab.

[0252] Example 9. Dose-dependent anti-tumor responses mediated by combination therapy of EGFRXCD28 bispecific antibody (REGN7075) and anti-PD-1 antibody (REGN2810) Materials and Methods NCI-H292

[0253] The NCI-H292 cell line is a human mucoepidermoid lung carcinoma cell line (ATCC CRL-1848) isolated from a 32-year-old female patient. The cell line was maintained in RPMI-1640 containing 10% FBS supplemented with PSG (penicillin, streptomycin, and glutamine).

[0254] Peripheral blood mononuclear cells (PBMC) Human PBMCs were obtained from ReachBio, catalog number 0500-401, donor number 0190205 was used in this study.

[0255] Procedures used in tumor testing: Female NSG mice (approximately 10 weeks old) were used in this study. Mice were inoculated with 2x10 6 Mixed with 4x10 PBMCs 6 Tumor cells were implanted subcutaneously. Tumor growth was monitored by caliper twice weekly for the duration of the study. Antibodies specified in Table 12 were administered by intraperitoneal injection at the prescribed doses as monotherapy or in combination therapy on the indicated days after tumor implantation. Experiments were terminated when mice developed ulcerated tumors or when tumor volumes reached 1500 cm, as per IACUC criteria. 3 The study was terminated when the mean age of the study exceeded 1. The statistical analysis is shown in Table 13. The data are summarized in Figures 11 and 12. Table 12 - Study Design [Table 12] Isotype control No. 1 and Isotype control No. 2 are negative control antibodies that bind to Feld1. REGN7075 is an anti-EGFRXCD28 bispecific antibody, also referred to as bsAb7075. REGN2810 is an anti-PD-1 antibody. Table 13: Statistical analysis at day 32: Mixed effects model (REML) analysis [Table 13-1] [Table 13-2]

[0256] summary As shown in Figures 11 and 12, the data demonstrated that when validated in this model, the most robust anti-tumor responses were observed when REGN7075 (anti-EGFRX anti-CD28) was combined with an anti-PD-1 antibody (REGN2810) compared to results obtained when either antibody was used alone.

[0257] Example 10A: Flow cytometry binding titration using REGN6323 Flow cytometric analysis was used to determine the binding of anti-EGFRX-anti-CD28 bispecific antibodies to several cancer cell lines (A375, 22RV1, PEO1, CAPAN2, SW1990, H292), human and cynomolgus monkey T cells, and Jurkat cells, and was detected with a labeled anti-human IgG secondary antibody. 5 Cells / wells were incubated for 30 min at 4°C with serially diluted EGFRxCD28 bispecific antibody or control antibody (human IgG4 antibody that binds to human antigen with no cross-reactivity to human or cynomolgus CD28). The antibodies ranged from 100 nM to 1.5 pM for human and cynomolgus T cells and 133 nM to 2 pM for EGFR expressing cells. After incubation, cells were washed twice with chilled PBS containing 1% filtered FBS and fluorophore-conjugated anti-human secondary antibody was added to the cells and incubated for an additional 30 min. Live / dead dye was added to human and cynomolgus T cell incubations. Wells containing only secondary antibody without test antibody were used as controls.

[0258] After incubation with EGFR-expressing cells, cells were washed and resuspended in 200 μL of chilled PBS containing 1% filtered FBS and analyzed by flow cytometry on a BD FACS Canto II.

[0259] After incubation with human or cynomolgus T cells, cells were washed and stained with a cocktail of anti-CD2, anti-CD16, anti-CD4, and anti-CD8 antibodies in Brilliant staining buffer for an additional 20 min incubation at 4° C. After washing, cells were resuspended in 200 μL of chilled PBS containing 1% filtered FBS, gated on Live / CD2+ / CD4+ / CD16- or Live / CD2+ / CD8+ / CD16-, and analyzed by flow cytometry on a BD FACS LSR-Fortessa-X20.

[0260] Flow Cytometry Binding Titer Results (See Tables 14A and 14B, and Figures 13A, 13B, and 13C) The binding of anti-EGFRX anti-CD28 bispecific antibody (REGN6323) to the surface of human and cynomolgus T cells was verified by flow cytometry as described above. The results showed that REGN6323 bound to both human and cynomolgus CD4+ and CD8+ T cells and did not reach saturation within the selected concentration range (Figure 13A).

[0261] The binding of anti-EGFRX anti-CD28 bispecific antibody (REGN6323) to the surface of other cell lines expressing EGFR was also verified by flow cytometry. The cell lines used were Jurkat cells, an immortalized line of human T lymphocyte cells (see FIG. 13C), A375 cells, an epithelial melanoma cell line, 22RV1 cells, an epithelial prostate cancer, PEO1 cells, an ovarian cancer cell line derived from ascites, CAPAN2, a pancreatic adenocarcinoma cell line, SW1990, a pancreatic adenocarcinoma derived from a metastatic site (spleen), and H292, a lung epithelial carcinoma cell line. The results showed that REGN6323 bound to A375 with an EC50 of 1.48E-09M. REGN6323 bound to 22RV1 with an EC50 of 5.54E-10M. REGN6323 bound to PEO1 with an EC50 of 6.67E-10. REGN6323 bound to CAPAN2 with an EC50 of 1.29E-09 M. REGN6323 bound to SW1990 with an EC50 of 2.96E-09 M. REGN6323 also bound to H292 cells with an EC50 of 6.74E-10 (see FIG. 13B).

[0262] The isotype control antibody did not bind to either human or cynomolgus T cells, nor to cell lines expressing EGFR.

[0263] These data suggest that REGN6323 has the ability to bind to a variety of cancer cell lines that express EGFR and therefore may be effective in treating multiple cancer indications.

[0264] Example 10B: Flow cytometry binding titration using REGN6321 and REGN6322 Experiments were also performed to determine whether other anti-EGFRX anti-CD28 bispecific antibodies could exhibit binding to multiple cell lines. Following the protocol described above, REGN6321 and REGN6322 were tested in a similar manner. REGN6323 was also included in this study.

[0265] As shown in Figure 13C, the results showed that REGN6321, REGN6322, and REGN6323 exhibited binding to the prostate cancer line 22RV1, as well as to a cell line named PEO1, which is an ovarian cancer cell line. These results showed that these additional anti-EGFRX anti-CD28 antibodies also have the ability to bind to other cancer cell lines that express EGFR. Table 14: Summary of flow cytometry binding studies with various EGFRXCD28 bispecific antibodies [Table 14-1] [Table 14-2] NC: EC50 not calculated; NT: not validated antibody.

[0266] Example 11: Cytotoxicity measured by flow cytometry To monitor the killing of cells expressing tumor-specific antigens (TSAs) of various sizes in the presence of a combination of anti-TSA x anti-CD3 and EGFRxCD28 antibodies, 22RV1 (PSMA+, STEAP2+), or PEO1 (MUC16+), or CAPAN2 (MUC16+), or SW1990 (MUC16+), or H292 (MUC16+) cells were labeled with 1 μM of the fluorescent tracking dye Violet Cell Tracker. After labeling, cells were incubated overnight at 37°C. Separately, human PBMCs were cultured at 1x10 6Cells were seeded at 10 ... 5E05 counting beads were added to each well immediately prior to flow cytometry analysis. 1E05 beads were collected for each sample. To assess specificity of killing, cells were gated on the live Violet labelled population. The percentage of live cells was recorded and used to calculate viability.

[0267] T cell activation was assessed by incubating cells with directly conjugated antibodies against CD2, CD4, CD8, and CD25 and recording the percentage of late-activated T cells among total T cell numbers (CD2+) or CD8+ T cells.

[0268] Flow Cytometry-Based Cytotoxicity Assay Results (See Table 15 and Figures 14 and 15) The costimulatory EGFRxCD28 bispecific antibody REGN6323 was tested for its ability to enhance the cytotoxicity of TSAxCD3 antibodies targeting cell surface antigens of various sizes and several cancer indications.

[0269] REGN6323 successfully enhanced the cytotoxicity of various TSAxCD3 bispecific antibodies targeting PSMA or MUC16 or STEAP2 in the presence of unstimulated human PBMC. Target cell killing was enhanced in the presence of the EGFRxCD28 and TSAxCD3 bispecific combination compared to TSAxCD3 single agent treatment, and cells were killed in a dose-dependent manner with an EC50 of pM.

[0270] The observed enhanced lysis of target cells was associated with enhanced CD25+ expression on T cells, again with an EC50 in pM. Table 15 [Table 15-1] [Table 15-2]

[0271] Example 12: Epitope Mapping on EGFR with REGN7075, REGN6323, and REGN6322 by HDX-MS Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of epidermal growth factor receptor (recombinant human EGFR, amino acid sequence in appendix) that interact with REGN7075 (mAb12999P2 x mAb14226P2), mAb13006P (REGN6323 EGFR arm parent), and mAb35193P (REGN6322 EGFR arm parent). General descriptions of HDX-MS are described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A. Results are shown in Tables 16, 17, and 18.

[0272] HDX-MS experiments were performed on an integrated MS platform consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical gradients, and a Thermo Q Exactive HF mass analyzer for peptide mass measurement.

[0273] The labeling solution was prepared in D2O at pD 7.0 as PBS buffer (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25 °C). For deuterium labeling, 11 μL of 52.0 μM hEGFR.mmh (Regeneron's in-house protein REGN355) or hEGFR.mmh (Ag-Ab complex) premixed with REGN7075 at a 1:1 molar ratio, 11 μL of 34.3 μM hEGFR.mmH (Regeneron's in-house protein REGN355) or hEGFR.mmH (Ag-Ab complex) premixed with mAb13006P2 at a 1:0.6 molar ratio, and 11 μL of 31.4 μM hEGFR.mmH (Regeneron's in-house protein REGN355) premixed with mAb35193P2 at a 1:0.6 molar ratio. hEGFR.mmH (REGN355) or hEGFR.mmH (Ag-Ab complex) were incubated in duplicate in 44 μL of DO labeling solution at 20° C. for various time points (e.g., non-deuterated control=0 s, deuterium labeling at 5 and 10 min). The deuterium reaction was quenched by adding 55 μL of pre-chilled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample with a 5 min incubation at 20° C. The quenched samples were then injected into a Waters HDX Manager and online pepsin / protease XIII digestion was performed. The digested peptides were separated on a C8 column (1.0 mm x 50 mm, NovaBioassays) with a 22 min gradient from 0% to 90% B (mobile phase A: 0.5% formic acid and 4.5% acetonitrile in water, mobile phase B: 0.5% formic acid in acetonitrile) at −9.5°C. The eluted peptides were analyzed by a Thermo Q Exactive HF mass spectrometer in LC-MS / MS or LC-MS mode.

[0274] LC-MS / MS data of non-deuterated EGFR samples were searched using the Byonic search engine (Protein Metrics, Inc.) against a database containing the hEGFR.mmH(REGN355) sequence and its reverse sequence. Search parameters were set as default with non-specific enzymatic digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into HDX Workbench software (version 3.3) to calculate the deuterium uptake of each peptide detected by LC-MS from all deuterated samples. For a given peptide, the centroid (intensity-weighted average mass) at each time point was used to calculate deuterium uptake (D) and the percentage of deuterium uptake (%D). Deuterium incorporation (D-incorporation) = average mass (deuterated) - average mass (non-deuterated)

number

[0275] A total of 271 peptides derived from hEGFR.mmH (REGN355) were identified from hEGFR.mmH alone or hEGFR.mmH samples complexed with REGN7075, representing 84.4% sequence coverage of hEGFR.mmH. All peptides exhibiting different percentage D-incorporation values ​​greater than 5% were defined as significantly protected. Peptides corresponding to amino acids 345-368 (LHILPVAFRGDSFTHTPPLDPQEL SEQ ID NO: 70) and 399-416 (LEIIRGRTKQHGQFSLAV SEQ ID NO: 71) on hEGFR.mmH were significantly protected by REGN7075 (hEGFR.mmH residues are numbered according to the hEGFR.mmH sequence).

[0276] A total of 341 peptides derived from hEGFR.mmH (REGN355) were identified from hEGFR.mmH alone or hEGFR.mmH samples complexed with mAb13006P, representing 85.8% sequence coverage of hEGFR.mmH. All peptides exhibiting different percentage D-incorporation values ​​greater than 5% were defined as significantly protected. Peptides corresponding to amino acids 345-368 (LHILPVAFRGDSFTHTPPLDPQEL SEQ ID NO: 70) and 399-416 (LEIIRGRTKQHGQFSLAV SEQ ID NO: 71) on hEGFR.mmH were significantly protected by mAb13006P (hEGFR.mmH residues are numbered according to the hEGFR.mmH sequence in the Appendix).

[0277] A total of 335 peptides derived from hEGFR.mmH (REGN355) were identified from hEGFR.mmH alone or hEGFR.mmH samples complexed with mAb35193P, representing 85.5% sequence coverage of hEGFR.mmH. All peptides exhibiting different percentage D-incorporation values ​​greater than 5% were defined as significantly protected. A peptide corresponding to amino acids 133-154 (CNVESIQWRDIVSSDFLSNMSM SEQ ID NO: 72) on hEGFR.mmH was significantly protected by mAb35193P (hEGFR.mmH residues are numbered according to the hEGFR.mmH sequence). Table 16: hEGFR.mmH peptides receive significant protection upon formation of hEGFR.mmH-REGN7075 complexes compared to hEGFR.mmH alone [Table 16] Table 17: hEGFR.mmH peptide confers significant protection upon formation of hEGFR.mmH-mAb13006P complex compared to hEGFR.mmH alone [Table 17-1] [Table 17-2] Table 18: hEGFR.mmH peptide confers significant protection upon formation of hEGFR.mmH-mAb35193P complex compared to hEGFR.mmH alone [Table 18]

[0278] hEGFR.mmh amino acid sequence hEGFR(ECD).CPGG.MMH6(REGN355): human EGFR (amino acids L25-A647, accession number NM_005228.4), containing a C-terminal CPGG.myc epitope(E1-L10).GlyGly.myc epitope(E1-L10).SerGly.6XHis.SSG tag (mmh tag is underlined) LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMD FQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPLMLYNPTTYQMDVNPEGKYSFGATCVKCCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRK VCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIA CPGGEQKLISEEDLGGEQKLISEEDLSGHHHHHHSSG (SEQ ID NO:69)

[0279] Example 13: Surface plasmon resonance derived binding affinity and kinetic constants of EGFRxCD28 bispecifics and related parent antibodies to EGFR and CD28 EGFR experimental procedures The equilibrium dissociation constant (K DValues) were determined using a real-time surface plasmon resonance biosensor using a Biacore4000 instrument. A CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567, lot no. REGN2567-L1) to capture purified anti-EGFRxCD28 bispecific Ab. Biacore binding experiments were performed in a buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Various concentrations of hEGFR.mmH (REGN355) containing a C-terminal myc-myc-hexahistidine tag were prepared in HBS-EP running buffer (3-fold serial dilutions ranging from 30 nM to 120 pM) and injected over the Ab capture surface at a flow rate of 30 μL / min. Binding was monitored for 5 min and dissociation of hEGFR.mmH in HBS-EP running buffer was monitored for 10 min. All binding kinetic experiments were performed at 25 °C. Binding (k a ) and dissociation (k d The reaction rate constant of the binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the reaction rate constants as follows: K D (M)=k d / k a and t 1 / 2 (min)=0.693 / k d / 60

[0280] The binding kinetic parameters of hEGFR.mmH to purified EGFRxCD28 Ab at 25° C. are shown in Table 19 below.

[0281] CD28 Experimental Procedure Equilibrium dissociation constants (K DValues) were determined using a real-time surface plasmon resonance biosensor using a Biacore T-200 instrument. A CM5 Biacore sensor surface was derivatized by amine coupling with a polyclonal rabbit anti-mouse Fc antibody (GE Healthcare, Cat. No. BR100838) and purified hCD28 with a C-terminal Fc tag (hCD28.mFc, REGN2012) was captured. Biacore binding experiments were performed in a buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Various concentrations of the bispecific Ab prepared in HBS-EP (3-fold serial dilutions ranging from 90 nM to 1.1 nM) were injected over the hCD28.mFc capture surface at a flow rate of 50 μL / min. Binding was monitored for 4 min and dissociation of the bispecific Ab in HBS-EP was monitored for 10 min. All binding kinetic experiments were performed at 25 °C. Binding (k a ) and dissociation (k d The reaction rate constant of the binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the reaction rate constants as follows: K D (M)=k d / k a and t 1 / 2 (min)=0.693 / k d / 60

[0282] The binding kinetic parameters of EGFRxCD28 Ab to hCD28.mFc at 25° C. are shown in Table 20 below.

[0283] Data summary in tabular format Table 19: Binding kinetics of hEGFR.mmH to anti-EGFRxCD28 bispecific mAb at 25°C [Table 19] Table 20: Binding kinetics of hCD28.mFc to anti-EGFRxCD28 bispecific mAb at 25°C [Table 20]

[0284] Example 14: Characterization of EGFRxCD28 bispecific antibodies in a ligand-mediated cell proliferation assay using BaF3 cells engineered to overexpress human EGFR. The EGFR (ErbB1, HER1) receptor is a member of the receptor tyrosine kinase (RTK) family that controls cell proliferation, survival, differentiation and migration in multicellular organisms. Activation of the receptor occurs via binding of its soluble ligands, such as EGF or TGFβ, inducing homodimerization and autophosphorylation of EGFR, ultimately resulting in the activation of multiple intracellular signaling cascades, such as the Ras / MAPK, PLCγ1 / PKC, PI3-kinase / Akt and STAT pathways (Wieduwilt et al. 2008; Cell Mol Life Sci. May; 65(10):1566-1584).

[0285] To test the blocking effect of anti-EGFRxCD28 antibodies on EGFR signaling, we developed a proliferation assay using an engineered IL-3-dependent Ba / F3 murine hematopoietic cell line genetically modified to stably overexpress human epidermal growth factor receptor (amino acids M1-A1210 of EGFR-Genbank accession number NP_005219.2) (Kong et al. 2017; Oncotarget,Vol.8,(No.22),pp:35488-35489). Engineered BaF3 / hEGFR cells were stimulated with ligands (human EGF or TGFβ) in the presence of titrated antibodies, and cell proliferation was measured by tritium uptake as a function of cell proliferation.

[0286] Experimental procedure Engineered BaF3 / hEGFR cells were grown in culture medium (RPMI1640+10% FBS+penicillin / streptomycin / L-glutamine+10μg / mL mouse IL-3+0.5μg / mL puromycin) and starved in IL-3-free medium (RPMI1640+1% FBS) for 24 hours before being used for cell proliferation assays. Briefly, 12.5×10^5 cells / well were plated in 96-well round-bottom tissue culture plates, and 1:3 serial dilutions of antibodies ranging from 15.2pM to 100nM were added in the presence of constant ligand hEGF (0.5nM) or hTGFβ (0.5nM) (including a control containing no antibody). Plates were incubated for 72 hours at 37°C / 5% CO2, 0.3 μCi / well tritiated thymidine was added to the cells, and plates were incubated for an additional 16 hours. Thymidine, or tritium, is incorporated in large amounts into newly synthesized DNA in dividing cells. After incubation, cells were harvested into 96-well UniFilter plates and 30 μL of scintillation fluid was added to each well. Tritium incorporation was measured as counts per minute (CPM) using a Microplate Scintillation & Luminescence Counter TopCount NXT instrument. Cells, antibodies, and ligands were prepared in assay medium (Opti-MEM + 0.1% bovine serum albumin) and all serial dilutions were tested in duplicate.

[0287] Antibody IC 50 / EC 50 Values ​​were determined from a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism™ software. Maximum inhibition of proliferation was determined by the following formula:

number

[0288] Summary of results: EGFR x CD28 bispecifics (REGN6322; REGN6323 and REGN7075) and their corresponding parental EGFR antibodies (mAb35193P2; mAb13006P2 and mAb12999P2) were validated in the presence of 0.5 nM hEGF or hTGFβ, along with an in-house produced comparator antibody (Erbitux) and an isotype-matched negative control (see Table 21).

[0289] In the presence of 0.5 nM EGF: None of the bispecific antibodies (REGN6322; REGN6323, and REGN7075) showed dose-dependent proliferation inhibition. The maximum inhibition values ​​ranged from 30.5 to -6.8% (negative values ​​indicate increased proliferation). On the other hand, mAb35193P2, mAb12999P2, and in-house manufactured Erbitux inhibited proliferation of BaF3 / hEGFR cells with IC 50 and maximum inhibition values ​​were 2.55 nM / 29.6%, 19.8 nM / 91.8%, and 6.69 nM / 97.6%, respectively. mAb13006P2 showed a maximum inhibition of 93.2%, with an IC 50 Values ​​could not be calculated. The negative isotype control antibody showed no inhibition as expected.

[0290] In the presence of 0.5 nM hTGFβ: REGN6322 demonstrated a 40.6% increase in proliferation and 50 The IC was 3.67 nM, whereas REGN6323 and REGN7075 showed no reaction, similar to the negative isotype control antibody. In contrast, mAb13006P2, mAb12999P2, and in-house manufactured Erbitux inhibited proliferation and had IC 50 and maximum inhibition values ​​were 6.55 nM / 97.2%, 7.79 nM / 95.9%, and 1.38 nM / 99.8%, respectively. mAb35193P2 showed an increase in proliferation by 32.7%, with an EC 50 The value was 914 pM. Table 21: Maximum inhibition and efficacy values ​​of antibodies [Table 21-1] [Table 21-2] Abbreviations ND: not determined; NC: not calculable; no significant value could be determined by PRISM. Example 15: Characterization of EGFRxCD28 Bispecific Antibodies in an Allogeneic T Cell Activation Assay Using NCI-H292 and Human Primary T Cells Table 22: Reagent / Antibody Information / Materials: [Table 22]

[0291] background Two signals, "signal 1" and "signal 2", are required for proper T cell activation. "Signal 1" is induced by the binding of the T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs), whereas "signal 2" occurs when the costimulatory CD28 receptor on T cells engages its ligand cluster of differentiation 80 or 86 (CD80 / CD86) on APCs (Martin et al. 1986; June et al. 1987; Harding et al. 1992). Thus, activation of CD28 signaling provides a targeted method to enhance existing TCR signaling.

[0292] EGFRxCD28 bispecific antibody inhibits EGFR in allogeneic T cell activation assays + Target cells and CD28 +It is designed to mimic the natural ligand CD28 by cross-linking T cells, generating a "signal 2" to enhance T cell activation in the presence of an existing "signal 1". However, T cell activation can be inhibited by ligation of the programmed cell death protein 1 receptor (PD-1) on T cells with its ligand, PD-L1 on APCs. Ligation of PD-1 results in the recruitment of phosphatases to the CD28 and TCR complex (Zou et al. 2008, Francisco et al. 2010, Hui et al. 2017), which then attenuates TCR signaling and CD28 stimulation. Thus, blocking the PD-1 / PD-L1 interaction with cemiplimab in combination with an EGFRxCD28 bispecific antibody may enhance T cell function and promote target cell killing, for example, in cancer.

[0293] Experimental procedure The ability of the EGFRxCD28 bispecific antibody to activate human primary T cells by engaging EGFR and CD28 to deliver "signal 2" was determined by IL-2 release and T cell proliferation, and demonstrated that EGFR was sufficient to generate an allogeneic TCR response to serve as "signal 1." + / PD-L1 + was evaluated in the presence of a human lung cancer cell line (NCI-H292).

[0294] Primary human CD3 + Isolation of T cells Human peripheral blood mononuclear cells (PBMCs) were isolated and frozen from healthy donor leukocyte packs from Precision for Medicine (donor 555015) using the EasySep™ Direct Human PBMC Isolation Kit according to the manufacturer's recommended protocol. CD3 + T cells were isolated by thawing a vial of frozen PBMCs. Donor PBMCs were isolated using StemCell Technologies' EasySep™ Human CD3 +Isolate CD3 T cells using a T cell isolation kit according to the manufacturer's recommended instructions. + T cells were enriched.

[0295] IL-2 release assay Enriched CD3 + T cells were resuspended in stimulation medium at 1 x 10 5 Growth-arrested NCI-H292 cells express endogenous EGFR and PD-L1 and were added at a concentration of 5 × 10 4 CD3 cells / well final concentration + T cells were added to the wells. Subsequently, REGN6322, REGN6323, REGN7075, non-TAAxCD28, and their isotype-matched controls (IsoC-2) were titrated in a 1:4 dilution from 0.76 pM to 50 nM and added to the wells. The end point of the 10-point dilution did not contain titrated antibodies. After addition of titrated antibodies, a fixed concentration of 20 nM of either cemiplimab or its isotype-matched control (IsoC-1) was added to the wells. The plates were incubated at 37 °C and 5% CO2 for 96 h, 50 μL of total supernatant was removed, and 5 μL from the collected supernatant was used for IL-2 measurement. The amount of IL-2 in the assay supernatant was determined using PerkinElmer's AlphaLisa kit according to the manufacturer's protocol. IL-2 measurements were acquired on a Perkin Elmer multilabel plate reader Envision, and values ​​were reported as pg / mL. All serial dilutions were tested in triplicate.

[0296] Antibody EC 50 Values ​​were determined from a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism™ software. Maximum IL-2 is given as the mean maximum response detected within the validated dose range.

[0297] T cell proliferation assay After 96 hours of incubation at 37°C and 5% CO2, the supernatant was removed (see IL-2 release assay), 0.25 mCi / well of titrated thymidine was added to the cells, and the plates were incubated for an additional 6–8 hours. Thymidine, or tritium, is abundantly incorporated into newly synthesized DNA in dividing cells. After incubation, cells were harvested into 96-well UniFilter plates and 30 μL of scintillation fluid was added to each well. Tritium incorporation was measured as counts per minute (CPM) using a Microplate Scintillation & Luminescence Counter TopCount NXT instrument. All serial dilutions were tested in triplicate.

[0298] Antibody EC 50 Values ​​were determined from a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism™ software. Maximum CPM is given as the mean maximum response detected within the dose range examined.

[0299] Summary of Results (Table 23) In the presence of cemiplimab, EGFRxCD28 antibody treatment (REGN6322, REGN6323, and REGN7075) elicited higher IL-2 and proliferative responses compared to non-TAAxCD28 and IsoC-2, CD28 bispecific isotype-matched controls, whereas in the absence of cemiplimab, EGFRxCD28 antibody treatment elicited higher IL-2 and proliferative responses compared to non-TAAxCD28 and IsoC-1, although the maximal values ​​were lower. Table 23: Maximum IL-2 release and proliferation, and antibody efficacy values [Table 23] Abbreviations ND: not determined; NC: not calculable; no significant value could be determined by PRISM.

[0300] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. The present invention provides, for example, the following items. (Item 1) 1. An isolated multispecific antigen-binding molecule comprising: a) Approximately 2x106 to human CD28 as measured by surface plasmon resonance at 37°C -7 K under M D a first antigen-binding domain (D1) that binds at b) Approximately 10 binding to human epidermal growth factor receptor (EGFR) on target tumor cells as measured by surface plasmon resonance at 37°C. -8 K under M D and a second antigen-binding domain (D2) that specifically binds to the antigen. (Item 2) The bispecific antigen-binding molecule has a binding affinity of about 10 -5 EC below M 50 2. The isolated multispecific antigen-binding molecule of item 1, which binds to the surface of a human T cell at (Item 3) The bispecific antigen-binding molecule has a binding affinity of about 10 -5 EC below M 50 2. The isolated polyspecific antigen-binding molecule according to item 1, which binds to the surface of a cynomolgus monkey T cell at (Item 4) The bispecific antigen-binding molecule has a binding affinity of about 2.5×10 -8 EC below M 50 2. The isolated multispecific antigen-binding molecule of item 1, which binds to the surface of a cell line expressing EGFR in (Item 5) The D2 domain is (a) an immunoglobulin chain comprising an HCDR of a heavy chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 2, 30, 40 and 50, or a variant thereof; and / or (b) an immunoglobulin chain comprising an LCDR of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof; or The D1 domain is (c) an immunoglobulin chain comprising an HCDR of a heavy chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 10, 59 and 63, or a variant thereof; and / or (d) the multispecific antigen-binding protein according to any one of items 1 to 4, comprising an immunoglobulin chain comprising an LCDR of a light chain variable region comprising an amino acid sequence set forth in one selected from the group consisting of SEQ ID NOs: 16 and 67, or a variant thereof. (Item 6) The D2 domain is (a) a heavy chain immunoglobulin or a variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2, 24, 30, 38, 40, 48, 50 and 58 or a variant thereof; and / or (b) a light chain immunoglobulin or a variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 16 and 28, or a variant thereof; or the D1 domain is (c) a heavy chain immunoglobulin or a variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 10, 26, 59, and 63, or a variant thereof; and / or (d) the multispecific antigen-binding protein of item 5, comprising a light chain immunoglobulin or a variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 16, 28 and 67 or a variant thereof. (Item 7) The D2 domain is (a) a heavy chain immunoglobulin or variable region thereof comprising HCDR1, HCDR2 and HCDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, 30, 40 or 50, each of which has at least 90% amino acid sequence identity to said amino acid sequence set forth in SEQ ID NO: 2, 30, 40 or 50, and / or (b) a light chain immunoglobulin or a variable region thereof comprising LCDR1, LCDR2 and LCDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, each of which has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; or the D1 domain is (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10, 59 or 63; a heavy chain immunoglobulin or a variable region thereof comprising HCDR1, HCDR2 and HCDR3, each having at least 90% amino acid sequence identity to said amino acid sequence set forth in SEQ ID NO: 10, 59 or 63, and / or (d) the multispecific antigen-binding protein of item 5, comprising a light chain variable region LCDR1, LCDR2 and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 or 67, and comprising a light chain immunoglobulin or a variable region thereof having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 or 67, respectively. (Item 8) (1) CDR-H1 comprising the amino acid sequence: GDSIITFY (SEQ ID NO: 4, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); CDR-H3 comprising the amino acid sequence: ARVSEDSYFHYGMDV (SEQ ID NO: 8, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and A D2 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); And CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); and CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and D1 domain including CDR-L3 including the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof) (2) CDR-H1 comprising the amino acid sequence: GFTFSTFI (SEQ ID NO: 32, or a variant thereof); CDR-H2 comprising the amino acid sequence: ISSNGGTI (SEQ ID NO: 34, or a variant thereof); and CDR-H3 comprising the amino acid sequence: TRGGDFWSGYYPFDY (SEQ ID NO: 36, or a variant thereof); CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and A D2 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); And CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and A D1 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); (3) CDR-H1 comprising the amino acid sequence: GFSFRDAW (SEQ ID NO: 42, or a variant thereof); CDR-H2 comprising the amino acid sequence: IRNKIDGGTT (SEQ ID NO: 44, or a variant thereof); and CDR-H3 comprising the amino acid sequence: TTDIWNYVLFYYYGLDV (SEQ ID NO: 46 or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and A D2 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); And CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); and CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and a D1 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); or (4) CDR-H1 comprising the amino acid sequence: DDSIISYY (SEQ ID NO: 52, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGRT (SEQ ID NO: 54, or a variant thereof); and CDR-H3 comprising the amino acid sequence: ARVSEDSYYHYGMDV (SEQ ID NO: 56, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and A D2 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof); And CDR-H1 comprising the amino acid sequence: GGSISSYY (SEQ ID NO: 12, or a variant thereof); CDR-H2 comprising the amino acid sequence: IYYSGIT (SEQ ID NO: 6, or a variant thereof); and CDR-H3 comprising the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO: 14, or a variant thereof); And CDR-L1 comprising the amino acid sequence: QSVSSSY (SEQ ID NO: 18, or a variant thereof); CDR-L2 comprising the amino acid sequence: GAS (SEQ ID NO: 20, or a variant thereof); and 8. The multispecific antigen-binding protein according to any one of items 5 to 7, comprising a D1 domain comprising a CDR-L3 comprising the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22, or a variant thereof). (Item 9) (1) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2 (or a variant thereof); and A D2 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); And A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); and D1 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (2) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); and A D2 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); And A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); and D1 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (3) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); and A D2 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); And A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); and A D1 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); or (4) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); and A D2 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); And A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); and 9. The multispecific antigen-binding protein according to any one of items 5 to 8, which binds to EGFR and CD28, comprising a D1 domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof). (Item 10) (1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); (2) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); (3) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); or (4) A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof); and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); And A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof); and 10. The multispecific antigen-binding protein according to any one of items 5 to 9, which binds to EGFR and CD28, comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof). (Item 11) 11. The antigen-binding protein according to any one of items 5 to 10, which is bispecific. (Item 12) A bispecific antigen-binding molecule that specifically binds to human EGFR and human CD28, wherein a first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, LCDR1 comprises the amino acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 22. (Item 13) A bispecific antigen-binding molecule that specifically binds to human EGFR and human CD28, wherein a first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein that comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 59 and 63, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 16. (Item 14) 1. A bispecific antigen-binding molecule that specifically binds to human EGFR and human CD28, wherein the second antigen-binding domain competes for binding to human EGFR with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 32, 42 and 52, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 34, 44 and 54, HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 36, 46 and 56, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 22. (Item 15) A bispecific antigen-binding molecule that specifically binds to human EGFR and human CD28, wherein a second antigen-binding domain competes for binding to human EGFR with a reference antigen-binding protein that comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 30, 40 and 50, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 16. (Item 16) A bispecific antigen-binding molecule that specifically binds to human EGFR and human CD28, wherein a first antigen-binding domain competes for binding to human CD28 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 16, and a second antigen-binding domain competes for binding to human EGFR with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 30, 40 and 50 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 16. (Item 17) (a) introducing into a host cell one or more polynucleotides encoding the immunoglobulin chains of said antigen binding protein; (b) culturing the host cell under conditions suitable for expression of the polynucleotide; and (c) optionally isolating said antigen binding protein or immunoglobulin chains from said host cells and / or from the medium in which said host cells are grown. (Item 18) 18. The method of claim 17, wherein the host cell is a Chinese hamster ovary cell. (Item 19) 19. An antigen-binding protein or immunoglobulin chain which is the product of the method according to any one of items 17 to 18. (Item 20) 20. A polynucleotide encoding an immunoglobulin chain of the antigen-binding protein according to any one of items 1 to 16 and 19. (Item 21) A vector comprising the polynucleotide according to item 20. (Item 22) A host cell comprising the antigen-binding protein or polynucleotide, and / or the vector, according to any one of items 1 to 16 and 19 to 21. (Item 23) 20. A composition or kit comprising one or more of the antigen-binding proteins according to any one of items 1 to 16 and 19, optionally in association with a further therapeutic agent. (Item 24) 20. A pharmaceutical formulation comprising the antigen-binding protein according to any one of items 1 to 16 and 19, and a pharma- ceutically acceptable carrier or excipient, and optionally a further therapeutic agent. (Item 25) 25. The antigen-binding protein, composition or formulation of any one of items 1 to 16, 19, 23 and 24, associated with a further therapeutic agent selected from the group consisting of a PD-1 inhibitor, a PD-1 antibody or antigen-binding fragment thereof, a PD-L1 inhibitor, an anti-PD-L1 antibody or antigen-binding fragment thereof, the platinum-based anti-cancer chemotherapeutic agents paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, oxaliplatin, the anti-cancer antibodies or antigen-binding fragments thereof pembrolizumab, nivolumab, trastuzumab, cetuximab, bevacizumab, and cemiplimab. (Item 26) 26. A container or injection device comprising the antigen-binding protein or composition or formulation according to any one of items 1 to 16, 19, 23, 24 or 25. (Item 27) 26. A method for administering to a subject the antigen-binding protein, composition or formulation according to any one of items 1 to 16, 19, 23, 24 or 25, comprising injecting the antigen-binding protein, composition or formulation into the body of the subject. (Item 28) 26. A method of treating or preventing a hyperproliferative disease in a subject in need thereof, comprising administering to said subject an effective amount of an antigen binding protein or composition or formulation according to any one of items 1 to 16, 19, 23, 24 or 25. (Item 29) 29. The method of claim 28, wherein the hyperproliferative disease is cancer, and further comprising, prior to administering the antigen binding protein, determining whether cells of the cancer express EGFR, or instructing another individual or entity to perform such a determination, and administering an EGFRxCD28 antigen binding protein to the subject if EGFR expression is present. (Item 30) 30. The method according to any one of items 28 to 29, wherein the hyperproliferative disease is an EGFR-expressing cancer. (Item 31) 31. The method of item 30, wherein the hyperproliferative disease is a cancer that is esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, breast cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer or ovarian cancer. (Item 32) 32. The method of any one of items 27 to 31, wherein the antigen binding protein is administered by subcutaneous, intravenous or intramuscular injection into the body of the subject. (Item 33) The antigen-binding molecule is 1 x 10 -12 M~1x10 -5 M's EC 50 17. The multispecific antigen-binding molecule according to any one of items 1 to 16, which binds to CD28-expressing human T cells at a value of (Item 34) The antigen-binding molecule is 1 x 10 -9 M~1x10 -5 M's EC 50 17. The multispecific antigen-binding molecule according to any one of items 1 to 16, which binds to CD28-expressing human T cells at a value of (Item 35) 35. The multispecific antigen-binding molecule of item 33 or 34, wherein the antigen-binding molecule binds to human cells expressing human CD28 and to cynomolgus monkey cells expressing cynomolgus monkey CD28. (Item 36) A pharmaceutical composition comprising the bispecific antigen-binding molecule according to any one of items 33 to 35 and a pharma- ceutically acceptable carrier or diluent. (Item 37) A nucleic acid comprising a nucleotide sequence encoding the bispecific antibody according to any one of Items 33 to 35. (Item 38) 38. An expression vector comprising the nucleic acid according to item 37. (Item 39) A host cell containing the expression vector according to item 38. (Item 40) A method for inhibiting the growth of cancer in a subject, comprising administering to the subject the isolated bispecific antibody according to any one of items 33 to 35, or the pharmaceutical composition according to item 36, thereby inhibiting the growth of the cancer in the subject. (Item 41) 41. The method of item 28 or 40, further comprising administering a second therapeutic agent. (Item 42) 42. The method of claim 41, wherein the antibody is an anti-tumor agent, a radiation therapy, an antibody drug binding agent, a checkpoint inhibitor, another different bispecific antibody comprising a first antigen-binding domain that binds to the same tumor target antigen and a second antigen-binding domain that binds to CD3 on T cells, another different bispecific antibody comprising a first antigen-binding domain that binds to a different tumor target antigen and a second antigen-binding domain that binds to CD3 on T cells, or a combination thereof. (Item 43) 37. A method for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition according to item 24 or 36, thereby treating the cancer in the subject. (Item 44) 44. The method of claim 43, wherein the cancer is an EGFR-expressing cancer. (Item 45) 45. The method of claim 44, wherein the EGFR-expressing cancer is selected from the group consisting of esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, breast cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer and ovarian cancer. (Item 46) 46. ​​The method of any one of items 43 to 45, further comprising administering to the subject a second therapeutic agent. (Item 47) 47. The method of item 46, wherein the second therapeutic agent is an antitumor agent, radiation therapy, an antibody drug binding agent, a checkpoint inhibitor, another different bispecific antibody comprising a first antigen-binding domain that binds to the same tumor target antigen and a second antigen-binding domain that binds to CD3 on T cells, another different bispecific antibody comprising a first antigen-binding domain that binds to a different tumor target antigen and a second antigen-binding domain that binds to CD3 on T cells, or a combination thereof. (Item 48) 48. The method of claim 42 or 47, wherein the checkpoint inhibitor is cemiplimab. (Item 49) 48. The method of claim 42 or 47, wherein the different bispecific antibodies comprise first antigen-binding domains that bind to different tumor antigens selected from the group consisting of PSMA, MUC16, and STEAP2, and second antigen-binding domains that bind to CD3 on T cells. (Item 50) 17. The bispecific antigen-binding molecule of any one of items 1 to 16, wherein the bispecific antigen-binding molecule interacts with one or more of amino acid residues 133-154 of EGFR set forth in SEQ ID NO: 72, or amino acid residues 345-368 of EGFR set forth in SEQ ID NO: 70, or amino acid residues 399-416 of EGFR set forth in SEQ ID NO: 71.

Claims

1. 1. A bispecific antibody, or an antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to human CD28, and a second antigen-binding domain that specifically binds to human epidermal growth factor receptor (EGFR); (a) the first antigen-binding domain that specifically binds to human CD28 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); the HCDR1 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, the HCDR3 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 14, the LCDR1 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, the LCDR2 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22; (b) the second antigen-binding domain that specifically binds to human EGFR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); (i) the HCDR1 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, the HCDR3 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 8, the LCDR1 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, the LCDR2 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22; (ii) the HCDR1 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 32, the HCDR2 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 34, the HCDR3 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 36, the LCDR1 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, the LCDR2 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22; (iii) the HCDR1 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 42, the HCDR2 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 44, the HCDR3 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 46, the LCDR1 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 18, the LCDR2 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 22; or (iv) the HCDR1 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 52, the HCDR2 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 54, the HCDR3 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 56, the LCDR1 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 18, the LCDR2 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO:

22. A bispecific antibody, or an antigen-binding fragment thereof.

2. The bispecific antibody, or antigen-binding fragment thereof, of claim 1 , wherein the antibody, or antigen-binding fragment thereof, binds to EGFR expressed on a cell.

3. The bispecific antibody, or antigen-binding fragment thereof, of claim 2 , wherein the cell is an EGFR-expressing cancer cell.

4. 4. The bispecific antibody, or antigen-binding fragment thereof, of claim 3, wherein the cancer cells are selected from the group consisting of cells of esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer, colorectal cancer, colon cancer, endometrial cancer, skin cancer, brain cancer, breast cancer, gastroesophageal cancer, prostate cancer and ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glioblastoma multiforme and gastroesophageal adenocarcinoma.

5. the first antigen-binding domain that specifically binds to human CD28 comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, HCDR3 comprising the amino acid sequence of SEQ ID NO: 14, LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; 5. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 4, wherein the second antigen-binding domain that specifically binds to human EGFR comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

22.

6. The first antigen-binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable region (LCVR) comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 16, and the second antigen-binding domain comprises (a) an HCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and an LCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO:16; (b) an HCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 30 and an LCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 16; (c) an HCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (d) an HCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 50 and an LCVR comprising an amino acid sequence having 90% sequence identity to the amino acid sequence of SEQ ID NO: 16; The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 4, comprising:

7. A bispecific antibody, or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human CD28 and a second antigen-binding domain that specifically binds to human EGFR, The first antigen-binding domain comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 16, and the second antigen-binding domain comprises (a) an HCVR comprising the amino acid sequence of SEQ ID NO:2 and an LCVR comprising the amino acid sequence of SEQ ID NO:16; (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 30 and an LCVR comprising the amino acid sequence of SEQ ID NO: 16; (c) an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising the amino acid sequence of SEQ ID NO: 16; or (d) an HCVR comprising the amino acid sequence of SEQ ID NO:50 and an LCVR comprising the amino acid sequence of SEQ ID NO:16; A bispecific antibody, or an antigen-binding fragment thereof, comprising:

8. 8. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 7, wherein the bispecific antibody, or antigen-binding fragment thereof, interacts with one or more of amino acid residues 133-154 of EGFR set forth in SEQ ID NO: 72, or amino acid residues 345-368 of EGFR set forth in SEQ ID NO: 70, or amino acid residues 399-416 of EGFR set forth in SEQ ID NO:

71.

9. The bispecific antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 8, which is a bispecific antibody comprising a heavy chain of the IgG1 isotype.

10. The bispecific antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 8, which is a bispecific antibody comprising a heavy chain of the IgG4 isotype.

11. (a) the first antigen-binding domain that specifically binds to human CD28 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26 and a light chain comprising the amino acid sequence of SEQ ID NO:28; (b) the second antigen-binding domain that specifically binds to human EGFR, (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 28; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 28; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 28; or (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain comprising the amino acid sequence of SEQ ID NO:

28. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 4 and 6 to 7, comprising:

12. A pharmaceutical composition comprising the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier or excipient.

13. A pharmaceutical composition comprising the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1 to 11 for treating cancer in a subject.

14. The pharmaceutical composition of claim 13, wherein the cancer is an EGFR-expressing cancer.

15. 15. The pharmaceutical composition of claim 14, wherein the EGFR-expressing cancer is selected from the group consisting of esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma, lung cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain tumor, glioblastoma multiforme, breast cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer and ovarian cancer.

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