Therapeutic methods using CTLA-4 and PD-1 bispecific antibodies

Abstract

The present disclosure provides methods of administering bispecific antibodies and antigen-binding fragments thereof that specifically bind human programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) to a subject in need thereof, e.g., a subject with cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 317,200, filed March 7, 2022, U.S. Provisional Application No. 63 / 347,748, filed June 1, 2022, and U.S. Provisional Application No. 63 / 374,815, filed September 7, 2022, each of which is incorporated herein by reference.

[0002] Sequence Listing Reference The contents of the electronically submitted Sequence Listing (Name: PDCT-200-WO-PCT.xml, Size: 26.6KB, and Creation Date: March 3, 2023) submitted with this application are incorporated by reference in their entirety into this specification.

[0003] 1.Technical Field The present disclosure relates generally to methods of using bispecific antibodies that specifically bind human programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and antigen-binding fragments thereof, to treat cancer, such as renal cell carcinoma and non-small cell lung cancer. [Background technology]

[0004] 2.Background technology Cancer remains a major health burden worldwide. Despite advances in cancer treatment, there continues to be an unmet medical need for more effective and less toxic therapies, especially for patients with advanced disease or cancers that are resistant to existing therapeutics.

[0005] The role of immune system, especially T cell-mediated cytotoxicity, in tumor control is well recognized. There is increasing evidence that T cells control tumor growth and survival in cancer patients at both early and late stages of disease. However, tumor-specific T cell responses are difficult to grow and sustain in cancer patients. The continued progress and success of cancer immunotherapy drugs that stimulate or enhance the innate immune response against cancer makes such therapeutic agents an attractive treatment option compared to therapies that use non-specific chemotherapeutic drugs and / or radiation.

[0006] Several molecular targets have been identified for their potential usefulness as cancer immunotherapy (IO) agents against cancer. In the field of cancer immunotherapy, some molecular targets that have been investigated for their therapeutic potential include cytotoxic T-lymphocyte antigen-4 (CTLA-4 or CD152), programmed death-ligand 1 (PD-L1 or B7-H1 or CD274), programmed death-1 (PD-1), OX40 (CD134 or TNFRSF4), and the T cell inhibitory receptor T cell immunoglobulin and mucin domain containing 3 (TIM3). Although some of these targets have been successfully exploited for treatment (e.g., PD-1 and CTLA-4), many patients are unresponsive to the therapeutic agents developed. Treatment regimens involving higher doses of immunotherapeutic agents and / or combinations of immunotherapeutic agents can also be considered, but such treatments may be associated with increased risk of side effects, which tend to increase with higher doses and cumulative exposure, and may be additive when used with combination immunotherapies. Some common side effects include hypophysitis, thyroiditis, adrenal insufficiency, enteritis, dermatitis, pneumonitis, hepatitis, pancreatitis, motor and sensory neuropathy, and arthritis. Furthermore, immunotherapeutic agents are typically associated with high costs, making treatments that include combination immunotherapeutic agents potentially cost-prohibitive for patients.

[0007] Thus, there remains a need to identify candidate targets for IO therapeutics, develop novel therapeutics against existing targets, and develop treatment strategies that avoid the shortcomings of currently used immunotherapeutic agents, such as lack of patient responsiveness and increased risk of side effects associated with combination treatments. IO therapeutics (e.g., binding proteins) with dual specificity for combinations of target molecules, especially those that exhibit greater binding affinity for the target molecules when compared to the binding affinity for the combination of individual monospecific binding proteins, provide a particularly desirable class of molecules for therapeutic potential. Summary of the Invention [Means for solving the problem]

[0008] 3. Overview of the Invention In some embodiments, disclosed herein is a method of treating renal cell carcinoma (RCC) in a subject, the method comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). In some embodiments, disclosed herein is a method of treating renal cell carcinoma (RCC) in a subject, the method comprising administering to the subject about 2.25 mg to about 2500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4).

[0009] In some embodiments, the methods comprise administering about 500 mg or about 750 mg of the bispecific antibody or antigen-binding fragment thereof.

[0010] In some embodiments, the methods further comprise administering one or more tyrosine kinase inhibitors.

[0011] In some embodiments, the method further comprises administering the tyrosine kinase inhibitor axitinib or lenvatinib.

[0012] In some embodiments, axitinib is administered orally at a dose of 5 mg twice daily starting on day −7 to day −1 prior to administration of the bispecific antibody or antigen-binding fragment thereof.

[0013] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered on day 1.

[0014] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered once per treatment cycle at a dose of 500 mg or 750 mg.

[0015] In some embodiments, the treatment cycle is three weeks.

[0016] In some embodiments, the methods further comprise a maintenance dose of the bispecific antibody or antigen-binding fragment thereof and / or one or more doses of a chemotherapeutic agent.

[0017] In some embodiments, disclosed herein is a method of treating non-small cell lung cancer (NSCLC) in a subject, the method comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds to programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). In some embodiments, disclosed herein is a method of treating non-small cell lung cancer (NSCLC) in a subject, the method comprising administering to the subject about 2.25 mg to about 2500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds to programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4).

[0018] In some embodiments, disclosed herein is a method of inhibiting the growth of a non-small cell lung tumor in a subject, the method comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). In some embodiments, disclosed herein is a method of inhibiting the growth of a non-small cell lung tumor in a subject, the method comprising administering to the subject about 2.25 mg to about 2500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4).

[0019] In some embodiments, the methods comprise administering about 500 mg or about 750 mg of the bispecific antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered once per treatment cycle.

[0021] In some embodiments, the treatment cycle is three weeks.

[0022] In some embodiments, the methods include administering one or more chemotherapeutic agents.

[0023] In some embodiments, the NSCLC or non-small cell lung tumor is non-squamous cell lung cancer.

[0024] In some embodiments, the NSCLC or non-small cell lung tumor is squamous cell lung carcinoma.

[0025] In some embodiments, the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, pemetrexed, and combinations thereof.

[0026] In some embodiments, the one or more chemotherapeutic agents are carboplatin and pemetrexed. In some embodiments, the NSCLC or non-small cell lung tumor is non-squamous cell lung cancer and the one or more chemotherapeutic agents are carboplatin and pemetrexed.

[0027] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every three weeks and carboplatin is administered at a dose of AUC 6 mg / mL·min or AUC 5 mg / mL·min every three weeks.

[0028] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 2000 mg, carboplatin is administered at a dose of AUC 5 mg / mL min, and pemetrexed is administered at a dose of 500 mg / m 2 is administered at a dose of

[0029] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 1500 mg, carboplatin is administered at a dose of AUC 5 mg / mL min, and pemetrexed is administered at a dose of 500 mg / m 2 every 3 weeks, where carboplatin is administered for 4 doses, followed by a maintenance dose of the bispecific antibody or antigen-binding fragment thereof every 3 weeks and pemetrexed every 3 weeks.

[0030] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 500 mg or about 750 mg, carboplatin is administered at a dose of AUC 5 mg / mL min, and pemetrexed is administered at a dose of 500 mg / mL. 2 every 3 weeks, where carboplatin is administered for 4 doses, followed by a maintenance dose of the bispecific antibody or antigen-binding fragment thereof every 3 weeks and a maintenance dose of pemetrexed every 3 weeks.

[0031] In some embodiments, the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, paclitaxel, nab-paclitaxel, and combinations thereof.

[0032] In some embodiments, the one or more chemotherapeutic agents are carboplatin and paclitaxel.

[0033] In some embodiments, the one or more chemotherapeutic agents are carboplatin and nab-paclitaxel. In some embodiments, the NSCLC or non-small cell lung tumor is squamous cell lung cancer and the one or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

[0034] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every 3 weeks, carboplatin is administered at a dose of AUC 6 mg / mL min every 3 weeks, and paclitaxel is administered at a dose of 200 mg / m 2 every 3 weeks in a dose of 100 mg / kg / day. In some embodiments, the carboplatin and paclitaxel are administered in 4 doses.

[0035] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every 3 weeks, carboplatin is administered at a dose of AUC 6 mg / mL min every 3 weeks, and nab-paclitaxel is administered at a dose of 100 mg / m body surface area (BSA). 2 on days 1, 8, and 15 of every 3 week cycle. In some embodiments, carboplatin and nab-paclitaxel are administered in 4 doses.

[0036] In some embodiments, the methods further comprise a maintenance dose of the bispecific antibody or antigen-binding fragment thereof and / or one or more chemotherapeutic agents.

[0037] In some embodiments, the subject is a human.

[0038] In some aspects, the subject has an advanced solid tumor.

[0039] In some aspects, administration results in the total disappearance and / or reduction of target lesions.

[0040] In some embodiments, the total disappearance and / or reduction of target lesions is determined by assaying a tumor biopsy sample.

[0041] In some embodiments, the sample is a fresh sample or a formalin-fixed paraffin-embedded (FFPE) sample.

[0042] In some embodiments, the sample is assayed by RT-PCR, in situ hybridization, RNase protection, an RT-PCR-based assay, immunohistochemistry (IHC), enzyme-linked immunosorbent assay, in vivo imaging, or flow cytometry.

[0043] In some embodiments, the bispecific antibody binds to cynomolgus PD-1 and CTLA-4.

[0044] In some embodiments, the bispecific antibody or antigen-binding fragment thereof binds to human PD-1 and CTLA-4.

[0045] In some aspects, the bispecific antibody or antigen-binding fragment thereof is a humanized bispecific antibody or antigen-binding fragment thereof.

[0046] In some aspects, the bispecific antibody or antigen-binding fragment thereof is monovalent.

[0047] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a DuetMab.

[0048] In some aspects, the bispecific antibody or antigen-binding fragment comprises an IgG heavy chain constant region.

[0049] In some aspects, the constant region comprises mutations at L234F, L235E and P331S.

[0050] In some embodiments, the constant region comprises a knob mutation and a hole mutation, optionally, the knob mutation is present in a heavy chain that includes a variable region that binds CTLA-4 and the hole mutation is present in a heavy chain that includes a variable region that binds PD-1.

[0051] In some embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region.

[0052] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises the anti-PD-1 and anti-CTLA-4 heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of the sequence of MEDI5752.

[0053] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:14, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:16, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0054] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3.

[0055] In some aspects, the bispecific antibody or antigen-binding fragment thereof is a full-length antibody.

[0056] In some embodiments, the bispecific antibody is MEDI5752.

[0057] In some aspects, the bispecific antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0058] In some embodiments, the antigen-binding fragment comprises a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, bispecific intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0059] In some aspects, provided herein is a method of treating progressive renal cell carcinoma in a subject, the method comprising administering to the subject about 750 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3.

[0060] In some aspects, provided herein is a method of treating progressive renal cell carcinoma in a subject, the method comprising administering to the subject about 500 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3.

[0061] In some aspects, provided herein are methods of treating non-small cell lung cancer in a subject, comprising administering to the subject: (1) about 750 mg or about 500 mg of a bispecific antibody that specifically binds PD-1 and CTLA-4, and comprises (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3; (2) carboplatin at a dose of AUC 5 mg / mL·min every 3 weeks for 4 cycles; and (3) pemetrexed at a dose of 500 mg / m2 every 3 weeks for cycles, followed by maintenance doses of the bispecific antibody and pemetrexed.

[0062] In some aspects, provided herein are methods of treating non-small cell lung cancer in a subject, comprising administering to the subject (1) about 750 mg or about 500 mg of a bispecific antibody that specifically binds PD-1 and CTLA-4, and comprises (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3, every three weeks; (2) carboplatin at a dose of 6 mg / mL·min AUC every three weeks; and (3) paclitaxel at a dose of 200 mg / m2 every three weeks, or (4) nab-paclitaxel at a dose of 100 mg / m2 of body surface area on days 1, 8, and 15 every three weeks for four cycles; and (5) a maintenance dose of a bispecific antibody.

[0063] In some embodiments, the non-small cell lung cancer and / or renal cell carcinoma comprises about 50% or more tumor cells that express PD-L1. In some embodiments, the non-small cell lung cancer and / or renal cell carcinoma comprises about 1-49% tumor cells that express PD-L1. In some embodiments, the non-small cell lung cancer and / or renal cell carcinoma comprises less than about 1% tumor cells that express PD-L1. In some embodiments, the non-small cell lung cancer and / or renal cell carcinoma comprises about 0% tumor cells that express PD-L1.

[0064] In some aspects, the disclosure also provides a method of expanding T cells in a subject in need thereof comprising administering a bispecific antibody or antigen-binding fragment thereof that specifically binds Programmed Cell Death-1 (PD-1) and Cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4), wherein the antibody comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:14, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:16, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, the bispecific antibody comprises (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the bispecific antibody is administered at a dose of about 225 mg to about 1,500 mg. In some embodiments, the bispecific antibody is administered at a dose of about 750 mg Q3W. In some embodiments, the bispecific antibody is administered every 3 weeks. In some embodiments, the subject has non-small cell lung cancer or renal cell carcinoma. In some embodiments, the proportion of new proliferating T cell clones is about 50%, about 60%, about 70%, or about 75% higher compared to the number of T cell clones before administration. In some embodiments, the bispecific antibody is MEDI5752. [Brief description of the drawings]

[0065] 4. Brief description of the drawings [Figure 1] [Figures 1A-1D] Study flow charts for the first-in-human dose-escalation study (Figure 1A) and dose-expansion study (Figures 1B-1D) of MEDI5752. [Diagram 2] 4 shows the predicted MEDI5752 concentration-time profile following IV infusion of MEDI5752 Q3W, including the predicted PD-1 EC20, EC50, and EC90. [Diagram 3] 4 shows the predicted MEDI5752 concentration-time profile following IV infusion of MEDI5752 Q3W, including the predicted EC20 and EC90 for CTLA-4, and the EC20 and EC90 for PD-1. [Figure 4] 4 shows the median pharmacokinetic profile of MEDI5752 following IV infusion of MEDI5752 Q3W. [Diagram 5] Shows exposure to CD4 T cell proliferation (Ctrough) following MEDI5752 treatment. [Figure 6] The percentage of free PD1 (PD1 receptor occupancy) on CD4 T cells is shown as measured longitudinally by flow cytometry following MEDI5752 treatment. [Figure 7] [Figures 7A-7C] Peripheral blood T cell proliferation (CD4+ Ki67+) (Figure 7A) and (Figure 7C) and T cell activation (ICOS expression on CD4 T cells) (Figure 7B) measured by flow cytometry after MEDI5752 monotherapy or in combination with chemotherapy. [Figure 8] [Figures 8A-8C] The total number of proliferating T cell clones (Figure 8A) and the percentage of newly proliferating T cell clones (Figures 8B and 8C) as measured by T cell receptor sequencing (TCRseq) after MEDI5752 monotherapy or in combination with chemotherapy. [Figure 9] Median duration of response for MEDI5752 monotherapy at various doses is shown. [Figure 10] Demonstrating objective responses to MEDI5752 monotherapy in a wide variety of IO naive tumors across a range of MEDI5752 doses. [Figure 11] The efficacy of 750 mg was similar to that of 1500 mg MEDI5752 monotherapy. [Figure 12]Responses in first-line renal cell carcinoma cohorts treated with MEDI5752 at 1,500 mg Q3W (top), 750 mg Q3W (middle), and 500 mg Q3W (bottom) are shown. [Figure 13] % change from baseline for first line RCC cohorts treated with MEDI5752 monotherapy at 750 mg and 1500 mg, as well as median duration of response and median PFS for first line RCC cohorts treated with 1500 mg are shown. [Figure 14] Progression-free survival (PFS) for patients treated with 1,500 mg MEDI5752 and chemotherapy compared to patients treated with pembolizumab and chemotherapy. Twenty patients were treated with MEDI5752 (11 events), with a median PFS of 15.1 months. Twenty-one patients were treated with pembrolizumab (16 events), with a median PFS of 8.9 months. [Figure 15] [Figure 15A] Waterfall plots of efficacy of treatment with 750mg MEDI5752 and chemotherapy in NSCLC patients with PD-L1 <1%, PD-L1 1-49%, PD-L1 >50%, and PD-L1 not assessable. [Figure 15B] Waterfall plots of efficacy of treatment in non-squamous NSCLC cohort 2 with subjects treated with 500mg MEDI5752 + carboplatin / pemetrexed and 750mg MEDI5752 + carboplatin / pemetrexed. [Figure 15C] Waterfall plots of squamous NSCLC expansion cohort 2 patients treated with 750mg MEDI5752 + carboplatin / pemetrexed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] 5. MODE FOR CARRYING OUT THEINVENTION In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly indicated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0067] 5.1 Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly indicated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0068] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, based on the identity of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. The antibodies may be naked antibodies or may be conjugated to other molecules, such as, for example, toxins, radioisotopes, etc.

[0069] Unless expressly stated and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific or multispecific antibodies, and single chain antibodies. In some embodiments, the antibody is a bispecific antibody. The term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes may be on the same target antigen or on different target antigens.

[0070] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment", "antigen-binding domain" or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. In the context of a bispecific antibody, an "antigen-binding fragment" binds to two antigens. An antigen-binding fragment may contain the antigen recognition site of an intact antibody (e.g., sufficient complementarity determining regions (CDRs) to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be obtained from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0071] A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to antibodies and antigen-binding fragments thereof produced in any number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0072] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are multivalent molecules. As used in this application, the term "valent" refers to the presence of a specified number of binding sites in an antibody molecule. For example, a natural antibody or a full-length antibody according to the invention has two binding sites and is "bivalent". The term "tetravalent" refers to the presence of four binding sites in an antigen-binding protein. The term "trivalent" refers to the presence of three binding sites in an antibody molecule. As used herein, the term "bispecific, tetravalent" refers to an antigen-binding protein according to the invention that has four antigen-binding sites, at least one of which binds to a first antigen and at least one of which binds to a second antigen or another epitope of the antigen.

[0073] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, broadly speaking, a portion of a light or heavy chain, typically about 110-120 amino acids or 110-125 amino acids at the amino terminus in a mature heavy chain and about 90-115 amino acids in a mature light chain, which differ in sequence between antibodies and are exploited for the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments of the present disclosure, the variable region is a human variable region. In some embodiments of the present disclosure, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain aspects of the disclosure, the variable region is a primate (e.g., non-human primate) variable region. In some aspects of the disclosure, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0074] The terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0075] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of an antibody heavy chain.

[0076] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues of the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In some embodiments, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acids 31-35 (optionally including one or two additional amino acids following 35, designated 35A and 35B in the Kabat numbering scheme) (CDR1), 50-65 (CDR2), and 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acids 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). In some aspects of the disclosure, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0077] Instead, Chothia refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0078] [Table 1]

[0079] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0080] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which gives rise to the IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects of the disclosure, the heavy chain is a human heavy chain.

[0081] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., κ (kappa) or λ (lambda), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects of the disclosure, the light chain is a human light chain.

[0082] As used herein, the terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-I" are used interchangeably. The complete PD-1 sequence can be found in NCBI Reference Sequence: NG_012110.1. The amino acid sequence of the human PD-1 protein is: [ka] It is.

[0083] Programmed Death-1 ("PD-1") is an approximately 31 kD type I membrane protein member of the extended CD28 / CTLA-4 family of T cell regulatory factors (see Ishida, Y. et al. (1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11:3887-3895).

[0084] PD-1 is expressed on activated T cells, B cells, and monocytes (Agata, Y. et al. (1996) "Expression of the PD-1 Antigen on the Surface of Stimulated Mouse T and B Lymphocytes," Int. Immunol. 8(5):765-772; Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation and Anti-Tumor Immunity," Semin. Cancer Biol. 17(4):288-298). PD-1 is a receptor responsible for downregulation of the immune system following activation by binding of PDL-1 or PDL-2 (Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation and Anti-Tumor Immunity," Semin. Cancer Biol. 17(4): 288-298) and functions as a cell death inducer (Ishida, Y. et al. (1992) "Induced Expression of PD-1, A Novel Member of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11: 3887-3895; Subudhi, S. K. et al. (2005) "The Balance of Immune Responses: Costimulation Verse Coinhibition," J. Molec. Med. 83: 193-202). This process is exploited by many tumors by overexpression of PD-L1, resulting in suppression of the immune response.

[0085] PD-1 is a well-validated target for immune-mediated therapy in oncology, with promising results from clinical trials, particularly in the treatment of melanoma and non-small cell lung cancer (NSCLC). Competitive inhibition of PD-1 / PD-L-1 interaction enhances T cell activation and enhances the recognition and elimination of tumor cells by the host immune system. It has been proposed to use anti-PD-1 antibodies to treat infections and tumors and to enhance adaptive immune responses (see, for example, U.S. Patent Nos. 7,521,051; 7,563,869; and 7,595,048).

[0086] Programmed death-ligand 1 (PD-L1) is also part of a complex system of receptors and ligands involved in the control of T-cell activation. In normal tissues, PD-L1 is expressed on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow-derived mast cells, as well as a variety of non-hematopoietic cells. Its normal function is to control the balance between T-cell activation and tolerance through interaction with its two receptors: Programmed Death 1 (also known as PD-1 or CD279) and CD80 (also known as B7-1 or B7.1). PD-L1 is also expressed on tumors and acts at multiple sites to help tumors avoid detection and elimination by the host immune system. PD-L1 is frequently expressed in a wide range of cancers. In some cancers, PD-L1 expression is associated with reduced survival and poor prognosis. Antibodies that block the interaction of PD-L1 with its receptor can reduce PD-L1-dependent immunosuppressive effects and enhance the cytotoxic activity of antitumor T cells in vitro. Durvalumab is a human monoclonal antibody against human PD-L1 that can block the binding of PD-L1 to both the PD-1 and CD80 receptors. It has been proposed to use anti-PD-L1 antibodies to treat infections and tumors and enhance adaptive immune responses (see, e.g., U.S. Patent Nos. 8,779,108 and 9,493,565, which are incorporated herein by reference in their entireties).

[0087] As used herein, the terms "cytotoxic T-lymphocyte-associated antigen-4", "CTLA-4", "CD152" and "hCTLA-4" are used interchangeably and include variants, isoforms and species homologs of human CTLA-4. The complete CTLA-4 sequence can be found in NCBI Reference Sequence: NG_011502.1. The amino acid sequence of the human CTLA-4 protein is: [ka] It is.

[0088] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on activated T cells and acts as a co-inhibitor that blocks T-cell responses after CD28-mediated T-cell activation. CTLA-4 is thought to be part of a central inhibitory pathway that regulates the amplitude of early activation of naive and memory T cells after TCR engagement and influences both antitumor immunity and autoimmunity. CTLA-4 is expressed exclusively on T cells, and expression of its ligands, CD80 (B7.1) and CD86 (B7.2), is primarily restricted to antigen-presenting cells, T cells, and other immune-mediating cells. Antagonist anti-CTLA-4 antibodies that block the CTLA-4 signaling pathway have been reported to enhance T-cell activation. One such antibody, ipilimumab, was approved by the FDA in 2011 for the treatment of metastatic melanoma. The use of anti-CTLA-4 antibodies has been proposed to treat infectious diseases and tumors and to enhance adaptive immune responses (see, e.g., U.S. Pat. Nos. 6,682,736; 7,109,003; 7,132,281; 7,411,057; 7,824,679; 8,143,379; 7,807,797; 8,491,895; 8,883,984; and U.S. Patent Application Publication No. 20150104409, which are incorporated by reference in their entireties).

[0089] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof from another species (usually human) to avoid eliciting an immune response in that species.

[0090] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues of the complementarity determining regions (CDRs) have been replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and capacity ("CDR-grafted") (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In some cases, certain Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody or antigen-binding fragment thereof can be further modified by substituting additional residues in the Fv framework regions and / or within the non-human CDR residues to refine and optimize the specificity, affinity, and / or capacity of the antibody or antigen-binding fragment thereof. Generally, a humanized antibody or antigen-binding fragment thereof comprises a variable domain that contains all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some aspects of the present disclosure, the "humanized antibody" is a resurfaced antibody.

[0091] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from the human immunoglobulin locus, and such an antibody or antigen-binding fragment thereof is made using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0092] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the inherent binding affinity that indicates a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium binding constant (KA). KD is k off / k on is calculated from the quotient of k off / k on It is calculated from the quotient of k on k refers to the binding rate constant of, for example, an antibody or an antigen-binding fragment thereof to an antigen, off refers, for example, to the dissociation of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0093] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or continuous epitope), or can be, for example, combined from two or more non-contiguous regions of a polypeptide (conformational, non-linear, discontinuous or non-contiguous epitope). In some aspects of the present disclosure, the epitope to which an antibody or antigen-binding fragment thereof specifically binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A(1990) Eur J Biochem 189:1-23; Chayen NE(1997) Structure 5:1269-1274; McPherson A(1976) J Biol Chem 251:6300-6303).Crystals of the antibody / antigen-binding fragment thereof:antigen can be examined using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; see U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art. For example, see Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0094] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that binds to the same amino acid residue as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined by hydrogen / deuterium exchange assay (see Coales et al. Rapid Commun. Mass Spectrom. 2009;23:639-647) or X-ray crystallography.

[0095] An antibody is said to "competitively inhibit" or "cross-compete" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0096] An "isolated" polypeptide, antibody, polynucleotide, vector, cell or composition is a polypeptide, antibody, polynucleotide, vector, cell or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some aspects of the disclosure, an isolated antibody, polynucleotide, vector, cell or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0097] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides that contain, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Because the polypeptides of this disclosure are based on antibodies, it is understood that in some aspects of this disclosure, the polypeptides can occur as single chains or associated chains.

[0098] As used herein, the term "MEDI5752" refers to an anti-PD-1 / CTLA-4 bispecific antibody composed of a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 2 (PD-1), and a light chain of SEQ ID NO: 3 and a heavy chain of SEQ ID NO: 4 (CTLA-4). MEDI5752 is disclosed in U.S. Patent No. 10,457,732, which is incorporated herein by reference in its entirety.

[0099] As used herein, the term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. The formulation may be sterile.

[0100] The terms "administer," "administering," "administration," and the like, as used herein, refer to methods (e.g., intravenous administration) that can be used to enable delivery of a drug, e.g., an anti-PD1 / CTLA-4 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0101] As used herein, the term "in combination" or "administered in combination" means that an antibody or antigen-binding fragment thereof described herein can be administered with one or more additional therapeutic agents. In some embodiments, an antibody or antigen-binding fragment thereof can be administered simultaneously or sequentially with one or more additional therapeutic agents. In some embodiments, an antibody or antigen-binding fragment thereof described herein can be administered with one or more additional therapeutic agents in the same composition or in different compositions.

[0102] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some aspects of the disclosure, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects of the disclosure, the subject is a cynomolgus monkey. In some aspects of the disclosure, the subject is a human.

[0103] The term "therapeutically effective amount" refers to an amount of a drug, e.g., an anti-PD1 / CTLA-4 antibody or antigen-binding fragment thereof, effective to treat a disease or disorder of interest. Terms such as "treat," "treatment," "treating," "ameliorating," and "ameliorating" refer to therapeutic measures that cure, slow, alleviate the symptoms, and / or halt the progression of a condition or disorder. Thus, those in need of treatment include those already diagnosed with the disorder or those suspected of having the disorder.

[0104] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0105] Whenever an embodiment of the present disclosure is described herein with the word "comprising," it is understood that similar embodiments that are separately described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0106] As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive. When the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include both "A and B," "A or B," "A" and "B." Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0107] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate deviations of more than 5%-10% and less than 5%-10% from that value or range that remain within the intended meaning of the recited value or range.

[0108] Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0109] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be obtained by reference to the specification as a whole. Thus, the terms defined immediately below are more particularly defined by reference to the specification as a whole.

[0110] 5.2 Methods of the Invention In one aspect, the invention relates to a method for treating renal cell carcinoma or non-small cell lung cancer in a subject in need thereof, wherein the therapy comprising an anti-PD-1 / CTLA-4 bispecific antibody or antigen-binding fragment thereof provides better therapeutic outcomes (e.g., objective response rates and disease control rates) to the affected subject.

[0111] In one aspect, the invention includes a method of selecting renal cell carcinoma or non-small cell lung cancer in a human patient for immunotherapy comprising determining the level of PD-L1 expression in a tumor sample. In some aspects, the tumor sample is PD-L1 positive. In some aspects, the tumor sample is PD-L1 negative.

[0112] In some embodiments, the invention includes a method of inhibiting the growth of renal cell carcinoma in a human patient comprising administering to the patient an anti-PD-1 / CTLA-4 bispecific antibody. In one embodiment, the invention includes a method of treating renal cell carcinoma in a human patient comprising administering to the patient an anti-PD-1 / CTLA-4 bispecific antibody. In some embodiments, the bispecific antibody is MEDI5752.

[0113] In some embodiments, the invention includes a method of inhibiting the growth of non-small cell lung cancer in a human patient comprising administering to the patient an anti-PD-1 / CTLA-4 bispecific antibody. In one embodiment, the invention includes a method of treating non-small cell lung cancer in a human patient comprising administering to the patient an anti-PD-1 / CTLA-4 bispecific antibody. In some embodiments, the bispecific antibody is MEDI5752.

[0114] In some embodiments, the method of treating renal cell carcinoma or non-small cell lung cancer includes administering to a subject about 100 mg to about 1500 mg of a bispecific antibody (e.g., MEDI5752) or an antigen-binding fragment thereof. In some embodiments, the method includes administering to a subject about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, about 1000 mg, about 1010 mg, about 1020 mg, about 1030 mg, about 1040 mg, about 1050 mg, In some embodiments, the method comprises administering a priming dose of about 750 mg, about 1000 mg, about 1250 mg, or about 1500 mg, followed by a maintenance dose of about 225 mg, about 500 mg, about 750 mg, about 1000 mg, or about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 225 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 500 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 750 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 1000 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 225 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 500 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 750 mg.In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 1000 mg. In some embodiments, the method comprises administering a priming dose of about 750 mg and a maintenance dose of about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 1000 mg and a maintenance dose of about 225 mg. In some embodiments, the method comprises administering a priming dose of about 1000 mg and a maintenance dose of about 500 mg. In some embodiments, the method comprises administering a priming dose of about 1000 mg and a maintenance dose of about 750 mg. In some embodiments, the method comprises administering a priming dose of about 1000 mg and a maintenance dose of about 1000 mg. In some embodiments, the method comprises administering a priming dose of about 1000 mg and a maintenance dose of about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 1250 mg and a maintenance dose of about 225 mg. In some embodiments, the method comprises administering a priming dose of about 1250 mg and a maintenance dose of about 500 mg. In some embodiments, the method comprises administering a priming dose of about 1250 mg and a maintenance dose of about 750 mg. In some embodiments, the method comprises administering a priming dose of about 1250 mg and a maintenance dose of about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 1250 mg and a maintenance dose of about 1250 mg. In some embodiments, the method comprises administering a priming dose of about 1500 mg and a maintenance dose of about 225 mg. In some embodiments, the method comprises administering a priming dose of about 1500 mg and a maintenance dose of about 500 mg. In some embodiments, the method comprises administering a priming dose of about 1500 mg and a maintenance dose of about 750 mg. In some embodiments, the method comprises administering a priming dose of about 1500 mg and a maintenance dose of about 1500 mg. In some embodiments, the method comprises administering a priming dose of about 1500 mg and a maintenance dose of about 1250 mg. In some embodiments, the dose of MEDI5752 administered can be decreased with continued administration.

[0115] In some embodiments, the method of treating renal cell carcinoma or non-small cell lung cancer comprises administering about 500 mg or 750 mg of the bispecific antibody (e.g., MEDI5752) or antigen-binding fragment thereof to a subject. In some embodiments, the method of treating renal cell carcinoma or non-small cell lung cancer comprises administering about 1000 mg of the bispecific antibody or antigen-binding fragment thereof. In some embodiments, the method of treating renal cell carcinoma or non-small cell lung cancer comprises administering about 1125 mg of the bispecific antibody or antigen-binding fragment thereof.

[0116] In some embodiments, a dose of the bispecific antibody or antigen-binding fragment thereof is administered to the subject once per treatment cycle. In some embodiments, the treatment cycle is three weeks. In some embodiments, a dose of the bispecific antibody or antigen-binding fragment thereof is administered every three weeks for about 12 months, about 24 months, about 36 months, or about 48 months.

[0117] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered in combination with one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is carboplatin. In some embodiments, the chemotherapeutic agent is pemetrexed. In some embodiments, the chemotherapeutic agent is axitinib.

[0118] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered in combination with two or more chemotherapeutic agents. In some embodiments, the method of treating non-small cell lung cancer further comprises administering the chemotherapeutic agents carboplatin and pemetrexed. In some embodiments, the method of treating non-small cell lung cancer further comprises administering the chemotherapeutic agents carboplatin and paclitaxel. The one or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

[0119] In some embodiments, carboplatin is administered at a dose of about AUC 4 mg / mL min to AUC 6 mg / mL min. In some embodiments, pemetrexed is administered at a dose of about 400 mg / mL 2 ~600mg / m2 In some embodiments, nab-paclitaxel is administered at a dose of about 50 mg / m 2 ~150mg / m 2 In some embodiments, paclitaxel is administered at a dose of about 150 mg / m 2 ~250mg / m 2 In some embodiments, axitinib is administered at a dose of about 4 mg to 6 mg. In some embodiments, lenvatinib is administered at a dose of about 8 mg to 20 mg. In some embodiments, lenvatinib is administered at a dose of about 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, or 20 mg. In some embodiments, lenvatinib is administered once a day.

[0120] In some embodiments, the method of treating non-small cell lung cancer comprises administering a bispecific antibody (e.g., MEDI5752) or an antigen-binding fragment thereof at a dose of about 500 mg or 750 mg, carboplatin is administered at a dose of 5 mg / mL min and pemetrexed is administered at a dose of 500 mg / mL. 2 In some embodiments, the carboplatin and pemetrexed are administered every 3 weeks for 4 cycles (i.e., 12 weeks). In some embodiments, the administration is followed by a maintenance dose. The maintenance dose comprises administering the bispecific antibody or antigen-binding fragment thereof in combination with pemetrexed once every 3 weeks. In some embodiments, the maintenance dose can be indefinite.

[0121] In some embodiments, the method of treating renal cell carcinoma further comprises administering the chemotherapeutic agent axitinib. In some embodiments, the chemotherapeutic agent (e.g., axitinib) is orally administered at a dose of 5 mg twice daily from day -7 to day -1 prior to administration of the bispecific antibody (e.g., MEDI5752) or antigen-binding fragment thereof. In such embodiments, the bispecific antibody or antigen-binding fragment thereof is administered on day 1. In some embodiments, axitinib is administered at a dose of 5 mg twice daily and the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 1500 mg once every three weeks. In such embodiments, axitinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 12 months, about 24 months, or about 36 months. In some embodiments, axitinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 12 months. In some embodiments, axitinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 24 months. In some embodiments, axitinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 36 months. In some embodiments, axitinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 48 months.

[0122] In some embodiments, the method of treating renal cell carcinoma further comprises administering the chemotherapeutic agent lenvatinib. In some embodiments, the chemotherapeutic agent (e.g., lenvatinib) is orally administered once daily at a dose of 14 mg or 18 mg from day -7 to day -1 prior to administration of the bispecific antibody (e.g., MEDI5752) or antigen-binding fragment thereof. In such embodiments, the bispecific antibody or antigen-binding fragment thereof is administered on day 1. In some embodiments, lenvatinib is administered once daily at a dose of 14 mg or 18 mg, and the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg once every 3 or 4 weeks. In such embodiments, lenvatinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 12 months, about 24 months, or about 36 months. In some embodiments, lenvatinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 12 months. In some embodiments, lenvatinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 24 months. In some embodiments, lenvatinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 36 months. In some embodiments, lenvatinib and the bispecific antibody or antigen-binding fragment thereof are administered for about 48 months.

[0123] In some embodiments, the method of treating non-small cell lung cancer comprises administering a bispecific antibody (e.g., MEDI5752) or an antigen-binding fragment thereof at a dose of 500 or 750 mg every 3 weeks, carboplatin at a dose of AUC 5 mg / mL min every 3 weeks for 4 cycles (i.e., 12 weeks), and pemetrexed at 500 mg / m 2every 3 weeks at a dose of 0.01 mg / kg / day. In such embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and pemetrexed are administered for about 12 months, about 24 months, about 36 months, or about 48 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and pemetrexed are administered for about 12 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and pemetrexed are administered for about 24 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and pemetrexed are administered for about 36 months or longer.

[0124] In some embodiments, the method of treating non-small cell lung cancer comprises administering a bispecific antibody (e.g., MEDI5752) or antigen-binding fragment thereof in combination with carboplatin and paclitaxel. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 or 750 mg every 3 weeks, carboplatin is administered at a dose of AUC 5 mg / mL min every 3 weeks for 4 cycles, and paclitaxel is administered at a dose of 200 mg / m body surface area (BSA) or less. 2 on days 1, 8, and 15 of every 3-week cycle for 4 cycles. In such embodiments, carboplatin is administered immediately after paclitaxel administration. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and paclitaxel are administered for about 12 months, about 24 months, or about 36 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and paclitaxel are administered for about 12 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and paclitaxel are administered for about 24 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and paclitaxel are administered for about 36 months or more.

[0125] In some embodiments, the method of treating non-small cell lung cancer comprises administering a bispecific antibody (e.g., MEDI5752) or antigen-binding fragment thereof in combination with carboplatin and nab-paclitaxel. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 or 750 mg every 3 weeks, carboplatin is administered at a dose of AUC 5 mg / mL min every 3 weeks for 4 cycles, and nab-paclitaxel is administered at a dose of 100 mg / m body surface area (BSA) or less. 2 on days 1, 8, and 15 of every 3-week cycle for 4 cycles. In such embodiments, carboplatin is administered immediately after administration of nab-paclitaxel. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and nab-paclitaxel are administered for about 12 months, about 24 months, or about 36 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and nab-paclitaxel are administered for about 12 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and nab-paclitaxel are administered for about 24 months. In some embodiments, the bispecific antibody or antigen-binding fragment thereof, carboplatin, and nab-paclitaxel are administered for about 36 months or more.

[0126] In some embodiments, the invention includes a method for extending progression-free survival of a human patient afflicted with renal cell carcinoma or non-small cell lung cancer over 12 months, comprising administering to the patient an immunotherapy as disclosed herein, wherein the patient exhibits progression-free survival over 12 months. In some embodiments, the progression-free survival of the patient can be extended by about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration, as compared to standard of care.

[0127] In some aspects, the invention includes methods for extending the overall response rate (ORR) by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more compared to standard of care.

[0128] In some embodiments, the invention includes methods for extending overall survival by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more compared to standard of care, hi some embodiments, patients treated with the methods of the invention have an overall survival of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more.

[0129] In yet other aspects, the invention includes methods for reducing tumor size by at least 10% in a human patient afflicted with renal cell carcinoma or non-small cell lung cancer comprising administering an immunotherapy as disclosed herein, where the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. In some aspects, the method includes identifying the patient as having a PD-L1 positive tumor prior to administration.

[0130] In some embodiments, the invention includes methods for increasing the objective response rate in a patient population to greater than 15%. In some embodiments, the objective response rate is greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75% or greater. In some embodiments, the method includes identifying the patient as having a PD-L1 positive tumor prior to administration.

[0131] In some embodiments, each patient in the method experiences (i) an extended progression-free survival of over 12 months, (ii) a reduction in tumor size of at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size prior to administration, or (iii) both.

[0132] The method of the present invention allows for treating renal cell carcinoma or non-small cell lung cancer, reducing tumor size, inhibiting tumor growth, making the patient tumor-free, preventing tumor recurrence, inducing remission in the patient, or any combination thereof, as a result of administering the immunotherapy disclosed herein. In certain embodiments, administering the immunotherapy disclosed herein induces a complete response. In other embodiments, administering the immunotherapy disclosed herein induces a partial response. In some embodiments, the response is assessed according to RECIST. In some embodiments, the response is assessed according to iRECIST. In some embodiments, the response is assessed according to pathological response.

[0133] In some embodiments, a PD-L1 positive tumor contains at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100% PD-L1 expressing cells. In some embodiments, a PD-L1 positive tumor contains between about 1% and about 49% PD-L1 expressing cells. In some embodiments, a PD-L1 positive tumor contains about 50% or more PD-L1 expressing cells.

[0134] In some embodiments, a PD-L1 positive tumor contains less than about 1% PD-L1 expressing cells. In some embodiments, a tumor contains 0% PD-L1 expressing cells. In some embodiments, the percentage of PD-L1 positive tumors can be determined by assays known to those of skill in the art. In some embodiments, Ventana PD-L1 (SP263) can be used.

[0135] In some embodiments, expression of PD-L1 is determined by receiving the results of an assay in which expression of PD-L1 can be determined.

[0136] In some embodiments, the methods of the invention alter the activation / proliferation and frequency of effector T cells, hi some embodiments, T cells are measured by flow cytometry-based assays or immunohistochemistry.

[0137] In some embodiments, the methods of the invention alter protein or gene expression of biomarkers, including, but not limited to, PD-1, PD-L1, CTLA-4, CD8, and IFN-γ.

[0138] To assess gene or protein expression (e.g., PD-L1), in one embodiment, a test tissue sample is obtained from a patient in need of therapy. In some embodiments, the test tissue sample includes, but is not limited to, any clinically relevant tissue sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, or a sample of a bodily fluid, such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the test tissue sample is from a primary tumor. In some embodiments, the test tissue sample is from a metastasis. In some embodiments, test tissue samples are taken from a subject at multiple time points, e.g., before, during, and / or after treatment. In some embodiments, the test tissue samples are taken from different sites of the subject, e.g., a sample from a primary tumor and a sample from a metastasis at a distant site.

[0139] In some embodiments, the test tissue sample is a paraffin-embedded fixed tissue sample. In some embodiments, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a frozen tissue sample. In some embodiments, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is cells isolated from a fluid. In some embodiments, the test tissue sample comprises circulating tumor cells (CTCs). In some embodiments, the test tissue sample comprises tumor infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample comprises tumor cells and tumor infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample comprises circulating lymphocytes. In some embodiments, the test tissue sample is an archived tissue sample. In some embodiments, the test tissue sample is an archived tissue sample with a known diagnosis, treatment and / or outcome history. In some embodiments, the sample is a tissue block. In some embodiments, the test tissue sample is dispersed cells. In some embodiments, the sample size ranges from about 1 cell to about 1 x 10 6 In some embodiments, the sample size is from about 1 cell to about 1 x 10 5 In some embodiments, the sample size is about 1 cell to about 10,000 cells. In some embodiments, the sample size is about 1 cell to about 1,000 cells. In some embodiments, the sample size is about 1 cell to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0140] In another embodiment, the evaluation of expression can be accomplished without obtaining a test tissue sample. In some embodiments, selecting a suitable patient includes (i) providing a test tissue sample, optionally comprising tumor cells and / or tumor-infiltrating inflammatory cells, obtained from a patient with cancer of the tissue; and (ii) evaluating the percentage of cells in the test tissue sample that express the gene / protein of interest based on evaluating that the percentage of cells in the test tissue sample is higher than a predetermined threshold level.

[0141] In certain embodiments of any of the methods, the percentage of cells expressing PD-L1 is assessed by performing an assay to detect the presence of PD-L1 RNA. In further embodiments, the presence of PD-L1 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In some embodiments, the presence of PD-L1 RNA is detected by an RT-PCR-based assay. In some embodiments, scoring the RT-PCR-based assay comprises assessing the RNA expression level of PD-L1 in the test tissue sample relative to a predetermined level.

[0142] In other embodiments, the percentage of cells expressing a gene / protein of interest (e.g., PD-L1) is assessed by performing an assay to detect the presence of a PD-L1 polypeptide. In further embodiments, the presence of the polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, protein expression is assayed by IHC.

[0143] 5.3 Patient population Provided herein are clinical methods for treating cancer (e.g., advanced renal cell carcinoma or non-small cell lung cancer) in human patients using any of the methods disclosed herein, e.g., a bispecific antibody (e.g., MEDI5752) or an antigen-binding fragment thereof, administered as a single agent or optionally in combination with one or more chemotherapeutic agents. In some aspects, the patient has an advanced solid tumor. In some aspects, the patient has a tumor that has not received prior immunotherapy exposure and is refractory to standard therapy or for which no standard therapy exists. In some aspects, the patient is an immunotherapy-naive patient with an advanced or metastatic solid tumor. In some aspects, the patient has an advanced clear cell renal cell carcinoma. In some aspects, the patient has first-line stage IIIB or IV non-squamous non-small cell lung cancer. In some aspects, the patient is eligible to receive platinum-based doublet chemotherapy.

[0144] In some aspects, the patient has squamous non-small cell lung cancer. In some aspects, the patient has non-squamous non-small cell lung cancer. In some aspects, the patient has an advanced solid tumor.

[0145] 5.4 Outcomes Patients treated according to the methods disclosed herein preferably experience an improvement in at least one symptom of cancer. In one embodiment, the improvement is measured by a reduction in the amount and / or size of measurable tumor lesions. In another embodiment, the lesions can be measured on chest x-rays or CT or MRI films. In another embodiment, cytological or histological examination can be used to assess responsiveness to therapy. In some embodiments, tumor response to administration of the bispecific antibody or antigen-binding fragment thereof can be determined by investigator review of tumor assessments, as defined by RECIST v1.1 guidelines. Further tumor measurements can be performed at the investigator's discretion or according to institutional practice.

[0146] In some embodiments, treated patients show complete response (CR), i.e., disappearance of all target lesions. In some embodiments, treated patients show partial response (PR), i.e., the sum of diameters of target lesions decreases by at least 30% based on the baseline sum of diameters. In some embodiments, treated patients show progressive disease (PD), i.e., the sum of diameters of target lesions increases by at least 20% based on the minimum sum in the study (including the baseline sum if it is the minimum value in the study). In addition to the relative increase of 20%, the sum must also show an absolute increase of at least 5 mm (Note: the appearance of one or more new lesions may be considered as progression). In some embodiments, treated patients show stable disease (SD), i.e., the minimum sum of diameters in the study does not shrink enough to qualify as PR, and does not increase enough to qualify as PD.

[0147] In another embodiment, the treated patient experiences a reduction in tumor size and / or a slower rate of growth, i.e., tumor growth is suppressed. In some embodiments, unwanted cell proliferation is reduced or inhibited. In some embodiments, one or more of the following may occur: the number of cancer cells may be reduced; tumor size may be reduced; cancer cell invasion into peripheral organs may be inhibited, prevented, delayed or stopped; tumor metastasis may be delayed or inhibited; tumor growth may be inhibited; tumor recurrence may be prevented or delayed; one or more symptoms associated with cancer may be alleviated to some extent.

[0148] In other aspects, administration of the bispecific antibody or antigen-binding fragment thereof by any of the methods provided herein results in at least one therapeutic effect selected from the group consisting of a reduction in tumor size, a reduction in the number of metastatic lesions appearing over time, a complete response, a partial response, or stable disease.

[0149] In some embodiments, one or more tumor biopsies can be used to determine tumor response to administration of a bispecific antibody or antigen-binding fragment thereof by any of the methods provided herein. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the sample is a fresh sample. The tumor sample (e.g., biopsy) can be used to identify predictive and / or pharmacodynamic biomarkers associated with immune and tumor microenvironment. Such biomarkers can be determined from assays including IHC, tumor mutation analysis, RNA analysis, and proteomic analysis. In certain embodiments, expression of tumor biomarkers is detected by RT-PCR, in situ hybridization, RNase protection, RT-PCR-based assays, immunohistochemistry, enzyme-linked immunosorbent assay, in vivo imaging, or flow cytometry.

[0150] 5.5 Bispecific antibodies and antigen-binding fragments thereof Provided herein are methods of treating cancer in a subject (e.g., a human subject), comprising administering to the subject an antibody (e.g., a bispecific monoclonal antibody, such as a chimeric, humanized or human antibody) that specifically binds PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4), and antigen-binding fragments thereof. In some aspects, PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4) antibodies and antigen-binding fragments thereof that can be used in the methods provided herein include MEDI5752, a monovalent, bispecific humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds PD-1 and CTLA-4 and contains a fragment crystallizable (Fc) domain that has been engineered to reduce Fc effector function.

[0151] MEDI5752 was constructed on the backbone of the DuetMab molecule. The design of DuetMab is described in Mazor et al., MAbs. 7(2):377-389, (2015 Mar-Apr 2015), which is incorporated herein by reference in its entirety. The "DuetMab" design involves knob-into-hole (KIH) technology for heterodimerizing two different heavy chains, replacing one native disulfide bond at the CH1-CL interface with an engineered disulfide bond to increase the efficiency of pairing of the cognate heavy and light chains.

[0152] The Fc domain of MEDI5752 has triple mutations (TM) (L234F, L235E and P331S) designed to reduce Fc-mediated immune effector functions (Oganesyan et al, Acta Crystallogr D Biol Crystallogr, 2008, 64(Pt 6);700-704). MEDI5752 contains anti-PD-1 and anti-CTLA-4 Fabs, an engineered inter-heavy chain disulfide at the CH1-CL interface of anti-CTLA-4, and an IgG1-TM Fc that is knob-into-hole. MEDI5752 contains knob mutations in the heavy chain that contains the variable region that binds CTLA-4 and hole mutations in the heavy chain that contains the variable region that binds PD-1.

[0153] MEDI5752 is described in US Pat. No. 10,457,732, which is incorporated herein by reference in its entirety.

[0154] In some embodiments of the disclosure, a bispecific antibody or antigen-binding fragment thereof for use in the methods described herein specifically binds to human PD-1 and human CTLA-4 and comprises the six CDRs of the MEDI5752 antibody listed as shown in Table 1.

[0155] [Table 2]

[0156] [Table 3]

[0157] In some aspects of the disclosure, a bispecific antibody or antigen-binding fragment thereof for use in the methods described herein specifically binds to human PD-1 and CTLA-4 and comprises the variable heavy chain (VH) and variable light chain (VL) of the MEDI5752 antibody.

[0158] In some aspects of the disclosure, a bispecific antibody or antigen-binding fragment thereof for use in the methods described herein specifically binds to human PD-1 and CTLA-4 and comprises the heavy chain (HC) of the MEDI5752 antibody listed in Table 3.

[0159] [Table 4]

[0160] In some embodiments of the disclosure, a bispecific antibody or antigen-binding fragment thereof for use in the methods described herein specifically binds to human PD-1 and CTL-4 and comprises the light chain (LC) of the MEDI5752 antibody listed in Table 4.

[0161] [Table 5]

[0162] In some embodiments, a bispecific antibody or antigen-binding fragment thereof that can be used in the disclosed methods comprises an isolated antibody that specifically binds to human PD-1 and CTLA-4 and cross-competes with MEDI5752 for binding to human PD-1 and CTLA-4. In some embodiments, a bispecific antibody or antigen-binding fragment thereof that can be used in the disclosed methods comprises an isolated antibody that binds to the same epitope on human PD-1 and CTLA-4 as MEDI5752.

[0163] In some aspects, the bispecific antibody or antigen-binding fragment thereof comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 13 Includes.

[0164] In some aspects, the bispecific antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3. Includes.

[0165] In some embodiments, the bispecific antibody or antigen-binding fragment comprises an IgG heavy chain constant region. In some embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region.

[0166] In some aspects, the bispecific antibody or antigen-binding fragment thereof is a humanized bispecific antibody or antigen-binding fragment thereof.

[0167] As provided herein, an antibody or antigen-binding fragment thereof that immunospecifically binds to PD-1 and CTLA-4 (e.g., human PD-1 and human CTLA-4) for use in the methods described herein can have reduced effector function, for example, as compared to an antibody or antigen-binding fragment thereof having a wild-type IgG1 sequence. Reduced effector function can result, for example, from the sequence of the constant region of the antibody or antigen-binding fragment thereof.

[0168] As provided herein, antibodies or antigen-binding fragments thereof that immunospecifically bind to PD-1 and CTLA-4 (e.g., human PD-1 and human CTLA-4) for use in the methods described herein can lack CDC and / or ADCC activity, e.g., as a result of the sequence of the constant region.

[0169] 5.6 Pharmaceutical Compositions Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example in a liquid carrier, or suitable for reconstitution in a solution or suspension for intravenous administration.

[0170] In general, such compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutical acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopeia or another generally recognized pharmacopoeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or aqueous saline solutions, as well as aqueous dextrose and glycerol solutions, may be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous.

[0171] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0172] 6. Working Example The examples in this section (ie, Section 6) are provided by way of illustration and not by way of limitation.

[0173] 6.1 Example 1: Phase 1 Clinical Evaluation of MEDI5752 To evaluate the safety and tolerability, and early evidence of efficacy of MEDI5752 in adult subjects with advanced solid tumors when administered as a single agent or in combination with chemotherapy, MEDI5752 was evaluated in a phase 1, first-in-human, multicenter, open-label, dose-escalation and dose-expansion study (Figures 1A-1D). The objective of the study was to provide a description of the safety profile, pharmacokinetics (PK), pharmacodynamics (PD), and early signs of antitumor activity. A maximum of approximately 396 subjects were to be enrolled in the study: approximately 86 subjects in the dose-escalation phase, approximately 92 subjects in the renal cell carcinoma (RCC) expansion cohort, approximately 198 subjects in the nonsquamous non-small cell lung cancer (NSCLC) expansion cohort, and approximately 20 subjects in the squamous NSCLC expansion cohort.

[0174] Approximately 50 sites worldwide were expected to be enrolled. At the time of data cutoff, a total of 178 participants with advanced solid tumors received at least one dose of MEDI5752 monotherapy at one of 10 dose levels (1 at 2.25 mg and 7.5 mg, 3 at 22.5 mg, 5 at 75 mg, 10 at 225 mg, 20 at 500 mg, 40 at 750 mg, 39 at 1500 mg, 34 at 2000 mg, and 7 at 2500 mg, respectively). A total of 152 participants with advanced non-squamous NSCLC received or were to receive at least one dose of MEDI5752 in combination with carboplatin and pemetrexed (approximately 35 at 500 mg, approximately 125 at 750 mg, 20 at 1500 mg, and 7 at 2000 mg). A total of 20 participants with advanced squamous NSCLC received at least one dose of MEDI5752 (750 mg) in combination with carboplatin and paclitaxel or nab-paclitaxel. Overall, 92 participants for 111 with renal cell carcinoma received at least one dose of MEDI5752 (33 at 500 mg, 32 at 750 mg, and 27 at 1500 mg).

[0175] The study included two phases: a dose escalation phase and a dose expansion phase (Figures 1A-1D). In the dose escalation phase, 10 dose levels were evaluated to identify the maximum tolerated dose (MTD), optimal biological dose (OBD), or highest protocol-defined dose (HPDD) dose. Following the dose escalation phase, a dose expansion phase was conducted to evaluate two cohorts of immunotherapy-naive subjects with advanced clear cell RCC (first-line to third-line in the RCC-C1 expansion cohort and first-line in the RCC-C2 expansion cohort), two cohorts of immunotherapy-naive subjects with first-line stage IIIB-IV non-squamous NSCLC (NSCLC expansion cohorts NSCLC-C1 and NSCLC-C2), and one cohort of immunotherapy-naive subjects with first-line stage IIIB-IV squamous NSCLC (NSCLC expansion cohort NSCLC-C3). Subjects in RCC-C2 and NSCLC-C1 expansion were randomized for treatment. Subjects continued treatment until progressive disease (PD) was confirmed, alternative cancer treatment was initiated, or unacceptable toxicity, withdrawal of consent, or other reason for discontinuing treatment occurred. All subjects were to be followed for survival until the end of the study (2 years after the last subject entered the study or when the study was discontinued, whichever occurred first).

[0176] Dose Escalation Phase Dose escalation consisted of 10 dose levels of MEDI5752 (2.25, 7.5, 22.5, 75, 225, 500, 750, 1500, 2000, and 2500 mg dose levels) administered by IV infusion. Once the OBD, MTD, or HPDD was determined, specific cohorts of up to 6 subjects with glomerular filtration rates (GFR) between 30 and 45 mL / min could be explored at the OBD, MTD, or HPDD (i.e., pharmacodynamic cohorts). A maximum of approximately 111 subjects were to be enrolled in the dose escalation phase.

[0177] Dose Expansion Phase Once MTD, OBD, or HPDD was established in the dose escalation phase, the dose expansion phase was initiated. Expansion cohorts enrolled only eligible metastatic subjects who were immunotherapy naïve.

[0178] The RCC and NSCLC expansion cohorts provided further safety, tolerability, efficacy, PK, and PD data for MEDI5752 at several dose levels in these target populations. The NSCLC cohort also evaluated MEDI5752 in combination with carboplatin plus pemetrexed and carboplatin plus paclitaxel or nab-paclitaxel. Additionally, the antitumor activity of MEDI5752 in combination with carboplatin plus pemetrexed was compared to the antitumor activity of pembrolizumab in combination with carboplatin plus pemetrexed in subjects with nonsquamous NSCLC.

[0179] Subjects in the RCC expansion cohorts (RCC-C1 and RCC-C2) received: MEDI5752 Q3W by IV infusion over 60 minutes (± 10 minutes) at 500 mg, 750 mg, and 1500 mg: Subjects at 500 mg and 750 mg were randomized in a 1:1 ratio with both cohorts open. MEDI5752 Q3W by IV infusion over 60 minutes (± 10 minutes): A single priming dose of 750 mg, 1000 mg, 1250 mg, or 1500 mg, followed by maintenance doses of 225 mg, 500 mg, 750 mg, 1000 mg, or 1250 mg.

[0180] Subjects in the non-squamous NSCLC expansion cohort (NSCLC-C1) received: Safety Run-in (NSCLC-C1S): MEDI5752 Q3W delivered by IV infusion over 60 minutes (±10 minutes) at 2000 mg in combination with carboplatin at area under the concentration-time curve (AUC) 5 mg / mL min and pemetrexed at 500 mg / m2 (Q3W) delivered in 4 doses, followed by maintenance therapy of MEDI5752 and pemetrexed Q3W maintained indefinitely Randomized 1:1 to receive one of the following (NSCLC-C1R): Arm A: MEDI5752 Q3W delivered by IV infusion over 60 minutes (± 10 minutes) at 1500 mg in combination with carboplatin at area under the concentration-time curve (AUC) 5 mg / mL·min and pemetrexed at 500 mg / m2 (Q3W) for 4 doses, followed by maintenance therapy of MEDI5752 and pemetrexed Q3W indefinitely; or Arm B: 200 mg pembrolizumab plus carboplatin AUC 5 mg / mL·min and pemetrexed 500 mg / m2 Q3W delivered in 4 doses, followed by 21 months (24 months total) of pembrolizumab and indefinite pemetrexed maintenance Q3W.

[0181] Subjects in the non-squamous NSCLC expansion cohort (NSCLC-C2) received: · Area under the concentration-time curve (AUC) 5 mg / mL·min in combination with carboplatin and pemetrexed 500 mg / m2 (Q3W) delivered by IV infusion over 60 minutes (± 10 minutes) at 500 mg or 750 mg Q3W for 4 doses, followed by maintenance therapy of MEDI5752 and pemetrexed Q3W indefinitely.

[0182] Subjects in the squamous NSCLC expansion cohort (NSCLC-C3) received: MEDI5752 Q3W by IV infusion over 60 minutes (± 10 minutes) at 500 mg or 750 mg for 4 doses, followed by indefinite maintenance therapy of MEDI5752 Q3W, in combination with carboplatin AUC 6 mg / mL min Q3W and either paclitaxel 200 mg / m2 Q3W or nab-paclitaxel 100 mg / m2 on days 1, 8, and 15 of every 3-week cycle.

[0183] Evaluation items The primary safety endpoints of the dose escalation phase were to assess the presence of AEs, SAEs, and DLTs, and to determine the MTD, OBD, or HPDD of MEDI5752 not exceeding the MTD.

[0184] The primary objective of the RCC and non-squamous NSCLC expansion cohorts was to evaluate the preliminary antitumor activity of MEDI5752 monotherapy in RCC and MEDI5752 + carboplatin + pemetrexed in non-squamous NSCLC (vs. pembrolizumab + carboplatin + pemetrexed in NSCLC-C1). The primary endpoint was OR, which is commonly used in oncology studies evaluating subjects with advanced solid tumors, including RCC or NSCLC. The primary objective of the squamous NSCLC expansion cohort was to evaluate the safety and tolerability of MEDI5752 in subjects with advanced solid tumors when combined with chemotherapy.

[0185] Secondary endpoints include safety assessment, further antitumor activity, PK, immunogenicity, and biomarker assessment.

[0186] PD-L1 status will be assessed in subjects who provided archived or fresh tumor biopsies. In the NSCLC expansion cohort, PD-L1 positivity is defined as baseline PD-L1 expression on ≥1% of tumor cells, and PD-L1 negativity is defined as baseline PD-L1 expression on <1% of tumor cells.

[0187] result Safety Data A total of 136 subjects were treated with MEDI5752. Table 5 shows the exposure and MEDI5752-related AEs observed at select MEDI5752 monotherapy and combination dose levels as of the data snapshot date.

[0188] [Table 6]

[0189] Table 6 shows the exposure and MEDI5752-related AEs observed in the RCC cohort.

[0190] [Table 7]

[0191] [Table 8]

[0192] Dose-limiting toxicities (DLTs) were reported in one subject each in the 2000 mg (grade 3 pneumonitis and grade 1 myocarditis in the same subject) and 2500 mg (grade 3 maculopapular rash) monotherapy cohorts. Two deaths were deemed related to treatment with MEDI5752: one subject died of diabetic ketoacidosis and hyperthyroidism, and another subject died of myocardial infarction.

[0193] Review of the benefit-risk profile of MEDI5752-treated subjects suggested that prolonged dosing at doses above 1500 mg was not suitable for further development due to high rates of treatment discontinuation (primarily driven by grade 3 / 4 hepatotoxicity).

[0194] Efficacy Data Among 86 subjects treated with MEDI5752 monotherapy in the dose escalation and maximum tolerated dose (MTD) / optimal biological dose (OBD) cohorts, the overall objective response rate (ORR) (by Response Evaluation Criteria in Solid Tumors [RECIST] version [v] 1.1) was 19.8% (17 / 86) with one complete response (CR) in the response-evaluable population, regardless of dose. Of the five subjects treated with MEDI5752 at 500 mg during dose escalation, two subjects had a best overall response (BOR) of partial response (PR), two subjects had stable disease (SD), and one subject had progressive disease (PD). Of the eight subjects treated with MEDI5752 at 750 mg during dose escalation, four subjects had a BOR of SD, three had PD, and one subject was not evaluable. The median duration of response with MEDI5752 monotherapy was 17.5 months (Figure 9), and MEDI5752 monotherapy demonstrated durable responses in a wide variety of IO-naive tumors across a range of MEDI5752 doses (Figure 10).

[0195] In the non-small cell lung cancer (NSCLC) expansion cohort (NSCLC-C1R), the ORR in the 1500 mg MEDI5752 + carboplatin / pemetrexed arm was 50.0% (10 / 20), while the ORR in the pembrolizumab + carboplatin / pemetrexed arm was 47.6% (10 / 21). Table 8 shows the efficacy data in this cohort. See also Figure 14.

[0196] [Table 9]

[0197] In the non-squamous non-small cell lung cancer (NSCLC) expansion cohort (NSCLC-C2), the ORR was 45.5% (30 / 66) in the first 66 response-evaluable subjects treated with 750 mg MEDI5752 + carboplatin / pemetrexed arm. In the subgroup with PD-L1 <1%, the ORR was 50% (24 / 48). Median follow-up was 7.2 months (range 0.3-16.4 months) (Figure 15B).

[0198] In the non-squamous non-small cell lung cancer (NSCLC) expansion cohort (NSCLC-C2), subjects treated with 500 mg MEDI5752 + carboplatin / pemetrexed arm and in the response-evaluable population had an ORR of 25% (8 / 32) with a preliminary response of CR+PR+undetermined PR in 34.4% (11 / 32). Median follow-up was 3.3 months (range 0.6-6.5 months) (Figure 15B).

[0199] In the squamous non-small cell lung cancer (NSCLC) expansion cohort (NSCLC-C2), subjects treated with 750 mg MEDI5752 + carboplatin / pemetrexed arm and in the response-evaluable population had an ORR of 22.2% (4 / 18) with a preliminary response of CR+PR+undetermined PR in 55.6% (10 / 18). Median follow-up was 2.8 months (range 0.7-7.6 months) (Figure 15C).

[0200] In the renal cell carcinoma (RCC) expansion cohort (RCC-C1) receiving 1500 mg MEDI5752, the ORR was 38.5% (10 / 26) with 2 CRs. In addition, the ORR in first-line RCC subjects was 58.3% (7 / 12) (Figure 12). In addition, the responses in the first-line RCC cohort were found to be durable (Figure 13). The efficacy of 500 mg and 750 mg is shown in Table 9 (Figure 12).

[0201] [Table 10]

[0202] Pharmacokinetic / pharmacodynamic data for MEDI5752 The data suggest that MEDI5752 exhibits non-linear PK due to potential saturation of target-mediated clearance at doses below 22.5 mg and possibly due to ADAs affecting the clearance of MEDI5752. The median PK profile of MEDI5752 over the first 84 days is shown in Figure 4.

[0203] Pharmacodynamic data suggested that MEDI5752 produced a dose-dependent increase in CD4+ T cell proliferation, reaching a plateau at 500 / 750 mg or greater (Figures 5 and 7A). At doses above 225 mg, MEDI5752 demonstrated sustained peripheral PD-1 receptor occupancy (>90%) (Figure 6). Pharmacodynamic data also suggested that in the monotherapy setting, MEDI5752 produced a dose-dependent increase in CD4+ T cell activation, reaching a plateau at 500 / 750 mg or greater (Figures 7B and 8A-B, respectively). Pharmacodynamic data in the combination chemotherapy setting demonstrated a higher increase in CD4+ T cell proliferation on day 8 of cycle 1 and a higher percentage of new expanding T cell clones in cycle 2 with MEDI5752 at doses of 750 mg and 1500 mg in combination with chemotherapy compared to pembrolizumab in combination with chemotherapy (Figures 7C and 8C, respectively).

[0204] 6.2 Example 2: Phase 1b Clinical Evaluation of MEDI5752 in Combination with Axitinib in Subjects with Advanced Renal Cell Carcinoma MEDI5752 was evaluated in a phase 1b, multicenter, open-label, dose-finding and dose-expansion study to assess the safety, tolerability, PK, immunogenicity, and antitumor activity of MEDI5752 in combination with axitinib and lenvatinib in previously untreated subjects with advanced RCC.

[0205] The study included a dose-finding phase followed by a dose-expansion phase. The dose-finding phase evaluated the safety and tolerability of MEDI5752 in combination with axitinib at up to two planned dose levels (Part A) and MEDI5752 in combination with lenvatinib at up to three planned dose levels plus dose levels (Part B). MEDI5752 in combination with axitinib at two dose levels was terminated after enrolling one subject each. Therefore, a phase 2 recommended dose (RP2D) was identified for MEDI5752 in combination with lenvatinib only. The RP2D was to be determined by evaluating the maximum tolerated dose (MTD) assessed in the dose-finding phase together with the entire remaining clinical data from the MEDI5752 program. Up to nine subjects were enrolled in each dose cohort. Additional subjects may have been required if additional cohorts, treatment schedules, or doses were explored based on new PK / pharmacodynamic and clinical data. Once the MTD or MAD of MEDI5752 in combination with lenvatinib was determined in the dose-finding phase, up to 50 subjects were enrolled in the dose-expansion phase.

[0206] Target Population Subjects aged 18 years or older with previously untreated histologically or cytologically proven RCC with clear cell component.

[0207] Treatment Groups and Regimens Up to approximately 70 subjects will be enrolled over two phases and assigned to study treatment as detailed below.

[0208] Dose exploration phase: The dose-finding phase, Part A (MEDI5752 + axitinib) included two planned dose levels: Dose level 1 (starting dose; n=max 9 subjects) (ended after 1 subject enrolled): Axitinib 5mg PO BID (Induction: Day -7 to Day -1) MEDI5752 1500 mg intravenously (IV) every 3 weeks (Q3W) and axitinib 5 mg PO BID (starting on Day 1 [i.e., Day 1 of Cycle 1]) Dose level-1 (tapering dose; n=max 9 subjects) (terminated after 1 subject enrolled): Axitinib 5mg PO BID (Induction: Day -7 to Day -1) MEDI5752 750 mg IV Q3W and axitinib 5 mg PO BID (starting on Day 1 [i.e., Day 1 of Cycle 1]) The dose-finding phase, Part B (MEDI5752 + lenvatinib) included 3 planned dose levels as follows: Dose level-3 LEN (starting dose; n=max 9 subjects) MEDI5752 500 mg IV Q3W and lenvatinib 14 mg PO QD (starting on Day 1 [i.e., Day 1 of Cycle 1]) Dose level-2 LEN (escalating doses of LEN; n=max 9 subjects) MEDI5752 500 mg IV Q3W and lenvatinib 18 mg PO QD (starting on Day 1 [i.e., Day 1 of Cycle 1]) Dose level-1 LEN (MEDI5752 dose escalation, LEN taper [if necessary]; n=max 9 subjects) MEDI5752 750 mg IV Q3W and lenvatinib (14 mg or 18 mg; dose informed by the 2 dose levels previously mentioned) PO QD (starting on Day 1 [i.e., Day 1 of Cycle 1]) Further dose levels MEDI5752 Q3W by IV infusion over 60 minutes (± 10 minutes): A single priming dose of 750 mg, 1000 mg, 1250 mg, or 1500 mg, followed by maintenance doses of 225 mg, 500 mg, 750 mg, 1000 mg, or 1250 mg, and lenvatinib (10 mg, 14 mg, or 18 mg) PO QD (starting on Day 1 of Cycle 1, Cycle 2, or Cycle 3) MEDI5752 Q3W: 500 mg or 750 mg by IV infusion over 60 minutes (± 10 minutes) and lenvatinib (10 mg) PO QD (starting on Day 1 of Cycle 1)

[0209] Dose expansion phase: MEDI5752 IV Q3W and lenvatinib PO QD (starting on Day 1 [i.e., Day 1 of Cycle 1]) at the combination dose determined in the dose-finding phase (starting on Day 1 [i.e., Day 1 of Cycle 1]) (n=up to 50 subjects).

[0210] Subjects received MEDI5752 in combination with axitinib or lenvatinib until disease progression, initiation of alternative cancer treatment, unacceptable toxicity, withdrawal of consent, or other reason to discontinue treatment. Subjects were to be followed for survival until the end of the study, defined as approximately 3 years after the last subject enrolled, unless the study was terminated prior to that time period.

[0211] Study Evaluation Items Safety: incidence of AEs / SAEs, dose-limiting toxicities, AEs leading to treatment discontinuation, abnormal laboratory parameters, vital signs, ECG results. Efficacy: Using RECIST v1.1, we will evaluate BOR, ORR, DCR, DoR, and TTR in the response-evaluable population at the interim analysis and in the as-treated population at the final analysis. PFS (by RECIST v1.1). Pharmacokinetics: Serum concentrations of MEDI5752 Immunogenicity: Incidence of ADAs against MEDI5752.

Claims

1. 1. A method of treating renal cell carcinoma (RCC) in a subject, comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4).

2. 1. A method of inhibiting renal carcinoma cell (RCC) tumor growth in a subject, comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4).

3. 3. The method of claim 1 or 2, comprising administering about 500 mg or about 750 mg of the bispecific antibody or antigen-binding fragment thereof.

4. The method of any one of claims 1 to 3, further comprising administering one or more tyrosine kinase inhibitors.

5. The method of any one of claims 1 to 4, further comprising administering the tyrosine kinase inhibitor axitinib or lenvatinib.

6. 6. The method of claim 5, wherein the axitinib is administered orally at a dose of 5 mg twice daily from day -7 to day -1 prior to administration of the bispecific antibody or antigen-binding fragment thereof.

7. 7. The method of claim 6, wherein the bispecific antibody or antigen-binding fragment thereof is administered on day 1.

8. The method of any one of claims 1 to 7, wherein the bispecific antibody or antigen-binding fragment thereof is administered once per treatment cycle at a dose of 500 mg or 750 mg.

9. 9. The method of claim 8, wherein the treatment cycle is three weeks.

10. 10. The method of any one of claims 4 to 9, further comprising a maintenance dose of said bispecific antibody or antigen-binding fragment thereof and / or one or more doses of said chemotherapeutic agent.

11. 1. A method of treating non-small cell lung cancer (NSCLC) in a subject, comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4).

12. 1. A method of inhibiting the growth of a non-small cell lung tumor in a subject, comprising administering to the subject about 250 mg to about 1500 mg of a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4).

13. 13. The method of claim 11 or 12, comprising administering about 500 mg or about 750 mg of the bispecific antibody or antigen-binding fragment thereof.

14. The method of any one of claims 11 to 13, wherein the bispecific antibody or antigen-binding fragment thereof is administered once per treatment cycle.

15. 15. The method of claim 14, wherein the treatment cycle is three weeks.

16. The method of any one of claims 11 to 15, further comprising administering one or more chemotherapeutic agents.

17. The method of any one of claims 11 to 16, wherein the NSCLC or non-small cell lung tumor is non-squamous cell lung cancer.

18. The method of any one of claims 11 to 16, wherein the NSCLC or non-small cell lung tumor is squamous cell lung carcinoma.

19. The method of any one of claims 11 to 17, wherein the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, pemetrexed, and combinations thereof.

20. 20. The method of claim 19, wherein the one or more chemotherapeutic agents are carboplatin and pemetrexed.

21. 17. The method of claim 16, wherein the NSCLC or non-small cell lung tumor is non-squamous cell lung cancer and the one or more chemotherapeutic agents are carboplatin and pemetrexed.

22. 20. The method of any one of claims 11 to 16 and 19, wherein the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every three weeks and the carboplatin is administered at a dose of AUC 6 mg / Lm min or AUC 5 mg / mL min every three weeks.

23. The bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 2000 mg, the carboplatin is administered at a dose of AUC 5 mg / mL min, and the pemetrexed is administered at a dose of 500 mg / mL. ２ The method of claim 20, wherein the dose is

24. The bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 1500 mg, the carboplatin is administered at a dose of AUC 5 mg / mL min, and the pemetrexed is administered at a dose of 500 mg / mL. ２ 21. The method of claim 20, wherein the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 0.1 mg / kg every 3 weeks for 4 doses, followed by maintenance doses of the bispecific antibody or antigen-binding fragment thereof every 3 weeks and pemetrexed every 3 weeks.

25. The bispecific antibody or antigen-binding fragment thereof is administered at a dose of about 500 mg or about 750 mg, the carboplatin is administered at a dose of AUC 5 mg / mL min, and the pemetrexed is administered at a dose of 500 mg / mL. ２ 21. The method of claim 20, wherein the bispecific antibody or antigen-binding fragment thereof is administered at a dose of 100 mg once every three weeks for four doses, followed by a maintenance dose of the bispecific antibody or antigen-binding fragment thereof every three weeks and a maintenance dose of the pemetrexed every three weeks.

26. The method of any one of claims 11 to 16 and 18, wherein the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, paclitaxel, nab-paclitaxel, and combinations thereof.

27. 27. The method of claim 26, wherein the one or more chemotherapeutic agents are carboplatin and paclitaxel.

28. 27. The method of claim 26, wherein the one or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

29. 17. The method of claim 16, wherein the NSCLC or non-small cell lung tumor is squamous cell lung cancer and the one or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

30. The bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every three weeks, the carboplatin is administered at a dose of AUC 6 mg / mL min every three weeks, and the paclitaxel is administered at a dose of 200 mg / mL. ２ 28. The method of claim 27, wherein the dose is administered every three weeks.

31. The bispecific antibody or antigen-binding fragment thereof is administered at a dose of 500 mg or 750 mg every three weeks, the carboplatin is administered at a dose of AUC 6 mg / mL min every three weeks, and the nab-paclitaxel is administered at a dose of 100 mg / m body surface area (BSA) or less. ２ on days 1, 8, and 15 of every three-week cycle.

32. The method of any one of claims 1 to 31, wherein administration occurs in 4, 3 week cycles.

33. 33. The method of any one of claims 4 to 10 and 16 to 32, further comprising a maintenance dose of said bispecific antibody or antigen-binding fragment thereof and / or said one or more chemotherapeutic agents.

34. The method of any one of claims 1 to 33, wherein the subject is a human.

35. The method of any one of claims 1 to 34, wherein the subject has an advanced solid tumor.

36. 36. The method of any one of claims 1 to 35, wherein said administration results in the total disappearance and / or reduction of target lesions.

37. The method of any one of claims 1 to 36, wherein said disappearance and / or reduction in the total number of target lesions is determined by assaying a tumor biopsy sample.

38. 38. The method of claim 37, wherein the sample is a fresh sample or a formalin-fixed paraffin-embedded (FFPE) sample.

39. 38. The method of claim 36 or 37, wherein the sample is assayed by RT-PCR, in situ hybridization, RNase protection, an RT-PCR based assay, immunohistochemistry (IHC), enzyme-linked immunosorbent assay, in vivo imaging, or flow cytometry.

40. 40. The method of any one of claims 1 to 39, wherein the bispecific antibody binds to cynomolgus PD-1 and CTLA-4.

41. The method of any one of claims 1 to 40, wherein the bispecific antibody or antigen-binding fragment thereof binds to human PD-1 and CTLA-4.

42. The method of any one of claims 1 to 41, wherein the bispecific antibody or antigen-binding fragment thereof is a humanized bispecific antibody or antigen-binding fragment thereof.

43. The method of any one of claims 1 to 42, wherein the bispecific antibody or antigen-binding fragment thereof is monovalent.

44. The method of any one of claims 1 to 43, wherein the bispecific antibody or antigen-binding fragment thereof is a DuetMab.

45. The method of any one of claims 1 to 44, wherein the bispecific antibody or antigen-binding fragment comprises an IgG heavy chain constant region.

46. 46. ​​The method of claim 45, wherein the constant region comprises mutations at L234F, L235E and P331S.

47. 47. The method of claim 45 or 46, wherein the constant region comprises a knob mutation and a hole mutation, optionally, the knob mutation is present in a heavy chain comprising a variable region that binds to CTLA-4 and the hole mutation is present in a heavy chain comprising a variable region that binds to PD-1.

48. The method of any one of claims 45 to 47, wherein the IgG heavy chain constant region is an IgG1 heavy chain constant region.

49. 49. The method of any one of claims 1 to 48, wherein the bispecific antibody or antigen-binding fragment thereof comprises anti-PD-1 and anti-CTLA-4 heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of the sequence of MEDI5752.

50. The bispecific antibody or antigen-binding fragment thereof, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

13. The method of any one of claims 1 to 49, comprising:

51. The bispecific antibody or antigen-binding fragment thereof, (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

3. The method of any one of claims 1 to 50, comprising:

52. The method of any one of claims 1 to 51, wherein the bispecific antibody or antigen-binding fragment thereof is a full-length antibody.

53. The method of any one of claims 1 to 52, wherein the bispecific antibody is MEDI5752.

54. The method of any one of claims 1 to 50, wherein the bispecific antibody or antigen-binding fragment thereof is an antigen-binding fragment.

55. 55. The method of claim 54, wherein the antigen-binding fragment comprises a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, bispecific intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

56. 1. A method of treating progressive renal cell carcinoma in a subject, comprising administering to the subject about 750 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

3.

57. 1. A method of treating progressive renal cell carcinoma in a subject, comprising administering to the subject about 500 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

3.

58. A method of treating non-small cell lung cancer in a subject, comprising: (1) about 750 mg or about 500 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3, every three weeks; (2) carboplatin at a dose of 5 mg / mL min AUC every three weeks for four cycles; and (3) 500 mg / mL every three weeks for four cycles. ２ a dose of pemetrexed followed by maintenance doses of said bispecific antibody and pemetrexed to said subject.

59. 1. A method of treating non-small cell lung cancer in a subject, comprising: (1) about 750 mg or 500 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, and comprises: (A) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3; and (2) carboplatin at a dose of 6 mg / mL min AUC every 3 weeks; and (3) 200 mg / m every 3 weeks ２ or (4) 100 mg / m2 of body surface area on days 1, 8, and 15 of every 3-week cycle ２ and nab-paclitaxel at a dose of (5) A maintenance dose of the bispecific antibody. to said subject.

60. The method of any one of claims 1 to 59, wherein the non-small cell lung cancer and / or the renal cell carcinoma comprises about 50% or more tumor cells that express PD-L1.

61. 61. The method of any one of claims 1 to 60, wherein the non-small cell lung cancer and / or the renal cell carcinoma comprises about 1-49% of tumor cells that express PD-L1.

62. 62. The method of any one of claims 1 to 61, wherein the non-small cell lung cancer and / or the renal cell carcinoma comprises less than about 1% tumor cells that express PD-L1.

63. The method of any one of claims 1 to 62, wherein the non-small cell lung cancer and / or the renal cell carcinoma comprises about 0% of tumor cells that express PD-L1.

64. 1. A method of expanding T cells in a subject in need thereof comprising administering a bispecific antibody or antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), wherein the antibody: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7; and (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

13. The method includes:

65. The bispecific antibody comprises: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

3.

65. The method of claim 64, comprising:

66. 66. The method of claim 64 or 65, wherein the bispecific antibody is administered at a dose of about 225 mg to about 1,500 mg.

67. 67. The method of any one of claims 64-66, wherein the bispecific antibody is administered at a dose of about 750 mg Q3W.

68. 67. The method of claim 66, wherein the bispecific antibody is administered every three weeks.

69. The method of any one of claims 64 to 68, wherein the subject has non-small cell lung cancer or renal cell carcinoma.

70. 70. The method of any one of claims 64 to 69, wherein the proportion of newly expanded T cell clones is about 50%, about 60%, about 70%, or about 75% higher compared to the number of T cell clones before administration.

71. The method of any one of claims 64 to 70, wherein the bispecific antibody is MEDI5752.

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