HK40117614A - Treatment methods using ctla-4 and pd-1 bispecific antibodies - Google Patents

Description

Treatment using CTLA-4 and PD-1 bispecific antibodies

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/317,200, filed March 7, 2022; 63/347,748, filed June 1, 2022; and 63/374,815, filed September 7, 2022, each of which is incorporated herein by reference.

References to sequence lists

The contents of the electronically submitted sequence list (name: PDCT-200-WO-PCT.xml, size: 26.6KB, creation date: March 3, 2023) submitted in this application are incorporated herein by reference in their entirety.

1. Technical Field

This disclosure generally relates to methods for treating cancers (e.g., renal cell carcinoma and non-small cell lung cancer) using bispecific antibodies that specifically bind to human programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and their antigen-binding fragments.

2. Background Technology

Cancer remains a major global health burden. Despite advances in cancer treatment, there remains an unmet medical need for more effective and less toxic therapies, especially for patients with advanced disease or cancer that is resistant to existing treatments.

The role of the immune system, particularly T cell-mediated cytotoxicity, in tumor control is well-established. Growing evidence suggests that T cells control tumor growth and survival in cancer patients, both in the early and late stages of the disease. However, tumor-specific T cell responses are difficult to elevate and maintain in cancer patients. The continued progress and success of cancer immunotherapy, which stimulates or enhances the innate immune response against cancer, makes this a compelling treatment option when compared to therapies utilizing non-specific chemotherapy and/or radiation.

Numerous molecular targets have been identified as potential targets for immuno-oncology (IO) therapy against cancer. Some of these targets, whose therapeutic potential in the field of immuno-oncology therapy is being investigated, 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-3 (TIM3). While some of these targets have been successfully utilized in therapy (e.g., PD-1 and CTLA-4), many patients do not respond to the developed therapies. Furthermore, while treatment regimens involving higher doses and/or combinations of immunotherapy can be considered, such therapies may be associated with an increased risk of side effects, which increase with higher doses and cumulative exposure, and appear to be additive when used in combination with other immunotherapies. Some common side effects include pituitary inflammation, thyroiditis, adrenal insufficiency, enterocolitis, dermatitis, pneumonia, hepatitis, pancreatitis, motor and sensory neuropathy, and arthritis. Furthermore, because immunotherapy is typically associated with high costs, treatments involving combinations of immunotherapy may be prohibitively expensive for patients.

Therefore, it remains necessary to continue identifying candidate targets for immunotherapy, developing new therapies targeting existing targets, and developing treatment strategies to avoid the drawbacks of currently used immunotherapies, including lack of patient response and increased risk of side effects associated with combination therapies. For combinations of target molecules that are bispecific (e.g., binding proteins), especially those that exhibit greater binding affinity to the target molecules compared to combinations of single-specific binding proteins, represent a particularly promising class of molecules in terms of therapeutic potential.

3. Summary of the Invention

In some aspects, this article discloses a method for treating a subject with renal cell carcinoma (RCC) 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 aspects, this article discloses a method for treating a subject with renal cell carcinoma (RCC) 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).

In some respects, these methods involve administering approximately 500 mg or 750 mg of a bispecific antibody or its antigen-binding fragment.

In some respects, these methods further include the administration of one or more tyrosine kinase inhibitors.

In some respects, these methods further include the administration of tyrosine kinase inhibitors such as axitinib or lenvatinib.

In some cases, axitinib was administered orally twice daily at a dose of 5 mg from day -7 to day 1 prior to the administration of the bispecific antibody or its antigen-binding fragment.

In some cases, bispecific antibodies or their antigen-binding fragments are administered on day 1.

In some cases, bispecific antibodies or their antigen-binding fragments are administered once per treatment cycle at a dose of 500 mg or 750 mg.

In some cases, the treatment cycle is three weeks.

In some respects, these methods further include maintaining administration of a bispecific antibody or its antigen-binding fragment and/or one or more doses of a chemotherapeutic agent.

In some aspects, this article discloses a method for treating a subject with non-small cell lung cancer (NSCLC) 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 aspects, this article discloses a method for treating a subject with non-small cell lung cancer (NSCLC) 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).

In some aspects, this article discloses a method for inhibiting the growth of non-small cell lung tumors 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 to programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). In other aspects, this article discloses a method for inhibiting the growth of non-small cell lung tumors in a subject, 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).

In some respects, the method involves administering approximately 500 mg or approximately 750 mg of a bispecific antibody or its antigen-binding fragment.

In some respects, bispecific antibodies or their antigen-binding fragments are administered once per treatment cycle.

In some cases, the treatment cycle is three weeks.

In some respects, the method involves the application of one or more chemotherapeutic agents.

In some respects, NSCLC, or non-small cell lung cancer, is non-squamous lung cancer.

In some respects, NSCLC, or non-small cell lung cancer, is squamous cell lung cancer.

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

In some cases, one or more chemotherapy agents are carboplatin and pemetrexed. In other cases, NSCLC or non-small cell lung cancer is non-squamous lung cancer, and one or more chemotherapy agents are carboplatin and pemetrexed.

In some respects, bispecific antibodies or their antigen-binding fragments are administered at doses of 500 mg or 750 mg every three weeks, and carboplatin is administered at doses of AUC 6 mg/mL·min or AUC 5 mg/mL·min every three weeks.

In some cases, bispecific antibodies or their antigen-binding fragments are administered at a dose of approximately 2000 mg, carboplatin at a dose of AUC 5 mg/mL·min, and pemetrexed at a dose of 500 mg/ ^m² .

In some cases, the bispecific antibody or its antigen-binding fragment is administered at a dose of approximately 1500 mg, carboplatin at a dose of AUC 5 mg/mL·min, and pemetrexed at a dose of 500 mg/ ^m² , every three weeks, with four doses of carboplatin followed by maintenance administration of the bispecific antibody or its antigen-binding fragment every three weeks and maintenance administration of pemetrexed every three weeks.

In some cases, the bispecific antibody or its antigen-binding fragment 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/ ^m² , once every three weeks, wherein carboplatin is administered for 4 doses, followed by maintenance administration of the bispecific antibody or its antigen-binding fragment once every three weeks, and maintenance administration of pemetrexed once every three weeks.

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

In some cases, one or more chemotherapeutic agents are carboplatin and paclitaxel.

In some cases, one or more chemotherapeutic agents are carboplatin and nab-paclitaxel. In other cases, NSCLC, or non-small cell lung cancer, is squamous cell lung cancer, and one or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

In some cases, bispecific antibodies or their antigen-binding fragments are administered at doses of 500 mg or 750 mg every three weeks, carboplatin at an AUC of 6 mg/mL·min is administered every three weeks, and paclitaxel at a dose of 200 mg/ ^m² is administered every three weeks. In some cases, carboplatin and paclitaxel are administered in four doses.

In some cases, the bispecific antibody or its antigen-binding fragment is administered at a dose of 500 mg or 750 mg every three weeks, carboplatin at an AUC of 6 mg/mL·min is administered every three weeks, and nab-paclitaxel is administered at a dose of 100 mg/ ^m² body surface area (BSA) on days 1, 8, and 15 of each 3-week cycle. In some cases, carboplatin and nab-paclitaxel are administered in four doses.

In some aspects, the method further includes maintaining administration of a bispecific antibody or its antigen-binding fragment and/or one or more chemotherapeutic agents.

In some respects, the subjects were human.

In some cases, the subjects had advanced solid tumors.

In some respects, application leads to the disappearance of target lesions and/or a reduction in their total amount.

In some respects, the disappearance of target lesions and/or their total reduction is determined by measuring tumor biopsy samples.

In some respects, the samples are either fresh or formalin-fixed paraffin-embedded (FFPE) samples.

In some respects, samples are measured by RT-PCR, in situ hybridization, RNase protection, RT-PCR-based assays, immunohistochemistry (IHC), enzyme-linked immunosorbent assays, in vivo imaging, or flow cytometry.

In some respects, the bispecific antibody binds to cynomolgus monkey PD-1 and CTLA-4.

In some respects, bispecific antibodies or their antigen-binding fragments bind to human PD-1 and CTLA-4.

In some respects, bispecific antibodies or their antigen-binding fragments are humanized bispecific antibodies or their antigen-binding fragments.

In some respects, bispecific antibodies or their antigen-binding fragments are monovalent.

In some respects, a bispecific antibody or its antigen-binding fragment is DuetMab.

In some respects, bispecific antibodies or antigen-binding fragments contain the constant region of the IgG heavy chain.

In some respects, the constant region includes mutations at L234F, L235E, and P331S.

In some respects, the constant region contains a pestle mutation and a mortar mutation, optionally wherein the pestle mutation is in the heavy chain containing the variable region that binds CTLA-4, and the mortar mutation is in the heavy chain containing the variable region that binds PD-1.

In some respects, the IgG heavy chain constant region is the IgG1 heavy chain constant region.

In some respects, bispecific antibodies or their antigen-binding fragments contain the sequences of MEDI5752 for anti-PD-1 and anti-CTLA-4 heavy chain variable regions (VH)CDR1, VH CDR2, VH CDR3, light chain variable regions (VL)CDR1, VL CDR2, and VLCDR3.

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

In some respects, a bispecific antibody or its antigen-binding fragment comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3.

In some respects, bispecific antibodies or their antigen-binding fragments are full-length antibodies.

In some respects, the bispecific antibody is MEDI5752.

In some respects, bispecific antibodies or their antigen-binding fragments are antigen-binding fragments.

In some respects, antigen-binding fragments include Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, bispecific intracellular antibody, IgGΔCH2, mini antibody, F(ab′)3, tetrasomal antibody, trisomal antibody, bisomal antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

In some respects, this article provides a method for treating a subject with advanced renal cell carcinoma, comprising administering to the subject approximately 750 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4 every three weeks, wherein the bispecific antibody comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3.

In some respects, this article provides a method for treating a subject with advanced renal cell carcinoma, the method comprising administering to the subject approximately 500 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4 every three weeks, and the bispecific antibody comprising: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3.

In some respects, this article provides a method for treating a subject with non-small cell lung cancer, the method comprising administering to the subject: (1) approximately 750 mg or approximately 500 mg every three weeks a bispecific antibody specifically binding to PD-1 and CTLA-4, wherein the bispecific antibody comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3; (2) carboplatin at a dose of AUC 5 mg/mL·min every three weeks for four cycles; and (3) pemetrexed at a dose of 500 mg/m2 every three weeks for multiple cycles, followed by maintenance administration of the bispecific antibody and pemetrexed.

In some respects, this article provides a method for treating a subject with non-small cell lung cancer, the method comprising administering to the subject: (1) approximately 750 mg or approximately 500 mg every three weeks a bispecific antibody that specifically binds to PD-1 and CTLA-4, and the bispecific antibody comprising: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3; and (2) carboplatin at a dose of AUC 6 mg/mL·min 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 body surface area on days 1, 8 and 15 of every three-week cycle for four cycles, and (5) maintenance administration of the bispecific antibody.

In some cases, non-small cell lung cancer and/or renal cell carcinoma contains approximately ≥50% of tumor cells expressing PD-L1. In some cases, non-small cell lung cancer and/or renal cell carcinoma contains approximately 1%–49% of tumor cells expressing PD-L1. In some cases, non-small cell lung cancer and/or renal cell carcinoma contains approximately <1% of tumor cells expressing PD-L1. In some cases, non-small cell lung cancer and/or renal cell carcinoma contains approximately 0% of tumor cells expressing PD-L1.

In some aspects, this disclosure also provides a method for expanding T cells in subjects in need, the method 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), the antibody comprising: (a) VHCDR1 containing the amino acid sequence of SEQ ID NO: 8, VHCDR2 containing the amino acid sequence of SEQ ID NO: 9, VHCDR3 containing the amino acid sequence of SEQ ID NO: 10, VLCDR1 containing the amino acid sequence of SEQ ID NO: 5, and (a) containing SEQ ID NO: 8. VLCDR2 containing the amino acid sequence of SEQ ID NO: 6, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 7; and (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14, VH CDR2 containing the amino acid sequence of SEQ ID NO: 15, VH CDR3 containing the amino acid sequence of SEQ ID NO: 16, VL CDR1 containing the amino acid sequence of SEQ ID NO: 11, VL CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 13. In some aspects, the bispecific antibody comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3. In some instances, the bispecific antibody was administered at a dose of approximately 225 mg to approximately 1,500 mg. In some instances, the bispecific antibody was administered at a dose of approximately 750 mg every 3 weeks. In some instances, the bispecific antibody was administered every three weeks. In some instances, the subject had non-small cell lung cancer or renal cell carcinoma. In some instances, the proportion of newly expanded T cell clones was approximately 50%, 60%, 70%, or 75% higher than the number of T cell clones prior to administration. In some instances, the bispecific antibody was MEDI5752.

4. Description of the attached drawings

Figures 1A-1D are flowcharts of the first human dose escalation (Figure 1A) and dose expansion (Figures 1B-1D) studies of MEDI5752.

Figure 2 shows the predicted MEDI5752 concentration-time curves after IV infusion of MEDI5752 Q3W, including the projected _EC20 , _EC50 , and _EC90 for PD-1.

Figure 3 shows the predicted concentration-time curves of MEDI5752 after IV infusion Q3W, including the predicted _EC20 and _EC90 for CTLA-4 and _EC20 and _EC90 for PD-1.

Figure 4 shows the average pharmacokinetic characteristics of MEDI5752 after IV infusion of MEDI5752 Q3W.

Figure 5 shows the exposure (C _-valley ) relative to CD4 T cell proliferation after MEDI5752 treatment.

Figure 6 shows the percentage of free PD1 on CD4 T cells (PD1 receptor occupancy measured longitudinally by flow cytometry after MEDI5752).

Figures 7A-7C show peripheral blood T cell proliferation (CD4+Ki67+) (Figure 7A) and (Figure 7C) and T cell activation (ICOS expression on CD4 T cells) as measured by flow cytometry after MEDI5752 monotherapy or combination chemotherapy (Figure 7B).

Figures 8A-8C show the total number of expanded T cell clones (Figure 8A) and the proportion of newly expanded T cell clones (Figures 8B and 8C) as measured by T cell receptor sequencing (TCRseq) after MEDI5752 monotherapy or combination chemotherapy.

Figure 9 shows the median duration of response for MEDI5752 monotherapy at different doses.

Figure 10 shows the objective response of MEDI5752 monotherapy in a variety of IO-naïve tumors at a range of MEDI5752 doses.

Figure 11 shows that the efficacy of 750 mg is similar to that of 1500 mg MEDI5752 monotherapy.

Figure 12 shows the response of a cohort of first-line renal cell carcinoma patients treated with MEDI5752 at doses of 1,500 mg Q3W (top), 750 mg Q3W (middle), and 500 mg Q3W (bottom).

Figure 13 shows the change (%) from baseline in the first-line RCC cohorts treated with 750 mg and 1500 mg MEDI5752 monotherapy, as well as the median duration of response and median PFS for those treated with 1500 mg in the first-line RCC.

Figure 14 shows the progression-free survival (PFS) of patients treated with MEDI5752 1,500 mg and chemotherapy compared to patients treated with pembrolizumab and chemotherapy. MEDI5752 treatment involved 20 subjects (11 events), with a median PFS of 15.1 months. Pembrolizumab treatment involved 21 subjects (16 events), with a median PFS of 8.9 months.

Figure 15A shows a waterfall plot of the efficacy of treatment with MEDI5752 750 mg and chemotherapy in NSCLC patients with PD-L1 < 1%, PD-L1 1%–49%, PD-L1 > 50%, and PD-L1 non-evaluable NSCLC. Figure 15B shows a waterfall plot of treatment efficacy in non-squamous NSCLC – Cohort 2, where subjects were treated with 500 mg MEDI5752 + carboplatin/pemetrexed and 750 mg MEDI5752 + carboplatin/pemetrexed. Figure 15C shows a waterfall plot of squamous NSCLC patients in the extended cohort 2 treated with 750 mg MEDI5752 + carboplatin/pemetrexed.

5. Detailed Implementation

To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below unless expressly specified herein. Further definitions are set forth throughout the application.

5.1 Definition

To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below unless expressly specified herein. Further definitions are set forth throughout the application.

The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecule that exhibits the desired biological activity. Antibodies can be any of the following five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ based on the characteristics of their heavy chain constant domains, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

Unless otherwise specified, and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific, or multispecific antibodies, as well as single-chain antibodies. In some respects, an antibody is a bispecific antibody. The term "bispecific antibody" refers to an antibody that binds to two different epitopes. Epitopes may be on the same target antigen or on different target antigens.

The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that binds to an antigen. In the context of bispecific antibodies, an "antigen-binding fragment" binds to two antigens. An antigen-binding fragment may contain the antigen recognition site of the complete antibody (e.g., a complementarity-determining region (CDR) sufficient to specifically bind the 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 can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially generated.

"Monoclonal" antibodies or their antigen-binding fragments refer to a group of homologous antibodies or their antigen-binding fragments that involve highly specific binding to a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically include different antibodies targeting different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragments encompass full-length and complete monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal" antibodies or their antigen-binding fragments refer to such antibodies and their antigen-binding fragments prepared in any number of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

In some respects, the antibodies or antigen-binding fragments thereof disclosed herein are multivalent molecules. As used herein, the term "valence" indicates 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" indicates the presence of four binding sites in an antigen-binding protein. The term "trivalent" indicates the presence of three binding sites in an antibody molecule. As used herein, the term "bispecific, tetravalent" indicates an antigen-binding protein according to the invention having four antigen-binding sites, at least one of which binds a first antigen and at least one of which binds a second antigen or another epitope of the antigen.

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, usually a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differs in sequence between antibodies and is responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some aspects of this disclosure, the variable region is a human variable region. In some aspects of this disclosure, the variable region comprises rodent or mouse CDRs and human frame regions (FRs). In certain aspects of this disclosure, the variable region is a primate (e.g., non-human primate) variable region. In some aspects of this disclosure, the variable region includes the rodent or rat CDR and the primate (e.g., non-human primate) frame region (FR).

The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to the variable region of the heavy chain of an antibody.

The term “Kabat numbering” and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci [Annals of the New York Academy of Sciences] 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within antibody heavy chain molecules are typically located at amino acid positions 31 to 35 (optionally including one or two additional amino acids following position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within antibody light chain molecules are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some aspects of this disclosure, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 196: 901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B exists, the loop endpoint is at 32; if only 35A exists, the loop endpoint is at 33; if both 35A and 35B exist, the loop endpoint is at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular.

As used herein, the terms “constant region” and “constant domain” are interchangeable and have their common meanings in the art. A constant region is a portion of an antibody, such as the carboxyl-terminal portion of the light and/or heavy chain, that is not directly involved in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence.

As used herein, the term "heavy chain," when used in relation to antibodies, can refer to any different type of amino acid sequence based on constant domains, such as α, δ, ε, γ, and μ, which generate IgA, IgD, IgE, IgG, and IgM antibodies, including IgG subclasses such as IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects of this disclosure, the heavy chain is the human heavy chain.

As used herein, the term "light chain" in relation to antibody use can refer to any different type of amino acid sequence based on a constant domain, such as κ or λ. Light chain amino acid sequences are well known in the art. In some aspects of this disclosure, the light chain is the human light chain.

As used herein, the terms “programmed cell 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 at NCBI reference sequence NG012110.1. The amino acid sequence of the human PD-1 protein is:

Programmed cell death-1 (“PD-1”) is a 31 kDa type I membrane protein member of the extended CD28/CTLA-4 family of T cell regulators (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. [European Organization for Molecular Biology Journal] 11: 3887-3895).

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 downregulating the immune system after activation via PDL-1 or PDL-2 binding (Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation and Anti-Tumor Immunity", Semin. Cancer Biol. 17(4): 288-298) and acts as an inducer of cell death (Ishida, Y. et al. (1992) "Induced Expression of PD-1, A Novel Member of The Immunog". "The lobulin gene superfamily, upon programmed cell death, induces the expression of PD-1 (a new member of the immunoglobulin gene superfamily)," 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 utilized in many tumors via overexpression of PD-L1, leading to suppression of the immune response.

PD-1 is a well-validated target for immuno-mediated therapy in oncology, with positive clinical trial results in the treatment of melanoma and non-small cell lung cancer (NSCLC). Antagonistic inhibition of the PD-1/PD-L-1 interaction increases T cell activation, enhancing the host immune system's recognition and elimination of tumor cells. The use of anti-PD-1 antibodies to treat infections and tumors and enhance adaptive immune responses has been proposed (see U.S. Patent Nos. 7,521,051, 7,563,869, and 7,595,048).

Programmed death-ligand 1 (PD-L1) is also part of a complex system of receptors and ligands involved in controlling 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, and various non-hematopoietic cells. Its normal function is to regulate the balance between T cell activation and tolerance through interactions with 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 by tumors and acts at multiple sites to help tumors evade detection and elimination by the host immune system. PD-L1 is expressed frequently 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 between PD-L1 and its receptors can alleviate PD-L1-dependent immunosuppression in vitro and enhance the cytotoxic activity of anti-tumor T cells. Devallumab is a human monoclonal antibody that targets human PD-L1 and blocks the binding of PD-L1 to both the PD-1 and CD80 receptors. The use of anti-PD-L1 antibodies to treat infections and tumors and to enhance adaptive immune responses has been proposed (see U.S. Patent Nos. 8,779,108 and 9,493,565, which are incorporated herein by reference in their entirety).

As used herein, the terms “cytotoxic T-lymphocyte-associated antigen-4,” “CTLA-4,” “CD152,” and “hCTLA-4” are used interchangeably and include variants, isotypes, and species homologs of human CTLA-4. The complete CTLA-4 sequence can be found at NCBI Reference Sequence: NG_011502.1. The amino acid sequence of the human CTLA-4 protein is:

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on activated T cells and acts as a co-inhibitor of T-cell activation following CD28-mediated activation to maintain checkpoints on T-cell responses. CTLA-4 is thought to regulate the early activation amplitude of naive and memory T cells after TCR conjugation and is part of a central suppressive pathway influencing both antitumor immunity and autoimmunity. CTLA-4 is expressed exclusively on T cells, and the expression of its ligands CD80 (B7.1) and CD86 (B7.2) is primarily limited to antigen-presenting cells, T cells, and other immune-mediated cells. Antagonistic 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 to treat infections and tumors and upregulate adaptive immune responses has been proposed (see U.S. Patent 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. Publication No. 20150104409, which are incorporated herein by reference in their entirety).

The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and ability, while the constant regions are derived from sequences in antibodies or antigen-binding fragments thereof derived from another species (usually human) to avoid evoking an immune response in that species.

The term “humanized” antibody or its antigen-binding fragment refers to a form of non-human (e.g., rodent) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing a minimal non-human (e.g., rodent) sequence. Typically, humanized antibodies or their antigen-binding fragments are human immunoglobulins in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) with the desired specificity, affinity, and ability (“CDR transplantation”) (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 human immunoglobulins are replaced by corresponding residues from antibodies or fragments derived from non-human species that possess the desired specificity, affinity, and capability. Humanized antibodies or their antigen-binding fragments can be further modified by replacing additional residues within the Fv framework region and/or within non-human CDR residues to improve and optimize the specificity, affinity, and/or capability of the antibody or its antigen-binding fragment. Generally, humanized antibodies or their antigen-binding fragments will contain variable domains containing all or substantially all CDR regions corresponding to non-human immunoglobulins, while all or substantially all FR regions are those shared by human immunoglobulins. Humanized antibodies or their antigen-binding fragments may also contain immunoglobulin constant regions or domains (Fc), typically at least a portion of the constant regions or domains of human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patent 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 this disclosure, “humanized antibody” is a surface-repair antibody.

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

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y is generally expressed as a dissociation constant (KD). Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (KD) and the equilibrium association constant (KA). KD is calculated as the quotient of _koff / _kon , while KA is calculated as the quotient of _koff / _kon . _Kon refers to, for example, the association rate constant of an antibody or its antigen-binding fragment with an antigen, while _koff refers to, for example, the dissociation of an antibody or its antigen-binding fragment with an antigen. _Kon and _koff can be determined by techniques known to those skilled in the art (such as or KinExA).

As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or an epitope can, for example, originate together from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or noncontinuous epitopes). In some aspects of this disclosure, epitopes to which antibodies or their antigen-binding fragments specifically bind can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen/deuterium exchange combined mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr [Acta Crystallographica Series D: Biocrystals] 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem [European Journal of Biochemistry] 189: 1-23; Chayen NE (1997) Structure [Structure] 5: 1269-1274; McPherson A (1976) J Biol Chem [Journal of Biochemistry] 251: 6300-6303). Antibody/its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.); see, for example, MethEnzymol [Enzymatic Methods] (1985), vols. 114 and 115, edited by Wyckoff HW et al.; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr [Acta Crystallography D: Biocrystalography] 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol [Enzymatic Methods] 276A: 361-423, edited by Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr [Acta Crystallography D: Biocrystalography] 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques (including alanine scanning mutagenesis), see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085.

An antibody that "binds to the same epitope" as a reference antibody is one 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. [Mass Spectrometry Letters] 2009; 23: 639-647) or X-ray crystallography.

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

"Isolated" peptides, antibodies, polynucleotides, carriers, cells, or compositions are peptides, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated peptides, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer exist in a form found in nature. In some aspects of this disclosure, isolated antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, "substantially pure" means material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers having amino acids of any length. Such polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover amino acid polymers that have been naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that, because the polypeptides disclosed herein are antibody-based, in some aspects of this disclosure, polypeptides may appear as single chains or associated chains.

As used herein, the term "MEDI5752" refers to an anti-PD-1/CTLA-4 bispecific antibody comprising the light chain of SEQ ID NO: 1 and the heavy chain of SEQ ID NO: 2 (PD-1), and the light chain of SEQ ID NO: 3 and the 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.

As used herein, the term "pharmaceutical formulation" refers to a preparation in a form that allows the bioactivity of the active ingredient to be effective and that contains no additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered. The formulation may be sterile.

As used herein, the terms “administer,” “administering,” “administration,” etc., refer to methods that enable the delivery (e.g., intravenous administration) of a drug, such as an anti-PD1/CTLA-4 antibody or its antigen-binding fragment, to a desired biological site of 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.

As used herein, the terms "combination" or "combination administration" mean that the antibody or antigen-binding fragment thereof described herein can be administered together with one or more other therapeutic agents. In some aspects, the antibody or antigen-binding fragment thereof can be administered simultaneously or sequentially with one or more other therapeutic agents. In some aspects, the antibody or antigen-binding fragment thereof described herein can be administered with one or more other therapeutic agents in the same or different compositions.

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

The term "therapeutic effective amount" refers to the amount of medication (e.g., anti-PD1/CTLA-4 antibody or its antigen-binding fragment) that is effective in treating a subject's disease or disorder. Terms such as "treating," "treatment," and "to treat," and "alleviating" and "to alleviate" refer to therapeutic measures that cure, slow, alleviate, or stop the progression of a pathological condition or disorder. Therefore, those requiring treatment include those already diagnosed with or suspected of having a disorder.

Unless the context clearly specifies otherwise, as used in this disclosure and claims, the singular forms “a” and “the” include the plural forms.

It should be understood that whenever an aspect of this disclosure is described herein with the language “comprising”, other similar aspects described with terms such as “consisting of” and/or “mainly composed of” are also provided.

Unless explicitly stated or obvious from the context, the term “or” as used herein is understood to be inclusive. The term “and/or” as used herein in phrases such as “A and/or B” is intended to include “A and B”, “A or B”, and “A and B”. Similarly, the term “and/or” as used in phrases such as “A, B and/or C” is intended to cover each of the following: 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).

As used herein, the terms “about” and “approximately” when used to modify numerical values or ranges indicate that deviations of 5% to 10% above or below the value or range are still within the intended meaning of the value or range stated.

Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

Units, prefixes, and symbols are represented in their forms accepted by the International System of Units (SI). Numerical ranges include the numbers that define the range. The headings provided herein are not limiting of the different aspects of this disclosure, which can be obtained by referring to this specification as a whole. Therefore, the terms defined immediately thereafter are defined more fully by referring to the specification in its entirety.

5.2 The method of the present invention

In one aspect, the present invention relates to a method for treating renal cell carcinoma or non-small cell lung cancer in subjects of need. Therapies comprising an anti-PD-1/CTLA-4 bispecific antibody or an antigen-binding fragment thereof produce better treatment outcomes (e.g., objective response rate and disease control rate) for the affected subjects.

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

In some aspects, the present invention includes a method for inhibiting the growth of renal cell carcinoma in a human patient, the method comprising administering an anti-PD-1/CTLA-4 bispecific antibody to the patient. In one aspect, the present invention includes a method for treating renal cell carcinoma in a human patient, the method comprising administering an anti-PD-1/CTLA-4 bispecific antibody to the patient. In some aspects, the bispecific antibody is MEDI5752.

In some aspects, the present invention includes a method for inhibiting the growth of non-small cell lung cancer in a human patient, the method comprising administering an anti-PD-1/CTLA-4 bispecific antibody to the patient. In another aspect, the present invention includes a method for treating non-small cell lung cancer in a human patient, the method comprising administering an anti-PD-1/CTLA-4 bispecific antibody to the patient. In some aspects, the bispecific antibody is MEDI5752.

In some aspects, methods of treating renal cell carcinoma or non-small cell lung cancer include administering to a subject approximately 100 mg to approximately 1500 mg of a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment. In some aspects, the method includes administering approximately 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, approximately 1000 mg, approximately 1010 mg, approximately 1020 mg, approximately 1030 mg, approximately 1040 mg, or approximately 10... 50 mg, approximately 1060 mg, approximately 1070 mg, approximately 1080 mg, approximately 1090 mg, approximately 1100 mg, approximately 1120 mg, approximately 1130 mg, approximately 1140 mg, approximately 1150 mg, approximately 1160 mg, approximately 1170 mg, approximately 1180 mg, approximately 1190 mg, approximately 1200 mg, approximately 1250 mg, approximately 1300 mg, approximately 1350 mg, approximately 1400 mg, approximately 1450 mg, or approximately 1500 mg. In some aspects, the method includes administering an initial dose of approximately 750 mg, approximately 1000 mg, approximately 1250 mg, or approximately 1500 mg, followed by a maintenance dose of approximately 225 mg, approximately 500 mg, approximately 750 mg, 1000 mg, or approximately 1250 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 225 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 500 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 750 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 1000 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 1250 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 225 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 500 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 750 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 1000 mg. In some aspects, the method includes administering an initial dose of about 750 mg and a maintenance dose of about 1250 mg. In some aspects, the method includes administering an initial dose of about 1000 mg and a maintenance dose of about 225 mg. In some aspects, the method includes administering an initial dose of about 1000 mg and a maintenance dose of about 500 mg. In some aspects, the method includes administering an initial dose of about 1000 mg and a maintenance dose of about 750 mg. In some aspects, the method includes administering an initial dose of about 1000 mg and a maintenance dose of about 1000 mg. In some aspects, the method includes administering an initial dose of about 1000 mg and a maintenance dose of about 1250 mg. In some aspects, the method includes administering an initial dose of about 1250 mg and a maintenance dose of about 225 mg. In some aspects, the method includes administering an initial dose of about 1250 mg and a maintenance dose of about 500 mg. In some aspects, the method includes administering an initial dose of about 1250 mg and a maintenance dose of about 750 mg. In some aspects, the method includes administering an initial dose of about 1250 mg and a maintenance dose of about 1250 mg. In some aspects, the method includes administering an initial dose of about 1250 mg and a maintenance dose of about 1250 mg. In some aspects, the method includes administering an initial dose of about 1500 mg and a maintenance dose of about 225 mg. In some aspects, the method includes administering an initial dose of about 1500 mg and a maintenance dose of about 500 mg. In some aspects, the method includes administering an initial dose of about 1500 mg and a maintenance dose of about 750 mg. In some aspects, the method includes administering an initial dose of about 1500 mg and a maintenance dose of about 1500 mg. In some aspects, the method includes administering an initial dose of about 1500 mg and a maintenance dose of about 1250 mg. In some aspects, the dosage of MEDI5752 may be reduced with continued administration.

In some aspects, methods of treating renal cell carcinoma or non-small cell lung cancer include administering approximately 500 mg or 750 mg of a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment to a subject. In some aspects, methods of treating renal cell carcinoma or non-small cell lung cancer include administering approximately 1000 mg of a bispecific antibody or its antigen-binding fragment. In some aspects, methods of treating renal cell carcinoma or non-small cell lung cancer include administering approximately 1125 mg of a bispecific antibody or its antigen-binding fragment.

In some respects, a dose of bispecific antibody or its antigen-binding fragment is administered to the subject once per treatment cycle. In some respects, the treatment cycle is three weeks. In some respects, a dose of bispecific antibody or its antigen-binding fragment is administered every three weeks for approximately 12 months, approximately 24 months, approximately 36 months, or approximately 48 months.

In some respects, a bispecific antibody or its antigen-binding fragment is administered in combination with one or more chemotherapeutic agents. In some respects, the chemotherapeutic agent is carboplatin. In some respects, the chemotherapeutic agent is pemetrexed. In some respects, the chemotherapeutic agent is axitinib.

In some aspects, a bispecific antibody or its antigen-binding fragment is administered in combination with more than one chemotherapeutic agent. In some aspects, methods of treating non-small cell lung cancer further include administering the chemotherapeutic agents carboplatin and pemetrexed. In some aspects, methods of treating non-small cell lung cancer further include administering the chemotherapeutic agents carboplatin and paclitaxel. One or more chemotherapeutic agents are carboplatin and nab-paclitaxel.

In some cases, carboplatin is administered at a dose between approximately AUC 4 mg/mL·min and AUC 6 mg/mL·min. In some cases, pemetrexed is administered at a dose between approximately 400 mg/ ^m² and 600 mg/ ^m² . In some cases, nab-paclitaxel is administered at a dose between approximately 50 mg/ ^m² and 150 mg/ ^m² . In some cases, paclitaxel is administered at a dose between approximately 150 mg/ ^m² and 250 mg/ ^m² . In some cases, axitinib is administered at a dose between approximately 4 mg and 6 mg. In some cases, lenvatinib is administered at a dose between approximately 8 mg and 20 mg. In some cases, lenvatinib is administered at doses of approximately 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, or 20 mg. In some cases, lenvatinib is administered once daily.

In some respects, treatment of non-small cell lung cancer involves administering a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment at a dose of approximately 500 mg or 750 mg, carboplatin at a dose of AUC 5 mg/mL·min, and pemetrexed at a dose of 500 mg/ ^m² . In some respects, carboplatin and pemetrexed are administered every three weeks for four cycles (i.e., twelve weeks). In some respects, maintenance dosing follows administration. Maintenance dosing involves administering a combination of the bispecific antibody or its antigen-binding fragment and pemetrexed every three weeks. In some respects, maintenance dosing may be indefinite.

In some aspects, the treatment of renal cell carcinoma further includes the administration of the chemotherapy agent axitinib. In some aspects, a chemotherapy agent (e.g., axitinib) is administered orally at a dose of 5 mg twice daily from day 7 to day 1, prior to the administration of a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment. In such aspects, the bispecific antibody or its antigen-binding fragment is administered on day 1. In some aspects, axitinib is administered at a dose of 5 mg twice daily, and the bispecific antibody or its antigen-binding fragment is administered at a dose of 1500 mg every three weeks. In such aspects, the administration of axitinib and the bispecific antibody or its antigen-binding fragment continues for approximately 12 months, approximately 24 months, or approximately 36 months. In some aspects, the administration of axitinib and the bispecific antibody or its antigen-binding fragment continues for approximately 12 months. In some aspects, the administration of axitinib and the bispecific antibody or its antigen-binding fragment continues for approximately 24 months. In some aspects, the administration of axitinib and the bispecific antibody or its antigen-binding fragment continues for approximately 36 months. In some cases, axitinib and bispecific antibodies or their antigen-binding fragments are administered for approximately 48 months.

In some aspects, the treatment of renal cell carcinoma further includes the administration of the chemotherapy agent lenvatinib. In some aspects, the chemotherapy agent (e.g., lenvatinib) is administered orally once daily at a dose of 14 mg or 18 mg from day 7 to day 1, prior to the administration of a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment. In such aspects, the bispecific antibody or its antigen-binding fragment is administered on day 1. In some aspects, lenvatinib is administered once daily at a dose of 14 mg or 18 mg, and the bispecific antibody or its antigen-binding fragment is administered every three or four weeks at a dose of 500 mg or 750 mg. In such aspects, lenvatinib and the bispecific antibody or its antigen-binding fragment are administered for approximately 12 months, approximately 24 months, or approximately 36 months. In some aspects, lenvatinib and the bispecific antibody or its antigen-binding fragment are administered for approximately 12 months. In some aspects, lenvatinib and the bispecific antibody or its antigen-binding fragment are administered for approximately 24 months. In some cases, administration of lenvatinib and a bispecific antibody or its antigen-binding fragment lasts approximately 36 months. In other cases, administration of lenvatinib and a bispecific antibody or its antigen-binding fragment lasts approximately 48 months.

In some aspects, treatment methods for non-small cell lung cancer include administration of a bispecific antibody (e.g., MEDI5752) or its antigen-binding fragment at a dose of 500 or 750 mg every three weeks, carboplatin at a dose of AUC 5 mg/mL·min every three weeks for four cycles (i.e., 12 weeks), and pemetrexed at a dose of 500 mg/ ^m² every three weeks. In some aspects, the bispecific antibody or its antigen-binding fragment, carboplatin, and pemetrexed are administered for approximately 12 months, 24 months, 36 months, or 48 months. In some aspects, the bispecific antibody or its antigen-binding fragment, carboplatin, and pemetrexed are administered for approximately 12 months. In some aspects, the bispecific antibody or its antigen-binding fragment, carboplatin, and pemetrexed are administered for approximately 24 months. In some aspects, the bispecific antibody or its antigen-binding fragment, carboplatin, and pemetrexed are administered for approximately 36 months or longer.

In some aspects, treatment of non-small cell lung cancer involves administering a bispecific antibody (e.g., MEDI5752) or its antigen-conjugated fragment in combination with carboplatin and paclitaxel. In some aspects, the bispecific antibody or its antigen-conjugated fragment is administered at a dose of 500 or 750 mg every three weeks, carboplatin at an AUC of 5 mg/mL·min is administered every three weeks for four cycles, and paclitaxel is administered at a dose of 200 mg/ ^m² body surface area (BSA) on days 1, 8, and 15 of each three-week cycle for four cycles. In these aspects, carboplatin is administered immediately after paclitaxel. In some aspects, the bispecific antibody or its antigen-conjugated fragment, carboplatin, and paclitaxel are administered for approximately 12 months, approximately 24 months, or approximately 36 months. In some aspects, the bispecific antibody or its antigen-conjugated fragment, carboplatin, and paclitaxel are administered for approximately 12 months. In some aspects, the bispecific antibody or its antigen-conjugated fragment, carboplatin, and paclitaxel are administered for approximately 24 months. In some cases, the administration of bispecific antibodies or their antigen-binding fragments, carboplatin, and nab-paclitaxel can last for approximately 36 months or longer.

In some aspects, treatment of non-small cell lung cancer involves the administration of a bispecific antibody (e.g., MEDI5752) or its antigen-conjugated fragment in combination with carboplatin and nab-paclitaxel. In some aspects, the bispecific antibody or its antigen-conjugated fragment is administered at a dose of 500 or 750 mg every three weeks, carboplatin at an AUC of 5 mg/mL·min every three weeks for four cycles, and nab-paclitaxel at a dose of 100 mg/ ^m² body surface area (BSA) on days 1, 8, and 15 of each three-week cycle for four cycles. In these aspects, carboplatin is administered immediately after nab-paclitaxel. In some aspects, the bispecific antibody or its antigen-conjugated fragment, carboplatin, and nab-paclitaxel are administered for approximately 12 months, approximately 24 months, or approximately 36 months. In some aspects, the bispecific antibody or its antigen-conjugated fragment, carboplatin, and nab-paclitaxel are administered for approximately 12 months. In some cases, administration of bispecific antibodies or their antigen-binding fragments, carboplatin, and nab-paclitaxel is continued for approximately 24 months. In other cases, administration of bispecific antibodies or their antigen-binding fragments, carboplatin, and nab-paclitaxel is continued for approximately 36 months or longer.

In some aspects, the present invention includes a method for prolonging progression-free survival for more than 12 months in human patients with renal cell carcinoma or non-small cell lung cancer, the method comprising administering the immunotherapy disclosed herein to the patient, wherein the patient exhibits progression-free survival for more than 12 months. In some aspects, after administration, the patient's progression-free survival may be prolonged for more than 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 compared to standard of care.

In some aspects, the present invention includes a method for prolonging 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.

In some aspects, the present invention includes a method for prolonging overall survival, wherein the overall survival is at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more longer than standard of care. In some aspects, patients treated with the method of the present 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.

In other aspects, the present invention includes a method for reducing the size of a tumor by at least 10% in a human patient with renal cell carcinoma or non-small cell lung cancer, the method comprising administering the immunotherapy disclosed herein, wherein 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 before administration. In some aspects, the method includes identifying the patient as having a PD-L1 positive tumor prior to administration.

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

In some respects, each patient in these methods experiences (i) prolonged progression-free survival of more than 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 before administration, or (iii) both.

As a result of administering the immunotherapy disclosed herein, the methods of the present invention can treat renal cell carcinoma or non-small cell lung cancer, reduce tumor size, inhibit tumor growth, eliminate tumors from a patient, prevent tumor recurrence, induce remission in a patient, or any combination thereof. In some aspects, administration of the immunotherapy disclosed herein induces a complete response. In other aspects, administration of the immunotherapy disclosed herein induces a partial response. In some aspects, the response is assessed according to RECIST. In some aspects, the response is assessed according to iRECIST. In some aspects, the response is assessed according to pathological response.

In some respects, PD-L1 positive tumors contain 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% of cells expressing PD-L1. In some respects, PD-L1 positive tumors contain about 1% to about 49% of cells expressing PD-L1. In some respects, PD-L1 positive tumors contain about ≥50% of cells expressing PD-L1.

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

In some respects, PD-L1 expression is determined by receiving the results of assays that can identify PD-L1 expression.

In some aspects, the method of the present invention alters the activation/proliferation and frequency of effector T cells. In some aspects, T cells are measured by flow cytometry-based assays or immunohistochemistry.

In some aspects, the methods of the present invention alter the protein or gene expression of biomarkers such as, but not limited to, PD-1, PD-L1, CTLA-4, CD8, and IFN-γ.

In some respects, to assess gene or protein expression (e.g., PD-L1), test tissue samples are obtained from patients requiring the therapy. In other respects, test tissue samples include, but are not limited to, any clinically relevant tissue sample, such as tumor biopsy, core needle biopsy tissue samples, fine needle aspirates, or bodily fluid samples, such as blood, plasma, serum, lymph, ascites, cystic fluid, or urine. In some respects, test tissue samples are derived from the primary tumor. In some respects, test tissue samples are derived from metastases. In some respects, test tissue samples are taken from the subject at multiple time points, e.g., before, during, and/or after treatment. In some respects, test tissue samples are taken from different locations within the subject; for example, one sample from the primary tumor and one sample from a metastasis at a distant location.

In some aspects, the test tissue sample is a paraffin-embedded, fixed tissue sample. In some aspects, the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some aspects, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a frozen tissue sample. In some aspects, the test tissue sample is a fresh-frozen (FF) tissue (e.g., tumor) sample. In some aspects, the test tissue sample is cells isolated from a fluid. In some aspects, the test tissue sample includes circulating tumor cells (CTCs). In some aspects, the test tissue sample includes tumor-infiltrating lymphocytes (TILs). In some aspects, the test tissue sample includes both tumor cells and tumor-infiltrating lymphocytes (TILs). In some aspects, the test tissue sample includes circulating lymphocytes. In some aspects, the test tissue sample is an archived tissue sample. In some aspects, the test tissue sample is an archived tissue sample with a known history of diagnosis, treatment, and/or outcome. In some aspects, the sample is a tissue block. In some aspects, the test tissue sample is dispersed cells. In some aspects, the sample size is about 1 cell to about 1 × ^10⁶ cells or more. In some aspects, the sample size is about 1 cell to about 1 × ^10⁵ cells. In some aspects, the sample size is about 1 cell to about 10,000 cells. In some aspects, the sample size is about 1 cell to about 1,000 cells. In some aspects, the sample size is about 1 cell to about 100 cells. In some aspects, the sample size is a single cell.

On the other hand, the assessment of expression can be performed without obtaining a test tissue sample. In some aspects, selecting an appropriate patient includes (i) optionally providing a test tissue sample obtained from a patient with cancer, the test tissue sample containing tumor cells and/or tumor-infiltrating inflammatory cells; and (ii) assessing the proportion of cells expressing the target gene/protein in the test tissue sample, based on an assessment that the proportion of such cells in the test tissue sample is above a predetermined threshold level.

In some aspects of any of the methods of the present invention, the proportion of cells expressing PD-L1 is assessed by performing an assay to detect the presence of PD-L1 RNA. In other aspects, the presence of PD-L1 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In some aspects, the presence of PD-L1 RNA is detected by an RT-PCR-based assay. In some aspects, scoring the RT-PCR-based assay includes assessing the level of PD-L1 RNA expression in the test tissue sample relative to a predetermined level.

In other respects, the proportion of cells expressing genes/proteins of interest (e.g., PD-L1) is assessed by performing assays to detect the presence of the PD-L1 peptide. Further, the presence of the peptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some respects, protein expression is measured by IHC.

5.3 Patient Group

This article provides clinical approaches to treating human patients with cancers (e.g., advanced renal cell carcinoma or non-small cell lung cancer) using any of the methods disclosed herein, such as bispecific antibodies (e.g., MEDI5752) or their antigen-binding fragments, as a single agent or optionally in combination with one or more chemotherapeutic agents. In some cases, the patient has an advanced solid tumor. In some cases, the patient has no prior immunotherapy exposure and has a tumor refractory to standard therapy or for which no standard therapy is available. In some cases, the patient has not received immunotherapy for advanced or metastatic solid tumors. In some cases, the patient has advanced clear cell renal cell carcinoma. In some cases, the patient has first-line stage IIIB or IV non-squamous non-small cell lung cancer. In some cases, the patient is eligible for platinum-based dual chemotherapy.

In some cases, the patient has squamous non-small cell lung cancer. In other cases, the patient has non-squamous non-small cell lung cancer. In some cases, the patient has an advanced solid tumor.

5.4 Ending

Patients treated according to the methods disclosed herein preferably experience improvement in at least one sign of cancer. In one aspect, improvement is measured by a measurable reduction in the number and/or size of tumor lesions. In another aspect, lesions can be measured on chest X-rays or CT or MRI films. In yet another aspect, cytological or histological evaluation of the response to therapy can be used. In some aspects, tumor response to administration of a bispecific antibody or its antigen-binding fragment can be determined by a researcher reviewing tumor assessments and defined according to RECIST v1.1 guidelines. Additional tumor measurements can be performed by researcher judgment or according to institutional practice.

In some respects, treated patients demonstrate a complete response (CR), meaning all target lesions disappear. In some respects, treated patients demonstrate a partial response (PR), meaning the sum of the target lesion diameters decreases by at least 30% relative to the baseline sum diameter. In some respects, treated patients demonstrate disease progression (PD), meaning the sum of the target lesion diameters increases by at least 20% relative to the minimum sum in the study (this includes the baseline sum, if it is the minimum sum in the study). In addition to a 20% relative increase, the sum must also show an absolute increase of at least 5 mm (note: the appearance of one or more new lesions can be considered progression). In some respects, treated patients demonstrate disease stability (SD), meaning there is neither sufficient shrinkage to meet PR nor sufficient increase to meet PD, with the minimum sum of diameters used as a reference at the time of the study.

On the other hand, treated patients experience tumor shrinkage and/or a slower growth rate, i.e., tumor growth is suppressed. In some respects, unwanted cell proliferation is reduced or suppressed. In some respects, one or more of the following may occur: the number of cancer cells may be reduced; the tumor size may be reduced; the invasion of cancer cells into peripheral organs may be inhibited, hindered, slowed, or stopped; tumor metastasis may be slowed or suppressed; tumor growth may be suppressed; tumor recurrence may be prevented or delayed; or one or more cancer-related symptoms may be alleviated to some extent.

In other respects, the administration of a bispecific antibody or its antigen-binding fragment according to any of the methods provided herein produces at least one therapeutic effect selected from the group consisting of: reduction in tumor size, reduction in the number of metastatic lesions over time, complete remission, partial remission, or disease stabilization.

In some respects, one or more tumor biopsies can be used to determine a tumor response to administration of a bispecific antibody or its antigen-binding fragment according to any of the methods provided herein. In some respects, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some respects, the sample is a fresh sample. Tumor samples (e.g., biopsies) can be used to identify predictive and/or pharmacodynamic biomarkers associated with immunity and the tumor microenvironment. Such biomarkers can be identified from assays including IHC, tumor mutation analysis, RNA analysis, and proteomics analysis. In some respects, the expression of tumor biomarkers is detected by RT-PCR, in situ hybridization, RNase protection, RT-PCR-based assays, immunohistochemistry, enzyme-linked immunosorbent assays, in vivo imaging, or flow cytometry.

5.5 Bispecific antibodies and their antigen-binding fragments

This article provides a method for treating cancer in a subject (e.g., a human subject), comprising administering to the subject an antibody (e.g., a bispecific antibody, a monoclonal antibody such as a chimeric antibody, a humanized antibody, or a human antibody) and its antigen-binding fragment that specifically binds to PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4). In some aspects, PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4) antibodies and their antigen-binding fragments that can be used in the methods provided herein include MEDI5752, which specifically binds to PD-1 and CTLA-4. This is a monovalent bispecific humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) having an engineered crystallizable fragment (Fc) domain to reduce Fc effector function.

MEDI5752 was constructed on the backbone of the DuetMab molecule. The DuetMab design was described in Mazor et al., MAbs [Monoclonal Antibodies]. 7(2): 377-389, (March-April 2015), which is hereby incorporated in its entirety by reference. The “DuetMab” design incorporates the kilo-honeypot (KIH) technique for heterodimerization of two different heavy chains and improves the efficiency of homologous heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond.

MEDI5752's Fc domain carries triple mutations (TM) (L234F, L235E, and P331S) designed to reduce Fc-mediated immune effector function (Oganesyan et al., Acta Crystallogr DBiol Crystallogr, 2008, 64(Pt 6): 700-704). MEDI5752 includes engineered interchain disulfide bonds at the anti-PD-1 and anti-CTLA-4Fab and anti-CTLA-4CH1-CL interfaces, as well as a club-and-mortar structure IgG1-TM Fc. MEDI5752 includes club mutations in the heavy chain containing the variable region for CTLA-4 binding and mortar mutations in the heavy chain containing the variable region for PD-1 binding.

MEDI5752 is described in U.S. Patent No. 10,457,732, which is incorporated herein by reference in its entirety.

In some aspects of this disclosure, the bispecific antibody or its antigen-binding fragment used in the methods described herein specifically binds to human PD-1 and human CTLA-4, and comprises six CDRs such as the MEDI5752 antibody provided in Table 1.

Table 1. VH CDR amino acid sequence ¹

The VH CDR in Table ¹ is determined based on Kabat.

Table 2. VL CDR amino acid sequence ²

The VL CDR in Table ² is determined based on Kabat.

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

In some aspects of this disclosure, the bispecific antibody or its antigen-binding fragment used in the methods described herein specifically binds to human PD-1 and human CTLA-4, and comprises the heavy chain (HC) of the MEDI5752 antibody listed in Table 3.

Table 3: Full-length heavy chain amino acid sequence

In some aspects of this disclosure, the bispecific antibody or its antigen-binding fragment used in the methods described herein specifically binds to human PD-1 and CTL-4 and comprises a light chain (LC) of the MEDI5752 antibody listed in Table 4.

Table 4: Full-length light chain amino acid sequence

In some aspects, the bispecific antibodies or antigen-binding fragments thereof that can be used in the disclosed methods include isolates that specifically bind to human PD-1 and CTLA-4 and cross-competitively bind to human PD-1 and CTLA-4 with MEDI5752. In some aspects, the bispecific antibodies or antigen-binding fragments thereof that can be used in the disclosed methods include isolates that bind to the same epitopes on human PD-1 and CTLA-4 as MEDI5752.

In some respects, bispecific antibodies or their antigen-binding fragments contain:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VH CDR2 containing the amino acid sequence of SEQ ID NO: 9, VHCDR3 containing the amino acid sequence of SEQ ID NO: 10, VLCDR1 containing the amino acid sequence of SEQ ID NO: 5, VL CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 7; and

(b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14, VH CDR2 containing the amino acid sequence of SEQ ID NO: 15, VH CDR3 containing the amino acid sequence of SEQ ID NO: 16, VL CDR1 containing the amino acid sequence of SEQ ID NO: 11, VL CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 13.

In some respects, bispecific antibodies or their antigen-binding fragments contain

(a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and

(b) A heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3.

In some respects, bispecific antibodies or antigen-binding fragments contain the constant region of the IgG heavy chain. In other respects, the constant region of the IgG heavy chain is the constant region of the IgG1 heavy chain.

In some respects, bispecific antibodies or their antigen-binding fragments are humanized bispecific antibodies or their antigen-binding fragments.

As provided herein, antibodies or antigen-binding fragments thereof that specifically bind to PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4) may have reduced effector function, for example, compared to antibodies or antigen-binding fragments thereof having a wild-type IgG1 sequence. This reduced effector function may be, for example, a result of the sequence of constant regions of the antibody or its antigen-binding fragment.

As provided herein, antibodies or antigen-binding fragments thereof that specifically bind to PD-1 and CTLA-4 (e.g., human PD-1 and CTLA-4) for the methods described herein may lack CDC and/or ADCC activity, for example, due to the sequence of the constant region.

5.6 Pharmaceutical Compositions

Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration (e.g., in a liquid carrier) or suitable for reconstitution into a liquid solution or suspension for intravenous administration.

Typically, these compositions contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceuticalally acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with the compound. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, polyethylene glycol ricinoleate, etc. Water or aqueous saline solutions, as well as aqueous dextran and glycerol solutions, can be used as carriers, particularly for injectable solutions. Liquid compositions intended 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.

The following examples are provided in an illustrative rather than restrictive manner.

6. Examples

The examples in this section (i.e., Section 6) are provided in an illustrative rather than restrictive manner.

6.1 Case 1: Phase I Clinical Evaluation of MEDI5752

MEDI5752 was evaluated in a Phase 1, first-in-human, multicenter, open-label, dose-escalation, and dose-expansion study in adult subjects with advanced solid tumors, as a single agent or in combination with chemotherapy (Figures 1A-1D). The aim of this study was to provide a description of early signs of safety profiles, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor efficacy. Up to approximately 396 subjects were enrolled: up to approximately 86 in the dose-escalation phase, approximately 92 in the renal cell carcinoma (RCC) expansion cohort, approximately 198 in the non-squamous non-small cell lung cancer (NSCLC) expansion cohort, and approximately 20 in the squamous NSCLC expansion cohort.

Participants were recruited at approximately 50 sites worldwide. As of the data cutoff date, a total of 178 participants with advanced solid tumors received at least one dose of MEDI5752 monotherapy at one of 10 dose levels (1 each of 2.25 mg and 7.5 mg, 3 of 22.5 mg, 5 of 75 mg, 10 of 225 mg, 20 of 500 mg, 40 of 750 mg, 39 of 1500 mg, 34 of 2000 mg, and 7 of 2500 mg). A total of 152 participants with advanced non-squamous NSCLC received or will 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 in combination with carboplatin and paclitaxel or nab-paclitaxel (750 mg). A total of 92 participants out of 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).

The study comprised two phases: dose escalation and dose expansion (Figures 1A–1D). The dose escalation phase evaluated 10 dose levels to determine the maximum tolerated dose (MTD), optimal biological dose (OBD), or highest protocol-defined dose (HPDD). Following the dose escalation phase was the dose expansion phase, which evaluated immunotherapy-naïve subjects with advanced clear cell RCC in two cohorts (first- to third-line in the RCC-C1 expansion cohort and first-line in the RCC-C2 expansion cohort), immunotherapy-naïve subjects with first-line stage IIIB–IV non-squamous NSCLC in two cohorts (NSCLC expansion cohorts NSCLC-C1 and NSCLC-C2), and immunotherapy-naïve subjects with first-line stage IIIB–IV squamous NSCLC in one cohort (NSCLC expansion cohort NSCLC-C3). Subjects in the RCC-C2 and NSCLC-C1 expansions were randomized for treatment. Subjects continued treatment until confirmed disease progression (PD), initiation of alternative cancer therapy, unacceptable toxicity, withdrawal of consent, or other reasons for discontinuation of treatment occurred. Survival of all subjects was monitored until the end of the study (either 2 years after the final subject's entry or at the end of the study, whichever occurred first).

Dosage escalation phase

The dose escalation consisted of 10 dose levels of MEDI5752 administered via IV infusion (dose levels 2.25, 7.5, 22.5, 75, 225, 500, 750, 1500, 2000, and 2500 mg). Once the OBD, MTD, or HPDD was determined, a specific cohort (i.e., the pharmacodynamic cohort) consisting of up to 6 subjects with a glomerular filtration rate (GFR) between 30 and 45 mL/min was explored under OBD, MTD, or HPDD. Up to approximately 111 subjects were recruited during the dose escalation phase.

Dose extension phase

Once the MTD, OBD, or HPDD is determined during the dose escalation phase, the dose expansion phase begins. In the expansion cohort, only eligible metastatic subjects who have not received prior immunotherapy are recruited.

The RCC and NSCLC extended cohorts provided additional safety, tolerability, efficacy, pharmacokinetic (PK), and PD data for MEDI5752 at multiple dose levels in these target populations. The NSCLC cohort also evaluated combinations of MEDI5752 with carboplatin plus pemetrexed and carboplatin plus paclitaxel or nab-paclitaxel. Furthermore, in subjects with non-squamous NSCLC, the antitumor activity of MEDI5752 in combination with carboplatin plus pemetrexed was compared to that of pembrolizumab in combination with carboplatin plus pemetrexed.

Subjects in the RCC extended cohort (RCC-C1 and RCC-C2) received the following:

• MEDI5752 Q3W was administered via IV infusion at doses of 500 mg, 750 mg, and 1500 mg over 60 minutes (±10 minutes); subjects receiving 500 mg and 750 mg were randomized in a 1:1 ratio when both cohorts were open.

• MEDI5752Q3W administered via IV over 60 minutes (±10 minutes): with a single initial dose of 750 mg, 1000 mg, 1250 mg or 1500 mg, followed by a maintenance dose of 225 mg, 500 mg, 750 mg, 1000 mg or 1250 mg.

Subjects in the non-squamous NSCLC extended cohort (NSCLC-C1) received:

• Safety-based delivery (NSCLC-C1 S): MEDI5752 Q3W was administered via IV infusion of 2000 mg over 60 minutes (±10 minutes) in combination with carboplatin, area under the concentration-time curve (AUC) of 5 mg/mL·min, and pemetrexed 500 mg/m2 (Q3W), for a total of 4 doses, followed by indefinite maintenance therapy with MEDI5752 and pemetrexed Q3W.

• Randomize to receive one of the following at a 1:1 ratio (NSCLC-C1R):

Group A: MEDI5752 1500 mg in IV infusion over 60 minutes (±10 minutes) Q3W combined with carboplatin, area under the concentration-time curve (AUC) 5 mg/mL·min, and pemetrexed 500 mg/m2 (Q3W), delivered for 4 doses, followed by indefinite maintenance therapy with MEDI5752 and pemetrexed Q3W; or

Group B: Pembrolizumab 200 mg plus carboplatin AUC 5 mg/mL·min and pemetrexed 500 mg/m2 Q3W, delivered 4 doses, followed by pembrolizumab for 21 months (24 months in total) and indefinite pemetrexed maintenance therapy Q3W.

Subjects in the non-squamous NSCLC extended cohort (NSCLC-C2) received:

• MEDI5752 Q3W, administered via IV infusion at 500 mg or 750 mg over 60 minutes (±10 minutes), combined with carboplatin (AUC) 5 mg/mL·min, and pemetrexed 500 mg/m2 (Q3W), for 4 doses, followed by indefinite maintenance therapy with MEDI5752 and pemetrexed Q3W.

Subjects in the squamous NSCLC extended cohort (NSCLC-C3) received:

• Administer MEDI5752 via IV infusion at 500 mg or 750 mg over 60 minutes (±10 minutes) for 3 weeks, combined with carboplatin AUC 6 mg/mL·min for 3 weeks and paclitaxel 200 mg/m2 for 3 weeks or nab-paclitaxel 100 mg/m2, for a total of 4 doses on days 1, 8, and 15 of each 3-week cycle, followed by indefinite MEDI5752 maintenance therapy for 3 weeks.

end

The primary safety endpoints during the dose escalation phase are to assess the presence of adverse events (AEs), severe adverse events (SAEs), and deep vein thrombosis (DLTs), and to determine the MTD, OBD, or HPDD of MEDI5752 without exceeding the MTD.

The primary objective of the RCC and non-squamous NSCLC expansion cohort was to evaluate the preliminary antitumor activity of MEDI5752 monotherapy in RCC, and the preliminary antitumor activity of MEDI5752 plus carboplatin plus pemetrexed (as opposed to pembrolizumab plus carboplatin plus pemetrexed in NSCLC-C1) in non-squamous NSCLC. The primary endpoint was OR, which is typically used in oncology studies to evaluate 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 combination with chemotherapy in subjects with advanced solid tumors.

Secondary endpoints included safety assessment, additional antitumor activity, PK, immunogenicity, and biomarker assessment.

PD-L1 status will be assessed in subjects who have provided archived or fresh tumor biopsies. For the NSCLC extended cohort, PD-L1 positivity is defined as baseline PD-L1 expression in ≥1% of tumor cells, while PD-L1 negativity is defined as baseline PD-L1 expression in <1% of tumor cells.

result

Security data

A total of 136 subjects received MEDI5752 treatment. Table 5 shows the exposures and MEDI5752-related adverse events observed at selected MEDI5752 monotherapy and combination dose levels up to the data snapshot date.

Table 5. Adverse Events Related to MEDI5752 as of January 25, 2023 (Data Snapshot Date)

AE, Adverse Events; N, Number; SAE, Serious Adverse Events.

Table 6 provides the exposures and MEDI5752-related adverse events observed in the RCC queue. Table 6 Adverse Events in the RCC Queue as of January 25, 2023 (Data Snapshot Date)

Table 7 Adverse Events in the NSCLC Queue as of January 25, 2023 (Data Snapshot Date)

*Related to MEDI5752, pembrolizumab, and/or CTx.

A patient treated with pembrolizumab plus CTx died of febrile neutropenia.

One patient treated with MEDI5752 750mg + CTx died of pneumonia, two died of febrile neutropenia, and one died of septic shock.

^A patient treated with MEDI5752 500mg + CTx died of febrile neutropenia.

^^A patient treated with MEDI5752 750mg + CTx died of respiratory sepsis.

One subject in each of the 2000 mg (grade 3 pneumonia and grade 1 myocarditis in the same subject) and 2500 mg (grade 3 maculopapular rash) monotherapy cohorts reported dose-limiting toxicities (DLTs). Two deaths were determined to be treatment-related to MEDI5752; one subject died from diabetic ketoacidosis and hyperthyroidism, and the other died from myocardial infarction.

A review of the benefit-risk profile of subjects treated with MEDI5752 indicated that long-term dosing at doses of 1500 mg and above is not suitable for further development due to the high rate of treatment discontinuation (primarily driven by grade 3/4 hepatotoxicity).

Efficacy data

In the dose-escalation and maximum tolerated dose (MTD)/optimal biological dose (OBD) cohorts, among the 86 subjects treated with MEDI5752 monotherapy, regardless of dose, the overall objective response rate (ORR) (according to the RECIST version [v] 1.1 criteria for response assessment in solid tumors) was 19.8% (17/86), with 1 subject achieving a complete response (CR). Among the 5 subjects treated with 500 mg of MEDI5752 during dose-escalation, 2 achieved a partial response (PR) with optimal overall response (BOR), 2 had stable disease (SD), and 1 had progressive disease (PD). Among the 8 subjects treated with 750 mg of MEDI5752 during dose-escalation, 4 had SD with BOR, 3 had PD, and 1 was not evaluable. The median duration of response for MEDI5752 monotherapy was 17.5 months (Figure 9). Across a range of MEDI5752 doses, MEDI5752 monotherapy also demonstrated durable responses in a variety of IO-naïve tumors (Figure 10).

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

Table 8. Efficacy of MEDI5752 1500mg + Carboplatin/Pemetrexed group relative to Pembrolizumab + Carboplatin/Pemetrexed group in first-line non-squamous NSCLC as of January 25, 2023 (data snapshot date).

NE, incalculable

In the extended cohort of non-squamous non-small cell lung cancer (NSCLC) (NSCLC-C2), the objective response rate (ORR) was 45.5% (30/66) in the first 66 subjects treated with MEDI5752 750 mg plus carboplatin/pemetrexed. In the PD-L1 <1% subgroup, the ORR was 50% (24/48). The median follow-up duration was 7.2 months (range, 0.3–16.4 months) (Figure 15B).

In the extended cohort of non-squamous non-small cell lung cancer (NSCLC) (NSCLC-C2), among subjects treated with MEDI5752 500 mg + carboplatin/pemetrexed, the evaluable population ORR was 25% (8/32), and the initial response rate of CR+PR+unconfirmed PR was 34.4% (11/32). The median follow-up duration was 3.3 months (range, 0.6–6.5 months) (Figure 15B).

In the expanded cohort of squamous non-small cell lung cancer (NSCLC) (NSCLC-C2), among subjects treated with MEDI5752 750 mg plus carboplatin/pemetrexed, the evaluable population ORR was 22.2% (4/18), and the initial response rate of CR+PR+unconfirmed PR was 55.6% (10/18). The median follow-up duration was 2.8 months (range, 0.7–7.6 months) (Figure 15C).

In the extended cohort (RCC-C1) of renal cell carcinoma (RCC) patients receiving MEDI5752 1500 mg, the ORR was 38.5% (10/26), with 2 complete remissions (CRs). Additionally, the ORR in first-line RCC patients was 58.3% (7/12) (Figure 12). Furthermore, the response in the first-line RCC cohort was found to be durable (Figure 13). The efficacy of 500 mg and 750 mg is given in Table 9 (Figure 12).

Table 9. Efficacy of MEDI 5752 750mg and 500mg in first-line RCC as of January 25, 2023 (data snapshot date)

MEDI5752 Pharmacokinetic/Pharmacodynamic Data

Data indicate that MEDI5752 exhibits nonlinear pharmacokinetic (PK) activity, possibly due to saturated target-mediated clearance at doses <22.5 mg, and possibly potentially due to the effect of ADA on MEDI5752 clearance. Figure 4 shows the mean MEDI5752 PK curves for the first 84 days.

Pharmacodynamic data showed that MEDI5752 led to a dose-dependent increase in CD4+ T cell proliferation, reaching a steady-state level at 500/750 mg and above (Figures 5 and 7A). At doses >225 mg, MEDI5752 showed sustained peripheral PD-1 receptor occupancy (>90%) (Figure 6). Pharmacodynamic data in monotherapy also showed that MEDI5752 led to a dose-dependent increase in CD4+ T cell activation, with T cell expansion reaching a steady-state level at 500/750 mg and above (Figures 7B and 8A-8B, respectively). Pharmacodynamic data in combination with chemotherapy showed that, compared to the combination of pembrolizumab and chemotherapy, the combination of MEDI5752 and chemotherapy at 750 mg and 1500 mg doses resulted in a greater increase in CD4+ T cell proliferation on day 8 of cycle 1 and a higher proportion of newly expanded T cell clones in cycle 2 (Figures 7C and 8C, respectively).

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-exploration and dose-expansion study to assess its safety, tolerability, pharmacokinetics, immunogenicity, and antitumor activity in previously untreated patients with advanced RCC.

This study included a dose-exploration phase, followed by a dose-expansion phase. The dose-exploration phase evaluated the safety and tolerability of up to two planned dose levels of MEDI5752 in combination with axitinib (Part A) and up to three or more additional planned dose levels of MEDI5752 in combination with lenvatinib (Part B). After recruiting one subject from each dose level, the MEDI5752 in combination with axitinib was discontinued. Therefore, a recommended phase 2 dose (RP2D) was determined only for the MEDI5752 in combination with lenvatinib. The RP2D was determined by evaluating the maximum tolerated dose (MTD) assessed during the dose-exploration phase, along with all clinical data from the remainder of the MEDI5752 program. A maximum of nine subjects were recruited per dose cohort. Additional subjects may be required if additional cohorts, treatment regimens, or doses are explored based on emerging PK/pharmacodynamic and clinical data. Once the MTD or MAD of the MEDI5752 in combination with lenvatinib was determined during the dose-exploration phase, up to 50 subjects were recruited for the dose-expansion phase.

Target Subject Group

Subjects aged ≥18 years, with histologically or cytologically confirmed advanced RCC, possessing clear cell components, and who have not previously received treatment.

Treatment group and regimen

Up to approximately 70 participants were recruited in two phases and assigned to study treatments detailed below.

Dosage exploration phase:

Part A of the dose exploration phase (MEDI5752 + axitinib) includes the following two planned dose levels:

• Dose Level 1 (starting dose; n = up to 9 subjects) (closed after recruiting 1 subject):

Axitinib 5mg PO BID (Implantation: Day 7 to Day 1)

MEDI5752 1500 mg IV every 3 weeks (Q3W) and axitinib 5 mg PO BID (starting from day 1 [i.e., day 1 of cycle 1]).

• Dose Level-1 (decreasing dose; n = up to 9 subjects) (turned off after recruiting 1 subject)

Axitinib 5mg PO BID (Implantation: Day 7 to Day 1)

MEDI5752 750mg IV Q3W and axitinib 5mg PO BID (starting from day 1 [i.e., day 1 of cycle 1])

Part B of the dose exploration phase (MEDI5752 + lenvatinib) includes the following three planned dose levels:

• Dose level - 3LEN (starting dose; n = up to 9 subjects)

MEDI5752 500mg IV Q3W and Lenvatinib 14mg PO QD (starting from day 1 [i.e., day 1 of cycle 1])

• Dose level - 2LEN (LEN dose increments; n = up to 9 subjects)

MEDI5752 500mg IV Q3W and Lenvatinib 18mg PO QD (starting from day 1 [i.e., day 1 of cycle 1])

• Dose level -1 LEN (MEDI5752 dose increments, LEN decrements [if needed]; n = up to 9 subjects)

MEDI 5752 750 mg IV Q3W and lenvatinib (14 mg or 18 mg; dosage determined by the previous two dose levels) PO QD (starting from day 1 [i.e., day 1 of cycle 1]).

• Other dosage levels

MEDI5752 Q3W administered via IV infusion over 60 minutes (±10 minutes): a single initial 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 from day 1 of cycle 1, cycle 2, or cycle 3).

MEDI5752 Q3W: 500 mg or 750 mg and lenvatinib (10 mg) PO QD over 60 minutes (±10 minutes) via IV infusion (starting from day 1 of cycle 1).

Dosage expansion phase:

• MEDI5752 IV Q3W and lenvatinib PO QD (starting from day 1 [i.e., day 1 of cycle 1]), administered at the combination dose determined during the dose exploration phase (starting from day 1 [i.e., day 1 of cycle 1]) (n = up to 50 subjects)

Subjects received MEDI5752 in combination with axitinib or lenvatinib until disease progression, initiation of alternative cancer therapy, unacceptable toxicity, withdrawal of consent, or other reasons for discontinuation of treatment. Subject survival was followed up until the end of the study, defined as approximately 3 years after the final subject's enrollment, unless the study ended before that time.

Study endpoints

Safety: incidence of adverse events/severe adverse events (AEs/SAEs), dose-limiting toxicities, AEs leading to treatment discontinuation, abnormal laboratory parameters, vital signs, and ECG results.

Efficacy: RECIST v1.1 will be used to assess response in the evaluable population for interim analysis of BOR, ORR, DCR, DoR, and TTR, and in the treated population for final analysis. PFS (according to RECIST v1.1).

Pharmacokinetics: Serum MEDI5752 concentration

Immunogenicity: Incidence of ADA against MEDI5752.

Claims (71)

1. A method for treating a subject with renal cell carcinoma (RCC), 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). 2. A method for inhibiting the growth of renal cell carcinoma (RCC) 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 programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). 3. The method of claim 1 or 2, wherein the method comprises administering about 500 mg or about 750 mg of the bispecific antibody or its antigen-binding fragment. 4. The method of any one of claims 1-3, further comprising administering one or more tyrosine kinase inhibitors. 5. The method of any one of claims 1-4, further comprising administering the tyrosine kinase inhibitor axitinib or lenvatinib. 6. The method of claim 5, wherein axitinib is administered orally twice daily at a dose of 5 mg from day 7 to day 1 prior to administration of the bispecific antibody or its antigen-binding fragment. 7. The method of claim 6, wherein the bispecific antibody or its antigen-binding fragment is administered on day 1. 8. The method according to any one of claims 1-7, wherein the bispecific antibody or its antigen-binding fragment is administered once per treatment cycle at a dose of 500 mg or 750 mg. 9. The method of claim 8, wherein the treatment period is three weeks. 10. The method of any one of claims 4-9, further comprising maintaining administration of the bispecific antibody or its antigen-binding fragment and/or one or more doses of a chemotherapeutic agent. 11. A method of treating a subject with non-small cell lung cancer (NSCLC), 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). 12. A method for inhibiting the growth of non-small cell lung tumors 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). 13. The method of claim 11 or 12, wherein the method comprises administering about 500 mg or about 750 mg of the bispecific antibody or its antigen-binding fragment. 14. The method of any one of claims 11-13, wherein the bispecific antibody or its antigen-binding fragment is administered once per treatment cycle. 15. The method of claim 14, wherein the treatment period is three weeks. 16. The method of any one of claims 11-15, further comprising administering one or more chemotherapeutic agents. 17. The method of any one of claims 11-16, wherein the NSCLC or non-small cell lung tumor is non-squamous lung cancer. 18. The method of any one of claims 11-16, wherein the NSCLC or non-small cell lung tumor is squamous cell lung cancer. 19. The method of any one of claims 11-17, wherein the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, pemetrexed, and combinations thereof. 20. The method of claim 19, wherein the one or more chemotherapeutic agents are carboplatin and pemetrexed. 21. The method of claim 16, wherein the NSCLC or non-small cell lung tumor is non-squamous lung cancer, and wherein the one or more chemotherapeutic agents are carboplatin and pemetrexed. 22. The method of any one of claims 11-16 and 19, wherein the bispecific antibody or its antigen-binding fragment is administered every three weeks at a dose of 500 mg or 750 mg, and the carboplatin is administered every three weeks at a dose of AUC 6 mg/mL·min or AUC 5 mg/mL·min. 23. The method of claim 20, wherein the bispecific antibody or its antigen-binding fragment 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/ ^m² . 24. The method of claim 20, wherein the bispecific antibody or its antigen-binding fragment 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/ ^m² , for a total of 4 doses, once every three weeks, followed by maintenance administration of the bispecific antibody or its antigen-binding fragment and pemetrexed once every three weeks. 25. The method of claim 20, wherein the bispecific antibody or its antigen-binding fragment 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/ ^m² , for a total of 4 doses, once every three weeks, followed by maintenance administration of the bispecific antibody or its antigen-binding fragment and pemetrexed once every three weeks. 26. The method of any one of claims 11-16 and 18, wherein the one or more chemotherapeutic agents are selected from the group consisting of carboplatin, paclitaxel, Nab-paclitaxel, and combinations thereof. 27. The method of claim 26, wherein the one or more chemotherapeutic agents are carboplatin and paclitaxel. 28. The method of claim 26, wherein the one or more chemotherapeutic agents are carboplatin and Nab-paclitaxel. 29. The method of claim 16, wherein the NSCLC or non-small cell lung tumor is squamous cell lung cancer, and wherein the one or more chemotherapeutic agents are carboplatin and Nab-paclitaxel. 30. The method of claim 27, wherein the bispecific antibody or its antigen-binding fragment 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/ ^m² every three weeks. 31. The method of claim 28, wherein the bispecific antibody or its antigen-binding fragment 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/ ^m² body surface area (BSA) on days 1, 8, and 15 of each 3-week cycle. 32. The method of any one of claims 1-31, wherein the application is performed in four 3-week cycles. 33. The method of any one of claims 4-10 and 16-32, further comprising maintaining administration of the bispecific antibody or its antigen-binding fragment and/or the one or more chemotherapeutic agents. 34. The method of any one of claims 1-33, wherein the subject is a human being. 35. The method of any one of claims 1-34, wherein the subject has an advanced solid tumor. 36. The method of any one of claims 1-35, wherein the application results in the disappearance of the target lesion and/or a reduction in its sum. 37. The method of any one of claims 1-36, wherein the disappearance of the target lesion and/or the sum of its reductions is determined by measuring the tumor biopsy sample. 38. The method of claim 37, wherein the sample is a fresh or formalin-fixed paraffin-embedded (FFPE) sample. 39. The method of any one of claims 36 or 37, wherein the sample is measured by RT-PCR, in situ hybridization, RNase protection, RT-PCR-based assay, immunohistochemistry (IHC), enzyme-linked immunosorbent assay, in vivo imaging, or flow cytometry. 40. The method of any one of claims 1-39, wherein the bispecific antibody binds to cynomolgus monkey PD-1 and CTLA-4. 41. The method of any one of claims 1-40, wherein the bispecific antibody or its antigen-binding fragment binds to human PD-1 and CTLA-4. 42. The method of any one of claims 1-41, wherein the bispecific antibody or its antigen-binding fragment is a humanized bispecific antibody or its antigen-binding fragment. 43. The method of any one of claims 1-42, wherein the bispecific antibody or its antigen-binding fragment is monovalent. 44. The method of any one of claims 1-43, wherein the bispecific antibody or its antigen-binding fragment is DuetMab. 45. The method of any one of claims 1-44, wherein the bispecific antibody or antigen-binding fragment comprises the constant region of the IgG heavy chain. 46. The method of claim 45, wherein the constant region includes mutations at L234F, L235E, and P331S. 47. The method of claim 45 or 46, wherein the constant region comprises a pestle mutation and a mortar mutation, optionally wherein the pestle mutation is in a heavy chain containing a variable region that binds CTLA-4, and the mortar mutation is in a heavy chain containing a variable region that binds PD-1. 48. The method of any one of claims 45-47, wherein the IgG heavy chain constant region is the IgG1 heavy chain constant region. 49. The method of any one of claims 1-48, wherein the bispecific antibody or its antigen-binding fragment comprises the anti-PD-1 and anti-CTLA-4 heavy chain variable regions (VH)CDR1, VH CDR2, VH CDR3, light chain variable regions (VL)CDR1, VL CDR2, and VL CDR3 of the MEDI5752 sequence. 50. The method of any one of claims 1-49, wherein the bispecific antibody or its antigen-binding fragment comprises: (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VHCDR2 containing the amino acid sequence of SEQ ID NO: 9, VH CDR3 containing the amino acid sequence of SEQ ID NO: 10, VLCDR1 containing the amino acid sequence of SEQ ID NO: 5, VLCDR2 containing the amino acid sequence of SEQ ID NO: 6, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 7; and (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14, VHCDR2 containing the amino acid sequence of SEQ ID NO: 15, VH CDR3 containing the amino acid sequence of SEQ ID NO: 16, VLCDR1 containing the amino acid sequence of SEQ ID NO: 11, VL CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 13. 51. The method of any one of claims 1-50, wherein the bispecific antibody or its antigen-binding fragment comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) A heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3. 52. The method of any one of claims 1-51, wherein the bispecific antibody or its antigen-binding fragment is a full-length antibody. 53. The method according to any one of claims 1-52, wherein the bispecific antibody is MEDI5752. 54. The method of any one of claims 1-50, wherein the bispecific antibody or its antigen-binding fragment is an antigen-binding fragment. 55. The method of claim 54, wherein the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, bispecific intracellular antibody, IgGΔCH2, mini antibody, F(ab′)3, tetrasomal antibody, trisomal antibody, bisomal antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. 56. A method of treating a subject with advanced renal cell carcinoma, the method comprising administering to the subject approximately 750 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4 every three weeks, the bispecific antibody comprising: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3. 57. A method of treating a subject with advanced renal cell carcinoma, the method comprising administering to the subject approximately 500 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4 every three weeks, the bispecific antibody comprising: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3. 58. A method of treating a subject with non-small cell lung cancer, the method comprising administering to the subject: (1) about 750 mg or about 500 mg of a bispecific antibody that specifically binds to PD-1 and CTLA-4 every three weeks, and the bispecific antibody comprising: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3; (2) carboplatin at a dose of AUC 5 mg/mL·min every three weeks for four cycles; and (3) pemetrexed at a dose of 500 mg/ ^m² every three weeks for multiple cycles, followed by maintenance administration of the bispecific antibody and pemetrexed. 59. A method for treating a subject with non-small cell lung cancer, the method comprising administering to the subject: (1) Approximately 750 mg or approximately 500 mg every three weeks of a bispecific antibody that specifically binds to PD-1 and CTLA-4, wherein the bispecific antibody comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3; and (2) Carboplatin at a dose of AUC 6 mg/mL·min, once every three weeks; and (3) Paclitaxel at a dose of 200 mg/m2, once every three weeks, or (4) Nab-paclitaxel at a dose of 100 mg/ ^m² body surface area on days 1, 8, and 15 of every three-week cycle, and (5) Maintain administration of the bispecific antibody. 60. The method of any of the preceding claims, wherein the non-small cell lung cancer and/or renal cell carcinoma comprises about ≥50% tumor cells expressing PD-L1. 61. The method of any of the preceding claims, wherein the non-small cell lung cancer and/or renal cell carcinoma comprises about 1% to 49% of tumor cells expressing PD-L1. 62. The method of any of the preceding claims, wherein the non-small cell lung cancer and/or renal cell carcinoma comprises about <1% of tumor cells expressing PD-L1. 63. The method of any of the preceding claims, wherein the non-small cell lung cancer and/or renal cell carcinoma comprises about 0% tumor cells expressing PD-L1. 64. A method for expanding T cells in a subject in need, the method comprising administering a bispecific antibody or an antigen-binding fragment thereof that specifically binds programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), the antibody comprising: (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 8, VHCDR2 containing the amino acid sequence of SEQ ID NO: 9, VH CDR3 containing the amino acid sequence of SEQ ID NO: 10, VLCDR1 containing the amino acid sequence of SEQ ID NO: 5, VL CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 7; and (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14, VHCDR2 containing the amino acid sequence of SEQ ID NO: 15, VH CDR3 containing the amino acid sequence of SEQ ID NO: 16, VLCDR1 containing the amino acid sequence of SEQ ID NO: 11, VL CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VLCDR3 containing the amino acid sequence of SEQ ID NO: 13. 65. The method of claim 64, wherein the bispecific antibody comprises: (a) a heavy chain containing the amino acid sequence listed in SEQ ID NO: 2 and a light chain containing the amino acid sequence listed in SEQ ID NO: 1; and (b) A heavy chain containing the amino acid sequence listed in SEQ ID NO: 4 and a light chain containing the amino acid sequence listed in SEQ ID NO: 3. 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. The method of claims 64 to 66, wherein the bispecific antibody is administered at a dose of about 750 mg Q3W. 68. The method of claim 66, wherein the bispecific antibody is administered once 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. 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 than the number of T cell clones prior to administration. 71. The method of any one of claims 64 to 70, wherein the bispecific antibody is MEDI5752.