Combination Therapies for the Treatment of Cancer

Abstract

Provided herein are methods of treating cancer by administering to a subject having cancer an antibody that specifically binds to programmed cell death protein 1 (PD1), or a functional fragment or functional variant thereof; and a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of transforming growth factor beta receptor II (TGFβRII).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to Indian Provisional Application No. IN202011054571, filed on 15 December 2020, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Immune checkpoint inhibitors are a type of cancer treatment that works by reversing T cell inhibition and tumor immune evasion. Examples of immune checkpoint inhibitors include antibodies that specifically bind to and inhibit immune checkpoint proteins such as programmed cell death protein 1 (PD1) or its ligand, programmed cell death ligand 1 (PDL1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4). However, antibody immune checkpoint inhibitors still present several clinical challenges regarding efficacy and patient-to-patient variability. Combination therapies targeting multiple non-redundant pathways that control the immune response can enhance the efficacy of immune checkpoint inhibitors. However, not all combinations provide synergistic effects superior to monotherapy components. Therefore, there is a need for combination therapies that promote an anti-tumor immune response, have an acceptable safety profile, and high efficacy compared to monotherapy and other immunotherapy combinations. [Overview of the Initiative]

[0003] This specification provides a method for treating cancer in a subject, comprising administering a drug that specifically binds to PD1 in combination with a fusion protein (e.g., the fusion protein described herein) that specifically binds to EGFR and TGFβ. The combination therapy disclosed herein may be particularly useful in the treatment of EGFR-induced cancer.

[0004] Accordingly, in one embodiment, the present disclosure provides a method for treating cancer in a human subject requiring treatment for cancer, the method comprising administering to the subject an antibody, or a functional fragment or functional variant thereof, that specifically binds to programmed cell death protein 1 (PD1); and administering to the subject a fusion protein comprising a targeting portion and an immunomodulatory portion, wherein (i) the targeting portion specifically binds to the epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain of transforming growth factor β receptor II (TGFβRII).

[0005] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0006] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 inhibits the binding of PD1 to PDL1. In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 inhibits PD1 signaling.

[0007] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 comprises VH including VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1; VH CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2; and VH CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

[0008] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 comprises VLs including VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4; VL CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5; and VL CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0009] In some embodiments, the antibody, or its functional fragment or functional variant, which specifically binds to PD1, includes a VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0010] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 includes a VL containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0011] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 includes a heavy chain region having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0012] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 includes a heavy chain region having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the antibody, or its functional fragment or functional variant, which specifically binds to PD1, includes a light chain region having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is pembrolizumab, nivolumab, semiprimab, spartalizumab, camrelizumab, tisrelizumab, dostallimab, cetrelimab, pidilizumab, MEDI0680, SSI-361, AMP-224, PDR001, PF-06801591, BGB-A317, TSR- Includes 042, AGEN-2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM-003, ENUM-388D4, IBI-308, JNJ-63723283, JS-001, JTX-4014, JY-034, CLA-134, STIA-1110, 244C8, and 388D4, or any functional fragment or functional variant of any of the above.

[0015] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 includes pembrolizumab, or any of the functional fragments or functional variants described above.

[0016] In some embodiments, the targeted portion that specifically binds to EGFR includes an antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof. In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0017] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to EGFR comprises VH including VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34; VH CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; and VH CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36.

[0018] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to EGFR comprises VLs including VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37; VL CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38; and VL CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39.

[0019] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to EGFR includes a VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40.

[0020] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to EGFR includes a VL containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[0021] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, comprises a heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42.

[0022] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, consists of a heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43.

[0023] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, comprises a heavy chain consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42.

[0024] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, consists of a heavy chain consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43.

[0025] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, comprises a light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0026] In some embodiments, the antibody that specifically binds to EGFR, or a functional fragment or functional variant thereof, consists of a light chain consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0027] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to EGFR includes cetuximab or panitumumab, or any of the functional fragments or functional variants described above.

[0028] In some embodiments, the immunomodulatory portion includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 56.

[0029] In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion. In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker. In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker of sufficient length to allow both the immunomodulatory portion and the targeting portion to bind to their respective targets simultaneously.

[0030] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 57, 58, 59, 60, or 61. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 57. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 57.

[0031] In some embodiments, the immunomodulatory portion is fused to the C-terminus of the targeting portion. In some embodiments, the immunomodulatory portion is fused to the N-terminus of the targeting portion.

[0032] In some embodiments, the targeting portion is an antibody comprising a light chain and a heavy chain, and the immunomodulatory portion is fused to the C-terminus of the heavy chain of the targeting portion.

[0033] In some embodiments, the targeted portion is an antibody that specifically binds to the epidermal growth factor receptor (EGFR), comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the heavy chain or the light chain via a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57.

[0034] In some embodiments, the targeted portion is an antibody that specifically binds to the epidermal growth factor receptor (EGFR), comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the light chain through a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57.

[0035] In some embodiments, the targeted portion comprises an antibody comprising a heavy chain containing an amino acid sequence which includes an amino acid sequence which is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43; and a light chain containing an amino acid sequence which is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0036] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of breast cancer, anal cancer, pancreatic cancer, thyroid cancer, liver cancer, ovarian cancer, lung cancer, skin cancer, brain cancer, spinal cord cancer, head cancer, neck cancer, and head and neck cancer.

[0037] In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is recurrent HNSCC. In some embodiments, the cancer is metastatic HNSCC. In some embodiments, the cancer is recurrent metastatic HNSCC.

[0038] In some embodiments, the cancer is anal canal squamous cell carcinoma (SCCAC). In some embodiments, the cancer is recurrent SCCAC. In some embodiments, the cancer is metastatic SCCAC. In some embodiments, the cancer is recurrent metastatic SCCAC.

[0039] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 is administered to the human subject in doses of approximately 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 200 mg.

[0040] In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 is administered to the human subject in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg. In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 is administered to the human subject in doses of approximately 200 mg. In some embodiments, the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 is administered to the human subject in doses of approximately 300 mg.

[0041] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is administered to the human subject every 1, 2, 3, or 4 weeks. In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is administered to the human subject every 3 weeks.

[0042] In some embodiments, the fusion protein is approximately 50mg-2000mg, 100mg-2000mg, 150mg-2000mg, 200mg-2000mg, 300mg-2000mg, 400mg-2000mg, 500mg-2000mg, 600mg-2000mg, 700mg-2000mg, 800mg-2000mg, 900mg-2000mg The fusion protein is administered to the human subjects in doses of g, 1000mg-2000mg, 1500mg-2000mg, 50mg-100mg, 50mg-500mg, 50mg-400mg, 50mg-300mg, 50mg-200mg, 50mg-100mg, 100mg-500mg, 100mg-400mg, 100mg-300mg, or 100mg-200mg. In some embodiments, the fusion protein is administered to the human subjects in doses of approximately 200mg-2000mg. In some embodiments, the fusion protein is administered to the human subject in doses of approximately 50 mg, 60 mg, 64 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, the fusion protein is administered to the human subject in doses of approximately 64 mg, 240 mg, 800 mg, or 1600 mg.

[0043] In some embodiments, the fusion protein is administered to the human subjects every 1, 2, 3, or 4 weeks. In some embodiments, the fusion protein is administered to the human subjects weekly. In some embodiments, the fusion protein is administered to the human subjects for 3 weeks.

[0044] In some embodiments, the fusion protein is administered co-administered with the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1, or administered before or after the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1. [Brief explanation of the drawing]

[0045] [Figure 1] This line graph shows the effects of cetuximab, BCA101 (fusion mAb anti-EGFR + TGFβRII ECD), anti-PD1 (pembrolizumab), and BCA101 + anti-PD1 (pembrolizumab) on tumor volume in huNOG-EXL mice with PC-3 xenograft tumors over 21 days. Values ​​are expressed as the mean ± SEM of 8-10 animals in each group. Statistical analysis was performed using two-way ANOVA and subsequent Bonferroni post-hoc test with GraphPad Prism (Version 8.3.0). ***Significant (p<0.001) difference when each treatment group was compared to the isotype control group at day 18. SSS significant (p<0.001) and # significant (p<0.05) difference when the combination treatment groups were compared to BCA101 and pembrolizumab, respectively, at day 18. [Figure 2A] Figure 2A is a line graph showing the tumor growth curves of each isotype in the huNOG-EXL mouse PC-3 tumor xenograft over a 21-day period. [Figure 2B] Figure 2B shows line graphs illustrating tumor growth in each cetuximab-treated group over a 21-day period. [Figure 2C] Figure 2C is a line graph showing the tumor growth in each BCA101 treatment group over a 21-day period. [Figure 2D] Figure 2D is a line graph showing the tumor growth in each group treated with anti-PD1 (pembrolizumab) over a 21-day period. [Figure 2E] Figure 2E is a line graph showing the tumor growth in each group treated with BCA101 + anti-PD1 (pembrolizumab) over a 21-day period. [Figure 3A] These are photographs of huNOG-EXL mice with PC-3 tumors from the control group (isotype control), the cetuximab-treated group, and the BCA101-treated group. The photographs were taken on day 19 of the study. [Figure 3B] These are photographs of huNOG-EXL mice with PC-3 tumors treated with anti-PD1 (pembrolizumab) and BCA101 + anti-PD1 (pembrolizumab). The photographs were taken on day 19 of the study. [Figure 4] This line graph shows the effect of cetuximab, BCA101, anti-PD1 (pembrolizumab), and BCA101 + anti-PD1 (pembrolizumab) on the percentage change in body weight in huNOG-EXL mice carrying PC-3 tumor xenografts. Values ​​are expressed as the mean ± SEM of 8-10 animals in each group. Gradual weight loss was observed in all groups. No visible clinical signs or abnormal behaviors were observed in any of the treatment groups. [Modes for carrying out the invention]

[0046] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each respective publication, patent, or patent application is specifically indicated as being incorporated by reference herein.

[0047] overview This disclosure provides novel combination therapies, particularly those comprising agents that specifically bind to and inhibit the function of PD1, and EGFR-targeted immunomodulatory fusion proteins that bind to TGFβ. The combination therapies described herein offer synergistic effects and improved efficacy that surpass each of the monotherapies. In some embodiments, the EGFR fusion protein comprises a targeting moiety that specifically binds to EGFR, and an immunomodulatory moiety that includes the amino acid sequence of the extracellular domain of transforming growth factor β receptor II (TGFβRII). The combination treatments disclosed herein may be particularly useful in the treatment of EGFR-mediated cancers, such as head and neck cancer and anal cancer.

[0048] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the claimed subject matter pertains. It should be understood that the preceding overview and the following detailed description are for illustrative and explanatory purposes only and do not limit any of the claimed subject matter. In this application, the use of singular forms includes plural forms unless otherwise specified.

[0049] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context makes clear. Furthermore, the use of the term “including,” as well as other forms such as “include,” “includes,” and “included,” is not limited to these.

[0050] In any aspect described herein with the word “comprising,” it is understood that other similar aspects described with respect to “consisting of” and / or “essentially consisting of” are also provided.

[0051] The headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0052] When used herein, the term "and / or" should be understood as a specific disclosure of each of two identified features or components, with or without the other. Thus, when used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art relating to this disclosure. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *the Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide those skilled in the art with many common dictionaries of the terms used herein.

[0054] Units, prefixes, and symbols are shown in their recognized forms within the International System of Units (SI). Numerical ranges include both ends of the digit defining the range. The headings provided herein are not limitations on any particular aspect of this disclosure, and may be obtained by referring to this specification as a whole. Thus, terms defined immediately thereafter are more fully defined by referring to the entire specification.

[0055] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer).

[0056] The terms “approximately” or “essentially include” mean a value or composition within an acceptable margin of error of a particular value or composition as determined by those skilled in the art, and this depends in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, “approximately” or “essentially include” may mean a standard deviation of 1 or greater than 1, according to the practice of the art. Alternatively, “approximately” or “essentially include” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to 10 times or 5 times the value. Where a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of “approximately” or “essentially include” should be assumed to be within an acceptable margin of error of the particular value or composition.

[0057] The terms “programmed cell death protein 1” and “PD1” are used interchangeably herein and refer to an immunosuppressive receptor belonging to the CD28 family. PD1 is predominantly expressed in vivo on pre-activated T cells and binds to two ligands, PDL1 and PDL2. As used herein, the term PD1 includes human PD1 (hPD1), variants, isoforms, and homologous species of hPD1, as well as analogs having at least one common epitope with hPD1. The complete hPD-1 sequence can be found in GenBank accession number U64863.

[0058] The terms “epidermal growth factor receptor” and “EGFR” are used interchangeably herein and refer to a transmembrane protein that is a receptor for the epidermal growth factor family (EGF family) of extracellular protein ligands. The term EGFR, as used herein, includes human EGFR (hEGFR), variants, isoforms, and homologous species of hEGFR, as well as analogs having at least one common epitope with EGFR. The complete hEGFR sequence can be found in GenBank gene ID:1956.

[0059] The terms “subject” and “patient” are used interchangeably herein and include any human or non-human animal. The term “non-human animal” includes, but is not limited to, non-human primates, sheep, dogs, and vertebrates such as rodents, mice, rats, and guinea pigs. In some embodiments, the subject is human.

[0060] As used herein, the term “administer” means the physical delivery of a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or altered in the body of the subject to produce a therapeutic agent in vivo) to a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. The term “parenteral administration,” as used herein, means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Therapeutic agents may be administered via non-parenteral routes, i.e., orally. Other non-parenteral routes of administration include, for example, intranasal, vaginal, rectal, sublingual, or topical, epithelial, or mucosal routes of administration. Administration may also be, for example, a single dose, multiple doses, and / or over one or more extended periods.

[0061] The terms “cancer” and “tumor” are used interchangeably herein and refer to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells within the body. Uncontrolled cell division and proliferation can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0062] The “therapeutic effective dose” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of disease, promotes disease regression as demonstrated by a reduction in the severity of symptoms, an increase in the frequency and duration of symptom-free periods, or prevents disability or incapacity due to the distress of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by analyzing the activity of the drug in in vitro assays.

[0063] The term "weight-based dose," as used herein, refers to the dose administered to a patient, calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-PD-1 antibody, an appropriate amount of anti-PD-1 antibody (i.e., 180 mg) may be calculated and used for administration.

[0064] As used herein, the term “fixed dose” means that two or more different proteins (e.g., an anti-PD-1 antibody and a fusion protein) in a single composition are present in the composition in a specific (fixed) ratio to one another. In some embodiments, the fixed dose is based on the weight of the protein (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the protein (e.g., mg / ml).

[0065] The term "constant dose," as used herein, refers to the dose administered to a patient without regard to the patient's weight or body surface area (BSA). Therefore, a constant dose is an absolute amount of the drug (e.g., fusion protein and / or anti-PD-1 antibody), not as a mg / kg dose. For example, a 60kg person and a 100kg person would receive the same dose of antibody.

[0066] The term “antibody” is used herein in its broadest sense and includes fully assembled antibodies; functional antibody fragments and functional variants capable of binding to antigens (e.g., Fab, F(ab')2, Fv, single-chain variable fragments (scFv), single-domain antibodies (e.g., VHH), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.); as well as non-antibody fragments that bind to antigens (e.g., recombinant fibronectin domains), and recombinant polypeptides including those described above. Unless otherwise specified, references to the numbering of specific amino acid residue positions in antibodies follow the EU numbering system, as described in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) ("Kabat").

[0067] As used herein, “variable region” refers to a domain of the antibody heavy or light chain involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions and three complementarity-determining regions.

[0068] As used herein, the term “complementarity-determining region” refers to each region of the antibody variable domain that is highly variable in the sequence and forms a structurally defined loop (“highly variable loop”). Generally, natural quadruple-chain antibodies contain six CDRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). CDRs are described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) ("Kabat") and Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include duplication or subsets of amino acid residues when compared to one another. Nevertheless, any definition of an antibody's CDR is intended to be within the scope of the term as defined and used herein. Those skilled in the art can routinely determine which residues constitute a particular CDR when given the variable region amino acid sequence of an antibody. Unless otherwise specified, CDRs are defined according to the Kabat system.

[0069] The term “fusion protein” and its grammatical equivalent, as used herein, means a protein comprising amino acid sequences derived from at least two separate proteins. The amino acid sequences of at least two separate proteins may be directly linked through peptide bonds; or they may be operably linked through amino acid linkers. Thus, the term, fusion protein, encompasses embodiments in which, for example, the amino acid sequence of protein A is directly linked to the amino acid sequence of protein B through peptide bonds (protein A-protein B), and embodiments in which, for example, the amino acid sequence of protein A is operably linked to the amino acid sequence of protein B through amino acid linkers (protein A-linker-protein B).

[0070] The term “fuse” and its grammatical equivalents, as used herein, refer to the operable linking of an amino acid sequence from one protein to an amino acid sequence from another protein. The term “fuse” encompasses both the direct linking of two amino acid sequences through peptide bonds and the indirect linking through amino acid linkers.

[0071] As used herein, the term “modified” means, with respect to a nucleic acid sequence, a nucleic acid sequence comprising at least one substitution, addition, or deletion of nucleotides compared to a reference nucleic acid sequence. As used herein, the term “modified” means, with respect to an amino acid sequence, an amino acid sequence comprising at least one substitution, addition, or deletion of amino acid residues compared to a reference amino acid sequence. For the purpose of determining the percentage of identity between two amino acid sequences, naturally occurring amino acid derivatives are not considered modified amino acids. For example, for the purpose of determining the percentage of identity between two amino acid sequences, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue would not be considered an amino acid modification. Furthermore, for example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue would not be considered an amino acid “modified” as defined herein. Modifications may include the inclusion of non-naturally occurring amino acid residues.

[0072] The term “identical” or “percent identity” refers to at least two nucleic acids or at least two amino acid sequences or subsequences that, with respect to a nucleic acid sequence or amino acid sequence, are identical when compared and aligned for the greatest match, either by measurement using a sequence comparison algorithm or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and subsequence coordinates and sequence algorithm program parameters are designed as needed. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence compared to the reference sequence based on the designed program parameters. Examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschuel et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without tolerance for gaps. In calculating the percentage of identity, typically only exact matches are counted. As mentioned above, the percentage of identity is based on amino acid matching between the smaller of the two proteins.

[0073] Anti-PD1 antibody In certain embodiments, methods for treating cancer are provided herein, comprising administering to a subject having cancer an antibody, or a functional fragment or functional variant thereof, that specifically binds to programmed cell death protein 1 (PD1) in combination with a fusion protein described herein. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD1, programmed cell death ligand 1 (PDL1) and programmed cell death ligand 2 (PDL2), have been identified, which are expressed on antigen-presenting cells and on many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD1.

[0074] In some embodiments, the antibody is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v), a single-domain antibody, a single-chain antibody, or VHH.

[0075] Exemplary human monoclonal antibodies that bind specifically and with high affinity to PD1 are disclosed in U.S. Patent No. 8,008,449. Each of the anti-PD-1 human monoclonal antibodies disclosed in U.S. Patent No. 8,008,449 has been demonstrated to exhibit one or more of the following characteristics: (a) 1 × 10⁻¹⁶ as determined by surface plasmon resonance using a Biocore biosensor system -7(b) binds to human PD1 with a KD of M or less; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PDL1 and / or PDL2 to PD1; (h) stimulates antigen-specific memory responses; (i) stimulates antibody responses; and / or (j) inhibits tumor cell proliferation in vivo. Anti-PD1 antibodies available in this disclosure include monoclonal antibodies that specifically bind to human PD1 and exhibit at least one, at least two, at least three, at least four, or at least five of the above features. In other embodiments, the anti-PD-1 antibody is selected from the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, or 5F4 described in U.S. Patent No. 8,008,449. Other anti-PD1 monoclonal antibodies are described, for example, in U.S. Patents No. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, as well as in PCT Public International Publication No. 2012 / 145493.

[0076] In some embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; KEYTRUDA®, lambrolizumab, and MK-3475; see, for example, International Publication No. 2008 / 156712), PDR001 (Novartis; Spartalis Also known as mab; see, for example, International Publication No. 2015 / 112900), MEDI0680 (AstraZeneca; also known as AMP-514; see, for example, International Publication No. 2012 / 145493), semiplimab (Regeneron; also known as REGN-2810; see, for example, International Publication No. 2015 / 112800), JS001 (Taizhou Junshi Pharma (see, e.g., Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), BGB-A317 (Tislelizumab Beigene; see, e.g., International Publication No. 2015 / 035606 and U.S. Patent Application No. 20150079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see, e.g., International Publication No. 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), TSR-042 (Tesaro Biopharmaceutical (also known as AB011; see, for example, International Publication No. 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see, for example, Si-Yang Liu et al., J. Hematol. Oncol.)The group consists of (see 70: 136 (2017)), AM-0001 (Armo), STI-1110 (SorrentoTherapeutics; see, e.g., International Publication No. 2014 / 194302), AGEN2034 (Agenus; see, e.g., International Publication No. 2017 / 040790), MGA012 (Macrogenics; see, e.g., International Publication No. 2017 / 019846), IBI308 (Innovent; see, e.g., International Publication Nos. 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540), and BCD-100 (Biocad).

[0077] In some embodiments, the anti-PD1 antibody is pembrolizumab, nivolumab, semiprimab, spartalizumab, camrelizumab, tisrelizumab, dostallimab, cetrelimab, pidilizumab, MEDI0680, SSI-361, AMP-224, PDR001, PF-06801591, BGB-A317, TSR-042, A The group is selected from GEN-2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM-003, ENUM-388D4, IBI-308, JNJ-63723283, JS-001, JTX-4014, JY-034, CLA-134, STIA-1110, 244C8, and 388D4.

[0078] In some embodiments, the anti-PD1 antibody is pembrolizumab, nivolumab, spartalizumab, semiprimab, camrelizumab, tisrelizumab, dostallimab, cetrelimab, pidilizumab, MEDI0680, SSI-361, AMP-224, PDR001, PF-06801591, BGB-A317, TSR-042, These are functional fragments of AGEN-2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM-003, ENUM-388D4, IBI-308, JNJ-63723283, JS-001, JTX-4014, JY-034, CLA-134, STIA-1110, 244C8, or 388D4.

[0079] In some embodiments, the anti-PD1 antibody is pembrolizumab, nivolumab, spartalizumab, semiprimab, camrelizumab, tisrelizumab, dostallimab, cetrelimab, pidilizumab, MEDI0680, SSI-361, AMP-224, PDR001, PF-06801591, BGB-A317, TSR-042, A It is a functional variant of GEN-2034, A-0001, BGB-108, BI-754091, CBT-501, ENUM-003, ENUM-388D4, IBI-308, JNJ-63723283, JS-001, JTX-4014, JY-034, CLA-134, STIA-1110, 244C8, or 388D4.

[0080] pembrolizumab In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®", lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody against the human cell surface receptor PD1. Pembrolizumab is described, for example, in U.S. Patent No. 8,900,587. In some embodiments, the anti-PD1 antibody cross-competes with pembrolizumab. In some embodiments, the anti-PD1 antibody binds to the same epitope as pembrolizumab. In some embodiments, the anti-PD1 antibody has the same CDR as pembrolizumab.

[0081] In some embodiments, the anti-PD1 antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-PD1 antibody comprises VH containing VH CDR1, which contains the amino acid sequence of SEQ ID NO: 1 having 0, 1, 2, or 3 amino acid modifications; VH CDR2, which contains the amino acid sequence of SEQ ID NO: 2 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3, which contains the amino acid sequence of SEQ ID NO: 3 having 0, 1, 2, or 3 amino acid modifications.

[0082] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 having 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2 having 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 3 having 1, 2, or 3 amino acid modifications.

[0083] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 having 0, 1, 2, or 3 amino acid modifications.

[0084] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence of SEQ ID NO: 4 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 6 having one, two, or three amino acid modifications.

[0085] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 1 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 3 having 0, 1, 2, or 3 amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 having 0, 1, 2, or 3 amino acid modifications.

[0086] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 having 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 having 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 having 1, 2, or 3 amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 having 1, 2, or 3 amino acid modifications.

[0087] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1; VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2; and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

[0088] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4; VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5; and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0089] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. Includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 6.

[0090] In some embodiments, the anti-PD1 antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-PD1 antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-PD1 antibody comprises VH, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7; and VL, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0091] In some embodiments, the anti-PD1 antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-PD1 antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0092] In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0093] Nivolumab In some embodiments, the anti-PD1 antibody is nivolumab. Nivolumab (also known as "OPDIVO®"; formerly called 5C4, BMS-936558, MDX-1106, or ONO-4538) is a complete human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD1 ligands (PDL1 and PDL2), thereby blocking the downregulation of antitumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9): 846-56; referred to as 5C4 in International Publication No. 2006 / 121168). In some embodiments, the anti-PD1 antibody cross-competes with nivolumab. In some embodiments, the anti-PD-1 antibody binds to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody has the same CDR as nivolumab.

[0094] In some embodiments, the anti-PD1 antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-PD1 antibody comprises VH containing VH CDR1, which includes the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 amino acid modifications; VH CDR2, which includes the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3, which includes the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2, or 3 amino acid modifications.

[0095] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13 having one, two, or three amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 14 having one, two, or three amino acid modifications.

[0096] In some embodiments, the anti-PD1 antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-PD1 antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15 having 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 16 having 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 having 1, 2, or 3 amino acid modifications.

[0097] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2, or 3 amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 amino acid modifications.

[0098] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13 having one, two, or three amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 14 having one, two, or three amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 16 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 having one, two, or three amino acid modifications.

[0099] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0100] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16; and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0101] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. Includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 17.

[0102] In some embodiments, the anti-PD1 antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-PD1 antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-PD1 antibody includes VH, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18; and VL, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0103] In some embodiments, the anti-PD1 antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-PD1 antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0104] In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0105] semiprimab In some embodiments, the anti-PD1 antibody is cemiprimab, which is a monoclonal antibody against the PD1 receptor. In some embodiments, the anti-PD1 antibody cross-competes with cemiprimab. In some embodiments, the anti-PD-1 antibody binds to the same epitope as cemiprimab. In some embodiments, the anti-PD-1 antibody has the same CDR as cemiprimab.

[0106] In some embodiments, the anti-PD1 antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-PD1 antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 23 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 24 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 25 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-PD1 antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 23, or the amino acid sequence of SEQ ID NO: 23 having 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 having 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25 having 1, 2, or 3 amino acid modifications.

[0107] In some embodiments, the anti-PD1 antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-PD1 antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 26 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 27 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 28 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-PD1 antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 26, or the amino acid sequence of SEQ ID NO: 26 having 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 27, or the amino acid sequence of SEQ ID NO: 27 having 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence of SEQ ID NO: 28 having 1, 2, or 3 amino acid modifications.

[0108] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 23 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 24 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 25 having 0, 1, 2, or 3 amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 26 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 27 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 28 having 0, 1, 2, or 3 amino acid modifications.

[0109] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 23, or the amino acid sequence of SEQ ID NO: 23 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 having one, two, or three amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25 having one, two, or three amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 26, or the amino acid sequence of SEQ ID NO: 26 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 27, or the amino acid sequence of SEQ ID NO: 27 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence of SEQ ID NO: 28 having one, two, or three amino acid modifications.

[0110] In some embodiments, the anti-PD1 antibody comprises VH, which includes VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23; VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0111] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27; and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28.

[0112] In some embodiments, the anti-PD1 antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. Includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 28.

[0113] In some embodiments, the anti-PD1 antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-PD1 antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-PD1 antibody includes VH, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29; and VL, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0114] In some embodiments, the anti-PD1 antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-PD1 antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0115] In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-PD1 antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0116] In some embodiments, the anti-PD1 antibody includes the antibodies listed in Table 1.

[0117] In some embodiments, the anti-PD1 antibody comprises VH including VH CDR1, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 1; VH CDR2, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 1; and VH CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 1.

[0118] In some embodiments, the anti-PD1 antibody comprises VLs including VL CDR1 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 1; VL CDR2 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR2 in Table 1; and VL CDR3 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 1.

[0119] In some embodiments, the anti-PD1 antibody includes VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 1; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 1; VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 1; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 1; VL CDR1 in Table 1 VLs include VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2, and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 1.

[0120] In some embodiments, the anti-PD1 antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain amino acid sequence in Table 1. In some embodiments, the anti-PD1 antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain amino acid sequence in Table 1. In some embodiments, the anti-PD1 antibody comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the heavy chain in Table 1; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the light chain in Table 1.

[0121] In some embodiments, the anti-PD1 antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 1. In some embodiments, the anti-PD1 antibody comprises VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the antibody in Table 1. In some embodiments, the anti-PD1 antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 1; and VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the antibody in Table 1.

[0122] [Table 1-1]

[0123] [Table 1-2]

[0124] [Table 1-3]

[0125] [Table 1-4]

[0126] Method for producing anti-PD1 antibodies The anti-PD1 antibodies described herein can be prepared by any conventional technique known in the art, such as recombinant techniques or chemical synthesis (e.g., solid-phase peptide synthesis). In one embodiment, the anti-PD1 antibody is prepared through recombinant expression in cells. Briefly, the anti-PD1 antibody can be prepared by synthesizing DNA encoding the anti-PD1 antibody and cloning the DNA into any suitable expression vector. Numerous cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of choice. The gene may be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator, so that the DNA sequence encoding the fusion protein is transcribed to RNA in host cells transformed with a vector containing this expression construct. The coding sequence may or may not include a signal peptide or a leader sequence. A heterologous leader sequence may be added to the coding sequence that causes the secretion of an expressed polypeptide derived from the host organism. Other regulatory sequences that enable the regulation of the expression of protein sequences associated with host cell proliferation may also be desirable. Such regulatory sequences are known to those skilled in the art, and examples include those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Other types of regulatory elements, such as enhancer sequences, may also be present in the vector. The control sequence and other regulatory sequences may be ligated into the coding sequence before insertion into a vector such as the cloning vector described above. Alternatively, the coding sequence may be directly cloned into an expression vector that already contains the control sequence and appropriate restriction sites.

[0127] Subsequently, an expression vector can be used to transform suitable host cells. Numerous mammalian cell lines are known in the art and are not limited to, but include immortalized cell lines available from the United States Cell Culture Lineage Preservation Center (ATCC), such as Chinese hamster ovary (CHO) cells, CHO suspension cells (CHO-S), HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkey kidney culture cells (COS), VERO, HepG2, Maidin Darby bovine kidney (MDBK) cells, NOS, U2OS, A549, HT1080, CAD, P19, NIH3T3, L929, N2a, MCF-7, Y79, SO-Rb50, DUKX-X11, and J558L. In some embodiments, anti-PD1 antibodies are produced in CHO or CHO-S cells.

[0128] Depending on the selected expression system and host, anti-PD1 antibodies are produced by growing host cells transformed with the aforementioned expression vector under conditions in which anti-PD1 antibodies are expressed. The anti-PD1 antibodies are then isolated from the host cells and purified. If the expression system secretes anti-PD1 antibodies into the growth medium, they can be purified directly from the medium. If anti-PD-1 antibodies are not secreted, they are isolated from the cell lysates. The selection of appropriate growth conditions and recovery methods is within the scope of the art. Once purified, the amino acid sequence of the anti-PD1 antibody can be determined by repeated cycles of Edman degradation and subsequent amino acid analysis by HPLC. Other methods for amino acid sequencing are also known in the art. Once purified, the functionality of the anti-PD1 antibody can be evaluated by any conventional method known in the art, such as ELISA.

[0129] Fusion protein In a particular embodiment, a method for treating cancer is provided herein, comprising administering to a subject having cancer an antibody, or a functional fragment or functional variant thereof, that specifically binds to programmed cell death protein 1 (PD1), such as those described herein, in combination with a fusion protein comprising a targeting portion and an immunomodulatory portion, wherein (i) the targeting portion specifically binds to the epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain of transforming growth factor β receptor II (TGFβRII).

[0130] EGFR targeting moiety In some embodiments, the EGFR-targeting moiety comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the antibody is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v), a single-domain antibody, a single-chain antibody, or a VHH. In some embodiments, the EGFR-targeting moiety binds to EGFR and inhibits downstream signaling through the bound EGF receptor.

[0131] In some embodiments, the anti-EGFR antibody is selected from the group consisting of cetuximab and panitumumab. In some embodiments, the anti-EGFR antibody is a functional fragment of cetuximab and panitumumab. In some embodiments, the anti-EGFR antibody is a functional variant of cetuximab and panitumumab.

[0132] Cetuximab In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR antibody cross-competes with cetuximab. In some embodiments, the anti-EGFR antibody binds to the same epitope as cetuximab. In some embodiments, the anti-EGFR antibody has the same CDR as cetuximab.

[0133] In some embodiments, the anti-EGFR antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-EGFR antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 34 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 35 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-EGFR antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 34, or the amino acid sequence of SEQ ID NO: 34 having 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 35 having 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36, or the amino acid sequence of SEQ ID NO: 36 having 1, 2, or 3 amino acid modifications.

[0134] In some embodiments, the anti-EGFR antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-EGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 37 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 38 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 39 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-EGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 37, or the amino acid sequence of SEQ ID NO: 37 having 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 38, or the amino acid sequence of SEQ ID NO: 38 having 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 39, or the amino acid sequence of SEQ ID NO: 39 having 1, 2, or 3 amino acid modifications.

[0135] In some embodiments, the anti-EGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 34 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 35 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36 having 0, 1, 2, or 3 amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 37 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 38 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 39 having 0, 1, 2, or 3 amino acid modifications.

[0136] In some embodiments, the anti-EGFR antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 34, or the amino acid sequence of SEQ ID NO: 34 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 35 having one, two, or three amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36, or the amino acid sequence of SEQ ID NO: 36 having one, two, or three amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 37, or the amino acid sequence of SEQ ID NO: 37 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 38, or the amino acid sequence of SEQ ID NO: 38 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 39, or the amino acid sequence of SEQ ID NO: 39 having one, two, or three amino acid modifications.

[0137] In some embodiments, the anti-EGFR antibody comprises VH, which includes VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34; VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36.

[0138] In some embodiments, the anti-EGFR antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37; VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38; and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39.

[0139] In some embodiments, the anti-EGFR antibody comprises VH, including VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38. Includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 39.

[0140] In some embodiments, the anti-EGFR antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-EGFR antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-EGFR antibody comprises VH, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and VL, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[0141] In some embodiments, the anti-EGFR antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-EGFR antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0142] In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0143] Panitumumab In some embodiments, the anti-EGFR antibody is panitumumab. In some embodiments, the anti-EGFR antibody cross-competes with panitumumab. In some embodiments, the anti-EGFR antibody binds to the same epitope as panitumumab. In some embodiments, the anti-EGFR antibody has the same CDR as panitumumab.

[0144] In some embodiments, the anti-EGFR antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-EGFR antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 45 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 46 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 47 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-EGFR antibody comprises VH containing VH CDR1 containing the amino acid sequence of SEQ ID NO: 45, or the amino acid sequence of SEQ ID NO: 45 having 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 46, or the amino acid sequence of SEQ ID NO: 46 having 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 47, or the amino acid sequence of SEQ ID NO: 47 having 1, 2, or 3 amino acid modifications.

[0145] In some embodiments, the anti-EGFR antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-EGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 48 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 49 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50 having 0, 1, 2, or 3 amino acid modifications. In some embodiments, the anti-EGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 48, or the amino acid sequence of SEQ ID NO: 48 having 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence of SEQ ID NO: 49 having 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50, or the amino acid sequence of SEQ ID NO: 50 having 1, 2, or 3 amino acid modifications.

[0146] In some embodiments, the anti-EGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 45 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 46 having 0, 1, 2, or 3 amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 47 having 0, 1, 2, or 3 amino acid modifications; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 48 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 49 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50 having 0, 1, 2, or 3 amino acid modifications.

[0147] In some embodiments, the anti-EGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 45, or the amino acid sequence of SEQ ID NO: 45 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 46, or the amino acid sequence of SEQ ID NO: 46 having one, two, or three amino acid modifications; and VH CDR3 containing the amino acid sequence of SEQ ID NO: 47, or the amino acid sequence of SEQ ID NO: 47 having one, two, or three amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 48, or the amino acid sequence of SEQ ID NO: 48 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence of SEQ ID NO: 49 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 50, or the amino acid sequence of SEQ ID NO: 50 having one, two, or three amino acid modifications.

[0148] In some embodiments, the anti-EGFR antibody comprises VH, which includes VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47.

[0149] In some embodiments, the anti-EGFR antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49; and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50.

[0150] In some embodiments, the anti-EGFR antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49. Includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 50.

[0151] In some embodiments, the anti-EGFR antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-EGFR antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-EGFR antibody includes VH, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51; and VL, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52.

[0152] In some embodiments, the anti-EGFR antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-EGFR antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0153] In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-EGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0154] In some embodiments, the anti-EGFR antibody includes the antibodies listed in Table 2.

[0155] In some embodiments, the anti-EGFR antibody comprises VH including VH CDR1, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 2; VH CDR2, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 2; and VH CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 2.

[0156] In some embodiments, the anti-EGFR antibody comprises VLs including VL CDR1 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 2; VL CDR2 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR2 in Table 2; and VL CDR3 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 2.

[0157] In some embodiments, the anti-EGFR antibody includes VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 2; VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 2; VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 2; VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 2; VL CDR1 in Table 2 VLs include VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2, and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 2.

[0158] In some embodiments, the anti-EGFR antibody includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain amino acid sequence in Table 2. In some embodiments, the anti-EGFR antibody includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain amino acid sequence in Table 2. In some embodiments, the anti-EGFR antibody comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain amino acid sequence of Table 2; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain amino acid sequence of Table 2.

[0159] In some embodiments, the anti-EGFR antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 2. In some embodiments, the anti-EGFR antibody comprises VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the antibody in Table 2. In some embodiments, the anti-EGFR antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 2; and VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the antibody in Table 2.

[0160] [Table 2-1]

[0161] [Table 2-2]

[0162] [Table 2-3]

[0163] TGFβ trap In a particular embodiment, a method for treating cancer is provided herein, comprising administering to a subject having cancer an antibody, or a functional fragment or functional variant thereof, that specifically binds to programmed cell death protein 1 (PD1), in combination with a fusion protein comprising a targeting portion and an immunomodulatory portion, as described herein, wherein (i) the targeting portion specifically binds to the epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain (ECD) of transforming growth factor β receptor II (TGFβRII).

[0164] In some embodiments, TGFβRII ECD binds to at least one TGFβ isoform. In some embodiments, TGFβRII ECD binds to TGFβ1. In some embodiments, TGFβRII ECD binds to TGFβ3. In some embodiments, TGFβRII ECD does not bind to TGFβ2.

[0165] In some embodiments, the TGFβRII ECD contains a sequence of naturally occurring TGFβRII ECD sufficient to enable the binding of the protein to TGFβ. In some embodiments, the TGFβRII ECD contains a sequence of naturally occurring TGFβRII ECD sufficient to enable the binding of the protein to TGFβ1. In some embodiments, the TGFβRII ECD contains a sequence of naturally occurring TGFβRII ECD sufficient to enable the binding of the protein to TGFβ3.

[0166] In some embodiments, the extracellular domain of TGFβRII includes a shortened portion of SEQ ID NO: 56 that can bind to TGFβ. The extracellular domain of TGFβRII may be shortened at the N-terminus, C-terminus, or both the N and C-terminus. The shortening may involve the deletion of 1 to 10 amino acids. The shortening may involve the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The shortening may involve the deletion of 1, 2, 3, 4, or 5 amino acids from the N-terminus, C-terminus, or both the N and C-terminus.

[0167] In some embodiments, the extracellular domain of TGFβRII contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the extracellular domain of TGFβRII consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the extracellular domain of TGFβRII consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56.

[0168] [Table 3]

[0169] Orientation In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the targeting portion. In some embodiments, the immunomodulatory portion is operably connected to the N-terminus of the targeting portion.

[0170] In some embodiments, the targeting portion is an antibody (or a functional fragment or functional variant thereof) comprising 1) VH or a heavy chain, and 2) VL or a light chain. In some embodiments, the immunomodulatory portion is operably linked to the C-terminus of the VH or heavy chain. In some embodiments, the immunomodulatory portion is operably linked to the C-terminus of the VL or light chain. In some embodiments, the immunomodulatory portion is operably linked to the C-terminus of the constant region of the heavy chain. In some embodiments, the immunomodulatory portion is operably linked to the C-terminus of the constant region of the light chain. In some embodiments, the immunomodulatory portion is operably linked to the N-terminus of the VH or heavy chain. In some embodiments, the immunomodulatory portion is operably linked to the N-terminus of the VL or light chain.

[0171] Linker In some embodiments, the targeting and immunomodulatory portions of the fusion protein are directly operably linked. In some embodiments, the targeting and immunomodulatory portions of the fusion protein are indirectly operably linked. In some embodiments, the targeting and immunomodulatory portions of the fusion protein are indirectly operably linked via a linker. In some embodiments, the linker is a peptide linker.

[0172] Any suitable peptide linker known in the art may be used to enable the binding of the immunomodulatory and targeting portions to their respective antigens. Exemplary peptide linkers, including glycine and serine amino acids, are provided in Table 4.

[0173] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 57–61. In some embodiments, the linker includes any one of the amino acid sequences of SEQ ID NOs. 57–61, or any one of the amino acid sequences of SEQ ID NOs. 57–61 having one, two, or three amino acid modifications.

[0174] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 57, or the amino acid sequence of SEQ ID NO: 57 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 57, or the amino acid sequence of SEQ ID NO: 57 having one, two, or three amino acid modifications.

[0175] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 58, or the amino acid sequence of SEQ ID NO: 28 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 58, or the amino acid sequence of SEQ ID NO: 58 having one, two, or three amino acid modifications.

[0176] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 59, or the amino acid sequence of SEQ ID NO: 59 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 59, or the amino acid sequence of SEQ ID NO: 59 having one, two, or three amino acid modifications.

[0177] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 60, or the amino acid sequence of SEQ ID NO: 60 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 60, or the amino acid sequence of SEQ ID NO: 60 having one, two, or three amino acid modifications.

[0178] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 61, or the amino acid sequence of SEQ ID NO: 61 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 61, or the amino acid sequence of SEQ ID NO: 61 having one, two, or three amino acid modifications.

[0179] [Table 4]

[0180] Exemplary fusion protein Exemplary fusion proteins of this disclosure are provided in Table 5. In one embodiment, the fusion protein comprises BCA101. BCA101 is a bifunctional fusion protein comprising an anti-EGFR antibody and the extracellular domain of TGFβRII fused to the C-terminus of the anti-EGFR antibody light chain.

[0181] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0182] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence identical to that of SEQ ID NO: 43. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence identical to that of SEQ ID NO: 62. In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence identical to that of SEQ ID NO: 43; and a light chain containing an amino acid sequence identical to that of SEQ ID NO: 62.

[0183] In some embodiments, the fusion protein includes a heavy chain, the amino acid sequence of which includes the amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein includes a light chain, the amino acid sequence of which includes the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein includes a heavy chain, the amino acid sequence of which includes the amino acid sequence of SEQ ID NO: 43; and a light chain, the amino acid sequence of which includes the amino acid sequence of SEQ ID NO: 62.

[0184] In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 43 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a light chain containing the amino acid sequence of SEQ ID NO: 62 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 43 having one, two, or three amino acid modifications; and a light chain containing the amino acid sequence of SEQ ID NO: 62 having one, two, or three amino acid modifications.

[0185] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0186] In some embodiments, the fusion protein includes a heavy chain having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein includes a light chain having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein includes a heavy chain having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 43; and a light chain having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0187] In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 43 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 62 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 43 having one, two, or three amino acid modifications; and a light chain consisting of the amino acid sequence of SEQ ID NO: 62 having one, two, or three amino acid modifications.

[0188] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0189] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0190] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0191] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0192] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0193] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0194] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65.

[0195] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53; and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65.

[0196] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0197] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0198] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65.

[0199] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65.

[0200] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0201] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67; and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55.

[0202] [Table 5-1]

[0203] [Table 5-2]

[0204] [Table 5-3]

[0205] [Table 5-4]

[0206] [Table 5-5]

[0207] [Table 5-6]

[0208] Method for creating fusion proteins The fusion proteins described herein can be prepared by any conventional technique known in the art, such as recombinant techniques or chemical synthesis (e.g., solid-phase peptide synthesis). In one embodiment, the fusion protein is prepared through recombinant expression in cells. Briefly, the fusion protein can be prepared by synthesizing the DNA encoding the fusion protein and cloning the DNA into any suitable expression vector. Numerous cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of choice. The gene may be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator, so that the DNA sequence encoding the fusion protein is transcribed into RNA in a host cell transformed with a vector containing this expression construct. The coding sequence may or may not include a signal peptide or a leader sequence. A heterologous leader sequence may be added to the coding sequence that causes the secretion of an expressed polypeptide derived from the host organism. Other regulatory sequences that enable the regulation of the expression of protein sequences associated with host cell proliferation may also be desirable. Such regulatory sequences are known to those skilled in the art, and examples include those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Other types of regulatory elements, such as enhancer sequences, may also be present in the vector. The control sequence and other regulatory sequences may be ligated into the coding sequence before insertion into a vector such as the cloning vector described above. Alternatively, the coding sequence may be directly cloned into an expression vector that already contains the control sequence and appropriate restriction sites.

[0209] The expression vector can then be used to transform suitable host cells. Numerous mammalian cell lines are known in the art and are not limited to those mentioned, including immortalized cell lines available from the United States Cell Culture Lineage Preservation Center (ATCC), such as Chinese hamster ovary (CHO) cells, CHO suspension cells (CHO-S), HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkey kidney culture cells (COS), VERO, HepG2, Maidin Darby bovine kidney (MDBK) cells, NOS, U2OS, A549, HT1080, CAD, P19, NIH3T3, L929, N2a, MCF-7, Y79, SO-Rb50, DUKX-X11, and J558L. In some embodiments, the fusion protein is produced in CHO or CHO-S cells.

[0210] Depending on the selected expression system and host, the fusion protein is produced by growing host cells transformed with the expression vector described above under conditions in which the fusion protein is expressed. The fusion protein is then isolated from the host cells and purified. If the expression system secretes the fusion protein into the growth medium, the fusion protein can be purified directly from the medium. If the fusion protein is not secreted, it is isolated from the cell lysate. The selection of appropriate growth conditions and recovery methods is within the scope of the art. Once purified, the amino acid sequence of the fusion protein can be determined, i.e., by repeated cycles of Edman degradation and subsequent amino acid analysis by HPLC. Other methods for amino acid sequencing are also known in the art. Once purified, the functionality of the fusion protein can be evaluated, for example, using a bifunctional ELISA, as described herein.

[0211] As described above, the functionality of the fusion protein can be tested by any method known in the art, such as ELISA. Each functionality can be measured in separate assays; for example, TGFβ binding and EGFR binding can be measured in two separate ELISAs. For example, one ELISA plate can be coated with an EGFR Fc chimeric protein and used to evaluate EGFR binding; another ELISA plate can be coated with TGFβ-1 and used to evaluate TGFβ-1 binding. Both functionalities can also be evaluated in a bifunctional ELISA. For example, BCA101 can be captured using an anti-idiotype mAb against cetuximab (for use with the BCA101 fusion protein), and the bound BCA101 can be detected by an enzyme-labeled polyclonal antibody against TGFβRIIECD. The concentration of BCA101 in the sample can be calculated from the BCA101 calibration curve. Target binding can also be evaluated via Biocore, where the EGFR and TGFβ1 targets are immobilized on an activated CM5 chip and then incubated with successive concentrations of the fusion protein. For example, the fusion protein may be evaluated by further in vitro functional assays, including cell surface binding by flow cytometry, inhibition of cell proliferation, ADCC assay, neutralization of TGFβ1-induced IL-11 release, and neutralization of TGFβ1-induced SMAD signaling.

[0212] How to use In one embodiment, a method for treating cancer in a subject having cancer is provided herein, which involves administering a fusion protein comprising a drug that specifically binds to PD1 (for example, a drug described herein) and a targeting portion and an immunomodulatory portion, wherein (i) the targeting portion specifically binds to the epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain of transforming growth factor β receptor II (TGFβRII).

[0213] In some embodiments, the methods disclosed herein are used as an alternative to standard treatment. In certain embodiments, standard treatment is used in combination with any of the methods disclosed herein. Standard treatments for different types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a coalition of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®), which provide detailed and up-to-date information on standard treatments for a variety of cancers. In some embodiments, the methods disclosed herein are used after standard treatment has failed.

[0214] In some embodiments, the fusion protein is administered co-administered with an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, or administered before or after the antibody that specifically binds to PD1, or a functional fragment or functional variant thereof.

[0215] In some embodiments, the fusion protein is administered co-administered with an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof. In some embodiments, the fusion protein is administered co-administered with an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof. In some embodiments, the fusion protein is administered within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours of administration of the antibody that specifically binds to PD1, or a functional fragment or functional variant thereof.

[0216] In some embodiments, the fusion protein is administered before administration of an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof. In some embodiments, the fusion protein is administered at least 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or 14 days before administration of an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof.

[0217] In some embodiments, the fusion protein is administered after administration of an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof. In some embodiments, the fusion protein is administered at least 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or 14 days after administration of an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof.

[0218] Exemplary cancer In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is both metastatic and recurrent. In some embodiments, the cancer is refractory to approved standard treatments. In some embodiments, the cancer is refractory to at least one approved standard treatment. In some embodiments, the cancer is refractory to at least all approved standard treatment agents.

[0219] In some embodiments, the cancer is EGFR-activated. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is selected from the group consisting of breast cancer, anal cancer, pancreatic cancer, thyroid cancer, liver cancer, ovarian cancer, lung cancer, skin cancer, brain cancer, spinal cord cancer, head cancer, neck cancer, and head and neck cancer.

[0220] In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is recurrent HNSCC. In some embodiments, the cancer is metastatic HNSCC. In some embodiments, the cancer is metastatic recurrent HNSCC. In some embodiments, the cancer is anal canal cancer. In some embodiments, the cancer is anal canal squamous cell carcinoma (SCCAC). In some embodiments, the cancer is recurrent SCCAC. In some embodiments, the cancer is metastatic SCCAC. In some embodiments, the cancer is metastatic recurrent SCCAC.

[0221] Dosage plan and schedule Anti-PD1 antibody In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer at a therapeutically effective dose. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer at a fixed dose. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer at a constant dose. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer at a weight-based dose.

[0222] In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer in doses of approximately 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 200 mg. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer in doses of approximately 200 mg. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer in doses of approximately 300 mg.

[0223] In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer approximately every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer approximately every week. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer approximately every 2 weeks. In some embodiments, an antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, is administered to a subject with cancer approximately every 3 weeks.

[0224] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is pembrolizumab. In some embodiments, pembrolizumab is administered to subjects in doses of approximately 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 200 mg. In some embodiments, pembrolizumab is administered in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg. In some embodiments, pembrolizumab is administered in a dose of approximately 200 mg. In some embodiments, pembrolizumab is administered in a dose of approximately 300 mg. In some embodiments, pembrolizumab is administered in a dose of approximately 400 mg.

[0225] In some embodiments, pembrolizumab is administered to subjects approximately every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, pembrolizumab is administered to subjects approximately every 2 weeks. In some embodiments, pembrolizumab is administered to subjects approximately every 3 weeks. In some embodiments, pembrolizumab is administered to subjects approximately every 4 weeks. In some embodiments, pembrolizumab is administered to subjects approximately every 5 weeks. In some embodiments, pembrolizumab is administered to subjects approximately every 6 weeks.

[0226] In some embodiments, pembrolizumab is administered to subjects at a dose of approximately 200 mg every three weeks. In some embodiments, pembrolizumab is administered to subjects at a dose of approximately 400 mg every six weeks.

[0227] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to PD1 is nivolumab. In some embodiments, nivolumab is administered to subjects in doses of approximately 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 200 mg. In some embodiments, nivolumab is administered in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg. In some embodiments, nivolumab is administered in a dose of approximately 200 mg. In some embodiments, nivolumab is administered in a dose of approximately 240 mg. In some embodiments, nivolumab is administered in a dose of approximately 300 mg. In some embodiments, nivolumab is administered in a dose of approximately 360 mg. In some embodiments, nivolumab is administered in a dose of approximately 400 mg. In some embodiments, nivolumab is administered in a dose of approximately 480 mg. In some embodiments, nivolumab is administered in a dose of approximately 500 mg.

[0228] In some embodiments, nivolumab is administered to subjects in doses of approximately 1 mg / kg to 5 mg / kg, 1 mg / kg to 4 mg / kg, 1 mg / kg to 3 mg / kg, or 1 mg / kg to 2 mg / kg. In some embodiments, nivolumab is administered in doses of approximately 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg. In some embodiments, nivolumab is administered in a dose of approximately 1 mg / kg. In some embodiments, nivolumab is administered in a dose of approximately 2 mg / kg. In some embodiments, nivolumab is administered in a dose of approximately 3 mg / kg.

[0229] In some embodiments, nivolumab is administered to subjects approximately every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, nivolumab is administered to subjects approximately every 2 weeks. In some embodiments, nivolumab is administered to subjects approximately every 3 weeks. In some embodiments, nivolumab is administered to subjects approximately every 4 weeks. In some embodiments, nivolumab is administered to subjects approximately every 5 weeks. In some embodiments, nivolumab is administered to subjects approximately every 6 weeks.

[0230] In some embodiments, nivolumab is administered to subjects at a dose of approximately 240 mg every two weeks. In some embodiments, nivolumab is administered to subjects at a dose of approximately 480 mg every four weeks. In some embodiments, nivolumab is administered to subjects at a dose of approximately 360 mg every three weeks. In some embodiments, nivolumab is administered to subjects at a dose of approximately 1 mg / kg every three weeks. In some embodiments, nivolumab is administered to subjects at a dose of approximately 3 mg / kg every two weeks.

[0231] Fusion protein In some embodiments, the fusion protein (i.e., a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to the epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of transforming growth factor β receptor II (TGFβRII)) is administered to a subject with cancer at a therapeutically effective dose. In some embodiments, the fusion protein is administered to a subject with cancer at a fixed dose. In some embodiments, the fusion protein is administered to a subject with cancer at a constant dose. In some embodiments, the fusion protein is administered to a subject with cancer at a weight-based dose.

[0232] In some embodiments, the fusion protein is approximately 50mg-2000mg, 100mg-2000mg, 150mg-2000mg, 200mg-2000mg, 300mg-2000mg, 400mg-2000mg, 500mg-2000mg, 600mg-2000mg, 700mg-2000mg, 800mg-2000mg, 900mg-2000mg The fusion protein is administered to the subject in doses of mg, 1000mg-2000mg, 1500mg-2000mg, 50mg-100mg, 50mg-500mg, 50mg-400mg, 50mg-300mg, 50mg-200mg, 50mg-100mg, 100mg-500mg, 100mg-400mg, 100mg-300mg, or 100mg-200mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 200mg-2000mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 50 mg, 60 mg, 64 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 64 mg, 240 mg, 800 mg, or 1600 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 64 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 240 mg. In some embodiments, the fusion protein is administered to the subject at a dose of approximately 800 mg. In other embodiments, the fusion protein is administered to the subject at a dose of approximately 1600 mg.

[0233] In some embodiments, the fusion protein is administered to the subject every 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, the fusion protein is administered to the subject weekly. In some embodiments, the fusion protein is administered to the subject every 2 weeks. In some embodiments, the fusion protein is administered to the subject every 3 weeks. In some embodiments, the fusion protein is administered to the subject every 4 weeks. In some embodiments, the fusion protein is administered to the subject every 5 weeks. In some embodiments, the fusion protein is administered to the subject every 6 weeks.

[0234] kit In one embodiment, a kit comprising an anti-PD-1 antibody and a fusion protein described herein for therapeutic use is provided herein. The kit typically includes labels and instructions for use indicating the intended use of the contents of the kit. Terms, labels, and any documents or recorded materials provided on, with, or accompanying the kit are included. Accordingly, this disclosure provides a kit for treating subjects suffering from cancer, and the kit includes: (a) a dose of anti-PD-1 antibody; and (b) a dose of a fusion protein described herein; and (c) instructions for use of the anti-PD-1 antibody and fusion protein in any of the combination therapy methods disclosed herein. In certain embodiments, the anti-PD-1 antibody and fusion protein may be co-packaged in unit dosage forms. In certain embodiments for treating human patients, the kit comprises an anti-human PD-1 antibody disclosed herein, such as pembrolizumab.

[0235] The present invention will be further illustrated by the following embodiments, which should not be construed as further limitations. The contents of all references cited throughout this specification are expressly incorporated herein by reference. [Examples]

[0236] [Example 1] Evaluation of the anticancer activity of BCA101 in combination with an anti-PD1 antibody in huNOG-EXL mice carrying subcutaneous PC-3 tumor xenografts. The purpose of this study was to evaluate the anticancer activity of BCA101 alone and in combination with the anti-PD1 antibody pembrolizumab in vivo.

[0237] material and method mouse The study utilized immunodeficient hGM-CSF / hIL3 gene-transformed NOG mice that exhibit a human-like immune system (human-derived lymphoid and myeloid lineages) upon transplantation of human hematopoietic stem cells. This model enabled the study of key innate mechanisms involved in the efficacy of immunotherapies and provided a suitable model for establishing human xenografts.

[0238] Male hGM-CSF / hIL3 NOG mice were obtained from Taconic Biosciences. hGM-CSF / hIL3 NOG mice transplanted with human CD34+ hematopoietic stem cells (HSCs) stably developed extensive cell lineages early, 6–8 weeks after injection. Both bone marrow and lymphoid cell lineages were present in peripheral blood, bone marrow, thymus and spleen, as well as in non-lymphatic tissues including the lungs and liver. huNOG-EXL mice with more than +25% hCD45 in peripheral blood were used. At the start of each study, the mice were 13–14 weeks old, and the average animal weight per group was approximately 26 g.

[0239] Preparation and subcutaneous injection of tumor cells All procedures were performed in a laminar flow hood according to sterile techniques. PC-3 (human prostate adenocarcinoma) cells with a viability >90% were used. Approximately 5 × 10 6 The cells were resuspended in 200 μl of serum-free medium containing 50% Matrigel, which was kept on ice.

[0240] The PC-3 cell line was grown in male huNOG-EXL mice by subcutaneous injection of cells into the right flank region of the mouse. Tumor growth in the transplanted area was monitored. When the tumor reached a palpable stage and a desired volume, the mice were randomized based on tumor volume (average tumor volume approximately 119 mm²).3 ) Administration was initiated. The research schedule is shown in Table 6.

[0241]

Table 6

[0242] Formulation of antibody dosages The necessary amounts of human IgG1 isotype control, cetuximab, BCA101, and pembrolizumab were diluted in the dilution buffer to obtain the appropriate working concentrations to deliver the intended dosages. A dosage volume of 10 mL / kg was maintained for all animals intraperitoneally. On the other hand, in the combination treatment on day 0 (Group V), for both BCA101 and pembrolizumab administrations, a dosage volume of 5 mL / kg was used to maintain a total dosage volume of 10 mL / kg per mouse.

[0243] observation Body weight and clinical signs During the experimental period, the respective body weights were measured every two days. The percentage change in body weight of each mouse was calculated and recorded. During the experimental period, the mice were observed for visible clinical signs every two days and recorded.

[0244] Tumor volume During the experimental period, on the day of randomization (day 0) and then every two days, the tumor volume was determined by two-dimensional measurement of the tumor length (L) and width (W) using a digital caliper. The following formula was used to calculate the tumor volume (TV): Tumor volume (mm 3 ) = (L×W 2 ) / 2, where L = length (mm) and W = width (mm). For each group, the mean, standard deviation (SD) or standard error (SEM) was calculated.

[0245] Antitumor activity The anti-tumor activity was evaluated as the maximum tumor growth inhibition against the isotype control group. Data evaluation was performed using standard calculations in Microsoft Excel.

[0246] Test / control values ​​in percentage (%T / C) The tumor inhibition (T / C in %) on a specific day was calculated using the following formula: %T / CX day = ((mean TV of the test group on day x - mean TV of the test group on day 0) / (mean TV of the control group on day x - mean TV of the control group on day 0)) × 100%; where TV = mm 3 Tumor volume at that stage. The minimum (or optimal) %T / C value recorded for a particular test group during the experimental period represents the maximum antitumor activity for each treatment.

[0247] Tumor growth inhibition (TGI) The TGI was calculated using the following formula: TGI = (1 - T / C) × 100%; where T = (mean TV of the test group on day X - mean TV of the test group on day 0) and C = (mean TV of the control group on day X - mean TV of the control group on day 0).

[0248] statistical analysis To evaluate the statistical significance of tumor inhibition, a two-way ANOVA and subsequent Bonferroni post-hoc test were performed using Graph Pad Prism V8.3.0. A p-value < 0.05 indicates a statistically significant difference between the groups.

[0249] Autopsy Based on ethical reasons and tumor endpoints, tumor necrosis / ulceration and tumor volume (tumor volume > 1500 mm) 3 Animals exhibiting the condition were humanely euthanized on the 21st day, and their corpses were dissected.

[0250] Sample collection On the 21st day, blood was collected from the remaining animals in all treatment groups. After collection, the blood was allowed to clot at room temperature for 20 minutes. Further, serum was separated by centrifugation at 2,000 x g for 10 minutes and stored at -80 °C for further use. All animals were humanely euthanized, and tumors (if available) were harvested and divided into two parts. One part of the tumor was snap-frozen, and the second part was fixed in 10% neutral buffered formalin (NBF).

[0251] result For the anti-tumor activity against PC-3 tumor xenografts, cetuximab, BCA101, and pembrolizumab were evaluated either as monotherapy or in combination therapy (BCA101 + pembrolizumab). Cetuximab, BCA101, and pembrolizumab were administered intraperitoneally at doses of 8.1, 10, and 10 mg / kg, respectively. The combination therapy of BCA101 + pembrolizumab was tested at the same doses and regimen. All animals from all treatment groups were alive on the 18th day. Therefore, the 18th day was selected for efficacy evaluation. Treatment with cetuximab, BCA101, pembrolizumab, and BCA101 + pembrolizumab demonstrated %T / C of 65%, 60%, 48%, and 22%, respectively, compared to the huIgG1 (isotype control) treatment group (Table 7).

[0252]

Table 7

[0253] The isotype control group had an average tumor volume of 1216 ± 89 mm 3 on the 18th day and showed progressive tumor growth throughout the experiment (Table 8). The average tumor volumes of the cetuximab, BCA101, pembrolizumab, and BCA101 + pembrolizumab treatment groups were 834 ± 175, 776 ± 127, 647 ± 145, and 362 ± 120 mm 3This was recorded (Table 8). The tumor growth profiles and their respective tumor growth curves during this period are shown in Figures 1 and 2A-2E, respectively. Photographs of animals with PC-3 tumors were taken on day 19 (Figures 3A and 3B).

[0254] [Table 8]

[0255] The %TGI values ​​for the cetuximab, BCA101, pembrolizumab, and BCA101+pembrolizumab groups were calculated as 35%, 40%, 52%, and 78% (day 18), respectively, compared to the isotype control group (Table 9). The treatment groups cetuximab, BCA101, pembrolizumab, and BCA101+pembrolizumab showed a significant reduction in tumor growth compared to the isotype control group (p<0.001; day 18). Partial tumor growth regression was observed in the BCA101 (1 / 10 mice), pembrolizumab (2 / 10 mice), and BCA101+pembrolizumab (4 / 10 mice) treatment groups. Tumor endpoint (tumor volume > 1500 mm²) 3 Based on ethical reasons (tumor necrosis / ulceration), the study was terminated on day 21, and all animals were humanely euthanized.

[0256] [Table 9]

[0257] Mortality rate and weight change During the experimental period, animal mortality was observed from isotype controls (Day 19 - D147 - A35 and Day 21 - D147 - A2), BCA101 (Day 19 - D147 - A38), combination treatment BCA101 + pembrolizumab (Day 20 - D148 - A9 and Day 21 - D149 - A65). No visible clinical signs of abnormal behavior were observed in any of the treatment groups. Furthermore, a progressive decrease in animal body weight was observed in all treatment groups. In all treated animals, severe weight loss (greater than -10%) was observed at the end of the 21-day study. The percentage change in animal body weight for all groups is summarized in Table 10 and Figure 4.

[0258]

Table 10

[0259] conclusion In this PC-3 tumor xenograft model, treatment with cetuximab, BCA101, pembrolizumab, and BCA101 + pembrolizumab at the given doses and regimens showed a significant decrease in tumor growth compared to the isotype control group. Among the doses and regimens tested, the combination treatment (BCA101 + pembrolizumab) showed more partial tumor regressions (4 / 10 mice) and the greatest tumor growth inhibition compared to single-agent treatment with pembrolizumab (partial regression - 2 / 10 mice) and BCA101 (partial regression - 1 / 10 mice) followed by cetuximab (no partial tumor regression). Severe weight loss could be largely attributed to the tumor burden in the isotype control and the tested doses in the treatment groups. In conclusion, BCA101 is a candidate for cancer treatment and may show better efficacy when combined with immune checkpoint inhibitors (e.g., anti-PD1 antibodies) against various xenograft models.

[0260] [Example 2] A Phase 1a / 1b dose-escalation and expanded cohort study evaluating the safety and tolerability of BCA101 monotherapy and its combination with the anti-PD1 antibody (pembrolizumab) in patients with EGFR-induced advanced solid tumors. In general, this study aims to evaluate the safety, tolerance, pharmacokinetics, pharmacodynamics, and efficacy of BCA101 monotherapy and in combination with pembrolizumab in patients with EGFR-driven advanced solid tumors. This is a phase 1 / 1b open-label study consisting of a dose-escalation part (Part A) and a subsequent expanded cohort (Part B) for both BCA101 monotherapy and the BCA101 + pembrolizumab combination.

[0261] the purpose The primary objectives of the study's dose escalation (Part A) are: 1) to evaluate the safety and tolerability of BCA101 monotherapy in patients with selected EGFR-driven advanced solid tumors refractory to standard treatment or in patients for whom no standard treatment is available; 2) to evaluate the safety and tolerability of BCA101 in combination with pembrolizumab in patients with either head and neck squamous cell carcinoma (HNSCC) or anal canal squamous cell carcinoma (SCCAC) whose tumors are refractory to standard treatment or for whom no standard treatment is available; and 3) to identify dose-limiting toxicities (DLTs) during the first cycle of treatment with BCA101 monotherapy or the combination of BCA101 and pembrolizumab. Patients will be enrolled in a sequential "3+3" design.

[0262] Once the maximum tolerated dose (MTD) / recommended dose (RD) is determined, dose escalation (Part B) begins. The primary objectives are to further evaluate 1) the safety and tolerability of BCA101 monotherapy in patients with selected cancers, and 2) the safety and tolerability of BCA101 in combination with pembrolizumab in patients with HNSCC and SCCAC. The patient cohort for the evaluation of BCA101 monotherapy includes 1) PD-L1-negative, EGFR-amplified squamous cell lung cancer (SqCLC); 2) RAS wild-type microsatellite-stable colorectal cancer (RAS wt, MSS CRC); 3) EGFR-amplified triple-negative breast cancer; and 4) any solid tumor with either KRAS G12D or G13D.

[0263] BCA101 will be administered intravenously every 7 days in the MTD or RD, based on the results of Part A of the study. Pembrolizumab will be administered according to the approved product label for use in specific instructions. Decisions to advance each cohort will aim to ensure that the ratio of risk to benefit in the MTD or RD justifies enrollment of the patient in the expanded cohort, based on a review of cumulative safety, PK, clinical data, and any PD data available. Patients will continue weekly infusions until disease progression, unacceptable toxicity, withdrawal of consent by the patient, or until the investigators determine that discontinuing treatment with the study drug is most beneficial to the patient.

[0264] The second objectives of this study are: 1) to determine the response rate in each part of the study using RECIST v1.1 and iRECIST; 2) to determine the clinical benefit rate in each part of the study using RECIST v1.1 and iRECIST; 3) to determine progression-free survival (PFS) in each part of the study using RECIST v1.1 and iRECIST; 4) RECIST The objectives of the study are: 5) determining the duration of response in each part of the study using v1.1 and iRECIST; 6) determining survival rates in each part of the study; 7) AUC of BCA101 and pembrolizumab; 8) Cmax of BCA101 and pembrolizumab; 9) Tmax of BCA101 and pembrolizumab; 10) concentration-versus-time profiles of BCA101 and pembrolizumab; 11) half-lives of BCA101 and pembrolizumab; and 12) immunogenicity of BCA101 and pembrolizumab through the incidence and titer of anti-drug antibodies.

[0265] The experimental objective of this study is to investigate the pharmacodynamic and biomarkers of BCA101. Experimental serum endpoints include levels of TGFβ1, TGFβ2, TGFβ3, soluble EGFR, VEGF, and other relevant cytokines, interleukins, and chemokines. Experimental hematological endpoints include immunophenotyping by flow cytometry for multi-parameter immunoprofiling of peripheral blood. Experimental tumor tissue endpoints include analysis of pre- and post-treatment biopsy specimens from preserved records for whole-exome sequencing and next-generation sequencing of DNA; and immunohistochemistry for relevant EGFR and TGFβ signaling pathway markers, tumor-infiltrating immune cells, and other relevant markers.

[0266] Research plan Overall design and planning of the research This is a phase 1 / 1b open-label study consisting of a dose-escalation part (Part A) and a subsequent expanded cohort (Part B) for both BCA101 monotherapy and the BCA101 plus pembrolizumab combination.

[0267] Dosage escalation (Part A) BCA101 monotherapy is administered to patients via intravenous infusion at weekly doses of 64 mg, 240 mg, 800 mg, or 1600 mg. Patients with the following tumor types are eligible: 1) Squamous cell carcinoma of the lung (SqCLC) 2) Squamous cell carcinoma of the head and neck (HNSCC) 3) RAS wild-type microsatellite stable colorectal cancer (RAT WT MSS CRC) 4) Triple-negative breast cancer (TNBC) 5) Chordoma 6) Squamous cell carcinoma of the anal canal (SCCAC) 7) Uveal melanoma 8) Glioblastoma (GBM) 9) Gastric cancer 10) Any solid tumor with KRAS G12D or G13D mutation 11) Any solid tumor with EGFR gene amplification 12) Epithelial ovarian cancer 13) Hepatocellular carcinoma (HCC) 14) Anaplastic carcinoma of the thyroid (ATC) 15) Pancreatic cancer 16) Other EGFR-driven advanced solid tumors (If there is convincing data or evidence to enroll patients with tumor types not listed in 1-15, the treating physician may discuss eligibility with the guarantor and the patient).

[0268] The combination of BCA101 and pembrolizumab is administered to patients via intravenous infusion every three weeks. Patients with the following tumor types are eligible: HNSCC and SCCAC.

[0269] Cohort expansion (Part B) Patients must have histologically or cytologically confirmed EGFR-driven advanced solid tumors that are refractory to current standard treatments. Patients with the following tumor types are eligible for monotherapy with BCA101: PD-L1 negative and EGFR gene-amplifying SqCLC, RAS WT, MSS, CRC, EGFR gene-amplifying TNBC, and any solid tumor with KRAS G12D or G13D mutations.

[0270] Patients with the following tumor types are eligible for treatment with combination therapy of BCA101 and pembrolizumab: 1) HNSCC 2) SCCAC.

[0271] Exclusion criteria Exposure to anti-EGFR antibodies within 4 weeks of initial administration of the study drug or any prior treatment with anti-TGFβ therapy.

[0272] A prior medical history of grade ≥2 intolerance or hypersensitivity to cetuximab, other anti-EGFR therapies, or other mouse proteins, or prior discontinuation of therapy in a toxic environment related to the treatment.

[0273] For Part B only: A prior medical history of Grade ≥2 intolerance or hypersensitivity to immune checkpoint inhibitors, or a history of discontinuation of any treatment in a toxic environment to immune checkpoint inhibitors.

[0274] Pregnant or breastfeeding women.

[0275] Any condition requiring systemic treatment with corticosteroids (prednisone or equivalent at >10 mg daily) or other immunosuppressive drugs within 14 days prior to the first dose of the study drug, excluding topical, intranasal, intrabronchial, or ocular steroids.

[0276] If you have a known history of human immunodeficiency virus (HIV), active hepatitis B (hepatitis B surface antigen; HBsAg), or hepatitis C.

[0277] Patients with a history of hepatitis B virus (HBV) infection or a resolved HBV infection (defined as having a negative HBsAg test and a positive antibody test for hepatitis B core antigen [anti-HBc]) are eligible. Patients who are positive for hepatitis C virus (HCV) antibodies are eligible only if their polymerase chain reaction is negative for HCV RNA.

Claims

1. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: the pharmaceutical composition comprises a fusion protein comprising a targeting moiety and an immunomodulatory moiety; (i) the targeting moiety specifically binds to epidermal growth factor receptor (EGFR); and (ii) the immunomodulatory portion comprises an amino acid sequence of the extracellular domain of transforming growth factor beta receptor II (TGFβRII); The pharmaceutical composition, wherein the treatment comprises administering a fusion protein in combination with an antibody that specifically binds to programmed cell death protein 1 (PD1), or a functional fragment or functional variant thereof.

2. 2. The pharmaceutical composition of claim 1, wherein the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

3. The pharmaceutical composition of claim 1 , wherein the antibody, or a functional fragment or functional variant thereof, that specifically binds to PD1 inhibits binding of PD1 to PDL1.

4. the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; a. VH CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1; b. VH CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2; and c. the VH CDR3 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:3; the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3; d. VL CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4; e. the VL CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and f. VL CDR3 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6; The pharmaceutical composition of claim 1.

5. the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; a. VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; b. VH CDR2 comprises the amino acid sequence of SEQ ID NO:2; and c. VH CDR3 comprises the amino acid sequence of SEQ ID NO:3; the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3; d. VL CDR1 comprises the amino acid sequence of SEQ ID NO:4; e. VL CDR2 comprises the amino acid sequence of SEQ ID NO:5; and f. VL CDR3 comprises the amino acid sequence of SEQ ID NO:6; The pharmaceutical composition of claim 1.

6. The antibody, or a functional fragment or functional variant thereof, which specifically binds to PD1, VH comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; A VL comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8; The pharmaceutical composition of claim 1 , comprising:

7. The antibody, or a functional fragment or functional variant thereof, which specifically binds to PD1, a heavy chain region comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; a light chain region comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:11; The pharmaceutical composition of claim 1 , comprising:

8. 2. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds to PD1, or a functional fragment or functional variant thereof, comprises pembrolizumab, or a functional fragment or functional variant of any of the foregoing.

9. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety that specifically binds to EGFR comprises an antibody or a functional fragment or variant thereof that specifically binds to EGFR.

10. 10. The pharmaceutical composition of claim 9, wherein the antibody, or functional fragment or functional variant thereof that specifically binds to EGFR is a full-length antibody, a single chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

11. the antibody, or functional fragment or functional variant thereof, that specifically binds to EGFR comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; a. VH CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:34; b. VH CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35; and c. the VH CDR3 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36; the antibody, or functional fragment or functional variant thereof, that specifically binds to EGFR comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3; d. VL CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:37; e. the VL CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:38; and f. The VL CDR3 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:39; The pharmaceutical composition according to claim 9.

12. the antibody, or functional fragment or functional variant thereof, that specifically binds to EGFR comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; a. VH CDR1 comprises the amino acid sequence of SEQ ID NO:34; b. VH CDR2 comprises the amino acid sequence of SEQ ID NO:35; and c. VH CDR3 comprises the amino acid sequence of SEQ ID NO:36; the antibody, or functional fragment or functional variant thereof, that specifically binds to EGFR comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3; d. VL CDR1 comprises the amino acid sequence of SEQ ID NO:37; e. the VL CDR2 comprises the amino acid sequence of SEQ ID NO:38; and f. VL CDR3 comprises the amino acid sequence of SEQ ID NO:39; The pharmaceutical composition according to claim 9.

13. The antibody, or a functional fragment or functional variant thereof, which specifically binds to EGFR, VH comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; A VL comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41; 10. The pharmaceutical composition of claim 9, comprising:

14. The antibody, or a functional fragment or functional variant thereof, which specifically binds to EGFR, a heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:42 or SEQ ID NO:43; a light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44; 10. The pharmaceutical composition of claim 9, comprising:

15. 2. The pharmaceutical composition of claim 1, wherein the immunomodulatory portion comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

56.

16. The pharmaceutical composition of claim 1 , wherein the immunomodulatory moiety is indirectly fused to the targeting moiety via a peptide linker.

17. The pharmaceutical composition of claim 16, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

57.

18. the targeting moiety is an antibody comprising a light chain and a heavy chain, and the immunomodulatory moiety is fused to the C-terminus of the heavy chain of the targeting moiety; or the immunomodulatory moiety is fused to the C-terminus of the light chain of the targeting moiety; The pharmaceutical composition of claim 1.

19. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety is an antibody that specifically binds to epidermal growth factor receptor (EGFR), comprising a heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43 and a light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the immunomodulatory moiety comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:56, and wherein the N-terminus of the immunomodulatory moiety is indirectly fused to the C-terminus of the light chain through a linker, wherein the linker comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

57.

20. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety comprises an antibody comprising: a heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43; and a light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

62.

21. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:43; and a light chain comprising the amino acid sequence of SEQ ID NO:

62.

22. The pharmaceutical composition of claim 1 , wherein the cancer is a solid tumor.

23. 23. The pharmaceutical composition of claim 22, wherein the cancer is selected from the group consisting of breast cancer, anal cancer, pancreatic cancer, thyroid cancer, liver cancer, ovarian cancer, lung cancer, skin cancer, brain cancer, spinal cancer, head cancer, neck cancer, and head and neck cancer.

24. 23. The pharmaceutical composition of claim 22, wherein the cancer is head and neck cancer.

25. 23. The pharmaceutical composition of claim 22, wherein the cancer is head and neck squamous cell carcinoma (HNSCC).

26. 23. The pharmaceutical composition of claim 22, wherein the cancer is recurrent HNSCC and / or metastatic HNSCC.

27. ​​The pharmaceutical composition of claim 22, wherein the cancer is anal cancer.

28. 28. The pharmaceutical composition of claim 27, wherein the cancer is squamous cell carcinoma of the anal canal (SCCAC).

29. 29. The pharmaceutical composition of claim 28, wherein the cancer is recurrent SCCAC and / or metastatic SCCAC.

30. 2. The pharmaceutical composition of claim 1, wherein the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 is pembrolizumab and is administered to the human subject at a dose of 100 mg to 200 mg.

31. 2. The pharmaceutical composition of claim 1, wherein the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 is pembrolizumab and is administered to the human subject at a dose of 200 mg.

32. 2. The pharmaceutical composition of claim 1, wherein the antibody, or functional fragment or functional variant thereof, that specifically binds to PD1 is pembrolizumab and is administered to the human subject every three weeks.

33. 10. The pharmaceutical composition of claim 1, wherein the fusion protein is administered to the human subject at a dose of 700 mg to 2000 mg.

34. 2. The pharmaceutical composition of claim 1, wherein the fusion protein is administered to the human subject at a dose of 1500 mg.

35. The pharmaceutical composition of any one of claims 1 to 34, wherein the fusion protein is administered to the human subject weekly.

36. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: the pharmaceutical composition comprises a fusion protein comprising a targeting moiety and an immunomodulatory moiety; the fusion protein comprises an antibody; the antibody comprises an antibody comprising a heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43; and a light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:62, the treatment comprising administering a fusion protein in combination with pembrolizumab; Pembrolizumab is administered to the human subject at a dose of 200 mg every three weeks; The fusion protein is administered to the human subject at a dose of between 700 mg and 2000 mg. Pharmaceutical compositions.

37. The pharmaceutical composition of claim 36, wherein the fusion protein is administered to the human subject at a dose of 1500 mg.

38. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: the pharmaceutical composition comprises a fusion protein comprising a targeting moiety and an immunomodulatory moiety; the fusion protein comprises an antibody; the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43; and a light chain comprising the amino acid sequence of SEQ ID NO: 62; the treatment comprising administering a fusion protein in combination with pembrolizumab; Pembrolizumab is administered to the human subject at a dose of 200 mg every three weeks; The fusion protein is administered to the human subject at a dose of between 700 mg and 2000 mg. Pharmaceutical compositions.

39. The pharmaceutical composition of claim 38, wherein the fusion protein is administered to the human subject at a dose of 1500 mg.

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