Methods of treating endometrial cancer with a bispecific anti-MUC16 x anti-CD3 antibody alone or in combination with an anti-PD-1 antibody

Abstract

This disclosure provides methods for treating endometrial cancer, reducing its severity, or inhibiting its growth. The methods of this disclosure comprise administering to a subject in need a single therapeutically effective amount of a bispecific antibody that specifically binds to mucin 16 (MUC16) and CD3, or administering said bispecific antibody in combination with a therapeutically effective amount of an antibody specifically binding to the programmed death 1 (PD-1) receptor or an antigen-binding fragment thereof.

Description

[0001] References to sequence lists

[0002] This application incorporates a computer-readable sequence list in ST.26 XML format, entitled 11640WO01_Sequence, created on December 4, 2024 and containing 54,668 bytes. Technical Field

[0003] This disclosure relates to a method of treating endometrial cancer using a bispecific antibody against mucin-16 (MUC16) and CD3 alone, or in combination with an anti-PD-1 antibody. Background Technology

[0004] Mucin 16 (MUC16), also known as cancer antigen 125, carcinogen 125, carbohydrate antigen 125, or CA-125, is a highly glycosylated integrated membrane glycoprotein with a single transmembrane domain expressed in many cancer types. MUC16 consists of three main domains: an extracellular N-terminal domain; a large tandem repeat domain containing domains of sea urchin sperm, enterokinase, and aggregate protein (SEA); and a C-terminal domain containing a transmembrane region and a short cytoplasmic tail. Proteolytic cleavage results in the detachment of the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in cancers (including endometrial cancer, ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma-mass-forming types, cervical adenocarcinoma, and gastrointestinal adenocarcinoma) and diseases and conditions including inflammatory bowel disease, cirrhosis, heart failure, peritonitis, and abdominal surgery. (Haridas, D. et al., 2014) FASEB J. , 28:4183-4199).

[0005] In a small series of 12 cases of endometrial cancer detailed in the Human Protein Atlas, 75% of the cases expressed MUC16 in 25% of the cells (The Human Protein Atlas). A similar MUC16 positivity rate was observed in a study of 485 patients undergoing hysterectomy for endometrial cancer assessment (Kakimoto, 2021). Another assessment showed that an estimated 73% of cases had ≥25% of tumor cells that were positive for MUC16 on immunohistochemical staining. Cleavage-type MUC16 levels, detected as serum CA125, are elevated in 70% of patients with stage 4 endometrial cancer, and circulating levels may be higher in patients with serous histological types of endometrial cancer (LyBarger, 2022), (Schmidt, 2018). Studies suggest that expression on cancer cells can protect tumor cells from attack by the immune system. (Felder, M. et al., 2014, ) Molecular Cancer, 13:129. Oregovomab and abavomab are anti-MUC16 antibodies, but their efficacy is limited. Felder, ibid., Das, S. Batra, SK 2015, Cancer Res. 75:4660-4674.

[0006] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, thereby inducing T cell activation in a manner similar to that of MHC molecules loaded with peptides participating in the TCR. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. Additionally, bispecific antibodies capable of binding to both CD3 and target antigens have been proposed for therapeutic applications involving T cell immune responses targeting tissues and cells expressing the target antigen.

[0007] Programmed death receptor-1 (PD-1) signaling in the tumor microenvironment plays a crucial role in immune surveillance, allowing tumor cells to escape the host's immune system. Blocking the PD-1 signaling pathway has demonstrated clinical activity in patients with various tumor types, and antibody therapies that block PD-1 (e.g., nivolumab and pembrolizumab) have been approved for the treatment of metastatic melanoma and metastatic squamous non-small cell lung cancer. Recent data have also demonstrated the clinical activity of PD-1 blockers in patients with aggressive NHL and Hodgkin's lymphoma (Lesokhin et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exposition, San Francisco, Calif.; Ansell et al. 2015). N. Engl. J. Med. 372(4):311-9).

[0008] In the United States, there are 66,000 new cases of endometrial cancer annually, resulting in 13,000 deaths each year (SEER, 2022). According to the National Cancer Database, 34.1% of Black women and 19.1% of White women are diagnosed at an advanced stage (Kucera, 2023). Until recently, the standard of care for first-line treatment of women with advanced endometrial cancer (including those whose cancer progressed after surgery and / or radiation therapy) was carboplatin and paclitaxel. This line of treatment was based on a study that showed no worse overall survival compared to the more toxic doxorubicin, cisplatin, and paclitaxel regimens (Miller, 2020). Currently, there are two FDA-approved anti-PD-1 antibodies, pembrolizumab (O'Malley, 2022) and dostarlimab (Oaknin, 2022), for second-line treatment of patients with dMMR endometrial cancer, which accounts for approximately 25% of endometrial cancer cases (Lorenzi, 2020). In this context, the overall response rate to anti-PD-1 is approximately 45%, with most patients experiencing durable responses. For patients with advanced pMMR and dMMR endometrial cancer, based on the KEYNOTE-775 study showing improved progression-free survival (PFS) compared to chemotherapy, the combination of pembrolizumab and lenvatinib was approved by the FDA in 2022 (Makker, 2022). Recent results from two randomized phase 3 studies have demonstrated that, in first-line treatment of advanced or recurrent advanced endometrial cancer, anti-PD-1 therapy with pembrolizumab or dostalimab in combination with carboplatin and paclitaxel significantly improves progression-free survival (PFS), particularly in women with dMMR endometrial cancer (Eskander, 2023) and (Mirza, 2023). Despite these successes, clinical needs remain unmet, necessitating the development of alternative therapies for endometrial cancer. Summary of the Invention

[0009] In one aspect, this disclosure includes a method of treating endometrial cancer in a subject of need, comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, said bispecific antibody or antigen-binding fragment comprising a first antigen-binding domain specifically binding to mucin 16 (MUC16) and a second antigen-binding domain specifically binding to human CD3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of at least 1 mg (e.g., weekly).

[0010] In some embodiments, the subject has previously been treated with platinum-based chemotherapy. In some embodiments, the subject has previously been treated with an antibody that binds to human programmed death receptor-1 (PD-1). In some embodiments, the subject has previously been treated with both platinum-based chemotherapy and an antibody that binds to human PD-1. In some embodiments, the subject has previously received at least two lines of therapy to treat the endometrial cancer. In some embodiments, the endometrial cancer is advanced or recurrent endometrial cancer. In some embodiments, the endometrial cancer is a cancer that expresses MUC16.

[0011] In some embodiments, this disclosure provides a method as discussed herein, wherein the bispecific antibody or its antigen-binding fragment comprises a first antigen-binding domain comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 1; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 2.

[0012] In some embodiments, the first antigen-binding domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first antigen-binding domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments of the methods disclosed herein, the first antigen-binding domain comprises HCVR comprising the amino acid sequence of SEQ ID NO: 1 and LCVR comprising the amino acid sequence of SEQ ID NO: 2.

[0013] In some embodiments, including those described above, the bispecific antibody or its antigen-binding fragment comprising a second antigen-binding domain comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 3; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the second antigen-binding domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 14, HCDR2 containing the amino acid sequence of SEQ ID NO: 15, and HCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the second antigen-binding domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 11, LCDR2 containing the amino acid sequence of SEQ ID NO: 12, and LCDR3 containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second antigen-binding domain comprises HCVR containing the amino acid sequence of SEQ ID NO: 3 and LCVR containing the amino acid sequence of SEQ ID NO: 2.

[0014] In some embodiments of the methods disclosed herein, the bispecific antibody comprises a human IgG heavy chain constant region. In some embodiments, the human IgG heavy chain constant region is isotype IgG1. In some embodiments, the human IgG heavy chain constant region is isotype IgG4. In some embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fc relative to a wild-type hinge of the same isotype. Receptor binding. In some embodiments, the first heavy chain or the second heavy chain contains a CH3 domain, but not both simultaneously contain a CH3 domain, the CH3 domain containing H435R (EU number) modification and Y436F (EU number) modification.

[0015] In some embodiments of the methods disclosed herein, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31, and a common light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the bispecific antibody is REGN4018 (ubamatamab).

[0016] In one aspect, this disclosure provides a method disclosed herein, wherein the subject has a serum CA-125 level equal to or greater than 60 U / ml. In some cases, the subject's serum CA-125 level is greater than 65 U / ml, 70 U / ml, 75 U / ml, 80 U / ml, 85 U / ml, 90 U / ml, or 95 U / ml.

[0017] In some embodiments, the method further comprises administering a second therapeutic agent or treatment regimen. In some embodiments, the second therapeutic agent or treatment regimen comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 33; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34.

[0018] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 37. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 38, LCDR2 containing the amino acid sequence of SEQ ID NO: 39, and LCDR3 containing the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises HCVR containing the amino acid sequence of SEQ ID NO: 33 and LCVR containing the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 41, and the light chain comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-PD-1 antibody is cimipril.

[0019] In one aspect, this disclosure provides a method in which the bispecific antibody (or a fragment thereof) is administered in a dosing regimen comprising a fractionated initial dose. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of 10 mg to 1000 mg weekly. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of about 250 mg weekly, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of about 800 mg weekly, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 750 mg. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of 10 mg to 1000 mg every three weeks. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of about 250 mg every three weeks, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg. In some embodiments, the bispecific antibody (or a fragment thereof) is administered to the subject at a dose of about 800 mg every three weeks, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 750 mg.

[0020] In one aspect, this disclosure provides a method in which the bispecific antibody or its antigen-binding fragment is administered in a dosing regimen comprising: (i) administering 1 mg of the bispecific antibody (or its fragment) in week 1, optionally wherein the dose is divided into a first portion of about 0.5 mg and a second portion of about 0.5 mg; (ii) administering 20 mg of the bispecific antibody (or its fragment) in week 2, optionally wherein the dose is divided into a first portion of about 10 mg and a second portion of about 10 mg; and (iii) administering 250 mg of the bispecific antibody (or its fragment) in week 3, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg. In some embodiments, the method further comprises administering the bispecific antibody (or its fragment) weekly at a dose of about 250 mg starting from week 4. In some embodiments, the method further comprises administering the bispecific antibody (or its fragment) every three weeks at a dose of about 250 mg starting from week 4. In some embodiments, the method further comprises administering the bispecific antibody (or its fragment) every three weeks at a dose of about 800 mg starting from week 4.

[0021] In one aspect, this disclosure provides a method in which an anti-PD-1 antibody is administered to a subject at a dose of 300 to 400 mg every three weeks. In some embodiments, the anti-PD-1 antibody is administered to the subject at a dose of 350 mg every three weeks.

[0022] In one aspect, this disclosure provides a method in which the subject has a stable disease, a partial response, or a complete response at least one week after administration of the bispecific antibody (or a fragment thereof) at a dose of 1-800 mg. In some embodiments, the subject has a stable disease, a partial response, or a complete response at least one week after administration of the bispecific antibody (or a fragment thereof) at a dose of 20-800 mg.

[0023] In one aspect, this disclosure provides a method in which the bispecific antibody (or a fragment thereof) is administered to the subject at a dose sufficient to achieve a serum concentration of at least 4 mg / L.

[0024] In one aspect, this disclosure provides a method in which MUC16 is expressed on ≥25% of tumor cells in the subject, as determined by immunohistochemical staining. In some embodiments, the subject has a baseline MUC16 immunohistochemical staining score of 2 in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 2+ in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 3 in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 3+ in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 4 in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 4+ in tumors expressing MUC16; or a baseline MUC16 immunohistochemical staining score of 5 in tumors expressing MUC16; or a tumor with ≥25% tumor cells expressing MUC16; or a tumor with ≥30% tumor cells expressing MUC16; or a tumor with ≥35% tumor cells expressing MUC16; or a tumor with ≥ Tumors with 40% or more of their tumor cells expressing MUC16; or tumors with ≥45% or more of their tumor cells expressing MUC16; or tumors with ≥50% or more of their tumor cells expressing MUC16; or tumors with ≥55% or more of their tumor cells expressing MUC16; or tumors with ≥60% or more of their tumor cells expressing MUC16; or tumors with ≥70% or more of their tumor cells expressing MUC16; or tumors with ≥75% or more of their tumor cells expressing MUC16.

[0025] In one aspect, this disclosure provides a method in which MUC16 is expressed on tumor cells in the subject, as determined by immunohistochemical staining. In some embodiments, the subject has an H score of ≥100; ≥105; ≥110; ≥115; ≥120; ≥125; ≥130; ≥135; ≥140; ≥145; ≥150; ≥155; ≥160; ≥165; ≥170; ≥175; ≥180; ≥185; ≥190; ≥195; or ≥200, as determined by the immunohistochemical staining described in Example 6. In some cases, the subject has a serous subtype of endometrial cancer. In some cases, the subject had endometrial cancer of the carcinosarcoma subtype. In some cases, the subject had grade 3 endometrial cancer. In some cases, the subject had mixed serous and endometrioid endometrial cancer. In some cases, the subject had mixed serous and clear cell endometrial cancer. In some cases, the subject had mixed clear cell and grade 3 endometrial cancer.

[0026] In one aspect, this disclosure provides a method in which the bispecific antibody (or a fragment thereof) is administered intravenously. In some embodiments, the bispecific antibody (or a fragment thereof) is administered subcutaneously. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment is administered intravenously. Attached Figure Description

[0027] Figure 1 This diagram shows the study design for monotherapy of REGN4018 administered intravenously.

[0028] Figure 2 This diagram shows the study design for a combination therapy of intravenous REGN4018 and cimiprimab.

[0029] Figure 3 This diagram shows a monotherapy study design where REGN4018 is administered subcutaneously initially and as a transitional IV dose, followed by a second transitional IV dose and a subsequent full IV dose. The second transitional IV dose can be omitted in additional patients if no CRS is observed in the initial patient.

[0030] Figure 4 The variability of MUC16 expression as measured by histology, and the H score in different pathological diagnostic categories, as determined in Example 6. Detailed Implementation

[0031] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as these methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be defined only by the appended claims. Any embodiment or feature of an embodiment may be combined with each other, and such combinations are expressly covered within the scope of this disclosure. Any particular value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range of values ​​having upper and lower ends indicating a range, and such ranges are covered within the scope of this disclosure.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value may differ from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0033] While any methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, preferred methods and materials are now described. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0034] Methods used to treat cancer or inhibit its growth

[0035] This disclosure includes methods for treating, improving, or reducing at least one symptom or indication of cancer (e.g., endometrial cancer) in a subject, or inhibiting the growth of cancer (e.g., endometrial cancer) in a subject. Methods according to this aspect of the disclosure include administering to a subject in need a therapeutically effective amount of a bispecific antibody against MUC16 and CD3 or an antigen-binding fragment thereof as monotherapy, or administering said bispecific antibody or an antigen-binding fragment thereof in combination with a therapeutically effective amount of an antibody specifically binding to PD-1 or an antigen-binding fragment thereof. As used herein, the terms “treat,” “treating,” etc., refer to temporarily or permanently relieving symptoms, eliminating the cause of symptoms, delaying or inhibiting tumor growth, reducing tumor cell burden or tumor load, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, and / or prolonging the survival duration of a subject.

[0036] As used herein, the term "subject in need" refers to a human or non-human mammal presenting with one or more symptoms or indications of cancer and / or having been diagnosed with cancer (including endometrial cancer) and requiring treatment for cancer. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may be diagnosed with a primary or metastatic tumor and / or have one or more symptoms or conditions, including (but not limited to) enlarged lymph nodes, abdominal distension, chest pain / chest pressure, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, splenomegaly, and itching. The term includes subjects with a primary or confirmed endometrial tumor. In a specific embodiment, the term includes human subjects with endometrial cancer or another tumor expressing MUC16 (e.g., endometrial cancer) and requiring treatment. In other specific embodiments, the term includes subjects with a MUC16+ tumor (e.g., a tumor expressing MUC16 as determined by flow cytometry or immunohistochemistry). In some embodiments, the representation includes human subjects with tumors that extensively express MUC16 in >50%, >55%, >60%, >65%, >70%, or >75% of their tumor cells. MUC16 expression can be determined and assessed by any method known in the art (see, for example, Shimizu et al. 2012, Cancer Sci. 103: 739-746). In some embodiments, the representation includes human subjects with a baseline MUC16 immunohistochemical staining score of 2+ (e.g., 2, 3, 4, or 5) in tumors expressing MUC16. In some embodiments, the representation includes human subjects with a baseline MUC16 immunohistochemical staining score of 2, 2+, 3, 3+, 4, 4+, or 5 in tumors expressing MUC16. In this context, immunohistochemical staining scores are based on the following criteria combined with cell percentage and staining intensity and pattern: score 1 (<5% strong or weak); score 2 (5-50% strong or weak); score 3 (51-75% strong or 51-100% weak); score 4 (76-99% strong); and score 5 (100% strong staining). In some embodiments, the statement "subjects in need" includes patients with endometrial cancer that is resistant, refractory, or poorly controlled to prior therapy (e.g., treatment with anticancer agents). For example, the statement includes subjects who have been treated with chemotherapy (platinum-based chemotherapeutic agents (e.g., cisplatin) or paclitaxel compounds (e.g., docetaxel)). The statement also includes subjects with endometrial tumors for whom conventional anticancer therapy is not desirable, for example, due to toxic side effects. For instance, the statement includes patients who have received one or more cycles of chemotherapy with toxic side effects.In some embodiments, the term "subject in need" includes patients with endometrial tumors who have received treatment but subsequently relapsed or metastasized. For example, the methods of this disclosure may be used to treat patients with endometrial tumors that may have previously received treatment with one or more anticancer agents and experienced tumor regression, but subsequently relapsed and developed resistance to said one or more anticancer agents (e.g., chemotherapy-resistant cancer).

[0037] The phrase “subjects in need” also includes subjects at risk of developing endometrial cancer, such as those with a family history of endometrial cancer, those with a history of endometrial cancer-related infections, those with BRCA1 / 2 gene mutations, or those with compromised immune systems due to HIV infection or immunosuppressive drugs.

[0038] In some embodiments, the methods of this disclosure can be used to treat patients exhibiting elevated levels of one or more cancer-related biomarkers, such as programmed death-ligand 1 (PD-L1), MUC16, CA125, human epididymal protein 4 (HE4), and / or carcinoembryonic antigen (CEA). For example, the methods of this disclosure comprise administering to a patient with elevated MUC16 and / or CA125 levels a combination of a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof with a bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof. Methods for determining MUC16 and / or CA125 expression are well known in the art. In some embodiments, MUC16 expression in tumor tissue is determined by immunohistochemistry (IHC) assay (see, for example, Bast et al. 1981, J. Clin. Invest. 68: 1331-1337). MUC16 expression can be assessed by any method known in the art (e.g., Shimizu et al. 2012, Cancer Sci. 103: 739-746). In some embodiments, MUC16 expression is determined based on imaging with labeled anti-MUC16 antibodies, such as by immunopositron emission tomography or iPET (described elsewhere herein).

[0039] In some embodiments, the methods of this disclosure are used in subjects with endometrial cancer. The terms “tumor,” “cancer,” and “malignancy” are used interchangeably herein. As used herein, the term “endometrial cancer” refers to a tumor of the uterus and includes adenocarcinoma (e.g., endometrioid), uterine carcinosarcoma, squamous cell carcinoma, small cell carcinoma, transitional cell carcinoma, and serous carcinoma. Endometrioid carcinoma includes adenocarcinoma, adenoacanthoma, adenosquamous carcinoma, secretory carcinoma, ciliated carcinoma, and choriocarcinoma. Advanced or recurrent endometrial cancer refers to those tumors that cannot be treated alone with surgery or radiation therapy or that have recurred after prior therapy.

[0040] According to some embodiments, this disclosure includes methods for treating or delaying or inhibiting tumor growth. In some embodiments, this disclosure includes methods for promoting tumor regression. In some embodiments, this disclosure includes methods for reducing tumor cell burden or reducing tumor load. In some embodiments, this disclosure includes methods for preventing tumor recurrence. According to this aspect of the disclosure, the method comprises administering to a subject in need a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof as monotherapy, or a combination of the bispecific antibody or its antigen-binding fragment with an anti-PD-1 antibody or its antigen-binding fragment, wherein each antibody is administered to the subject in multiple doses, for example as part of a specific therapeutic dosing regimen. For example, the therapeutic dosing regimen may comprise administering one or more doses of the anti-MUC16×CD3 antibody or its antigen-binding fragment to the subject at a frequency of approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every two months, once every three months, once every four months, or less. In some embodiments, one or more doses of an anti-PD-1 antibody or its antigen-binding fragment are administered in combination with one or more therapeutically effective doses of a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, wherein the one or more doses of the anti-PD-1 antibody or its antigen-binding fragment are administered to the subject at a frequency of approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every two months, once every three months, once every four months, or less.

[0041] In some embodiments, each dose of the anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment is administered in more than one fraction (e.g., in 2-5 fractions (“fractionated administration”)) during a given dosing period. The anti-MUC16 / anti-CD3 bispecific antibody or its antigen-binding fragment may be administered in fractionated doses to reduce or eliminate cytokine “surges” induced in response to antibody administration. Cytokine surges refer to clinical symptoms of cytokine release syndrome (“cytokine storm”) and infusion-related reactions. In some embodiments, the method of this disclosure comprises administering to a subject in need one or more doses of a combination of an anti-PD-1 antibody or its antigen-binding fragment and one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, wherein, during a given dosing period, the dose of the bispecific antibody or its antigen-binding fragment is administered in fractionated doses or in more than one fraction (e.g., in 2 fractions, 3 fractions, 4 fractions, or 5 fractions). In some embodiments, the dose of a bispecific antibody or its antigen-binding fragment is divided into two or more fractions, each fraction containing an amount of antibody or its antigen-binding fragment equal to that of the other fractions. For example, a dose containing 1000 micrograms of anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment may be administered once weekly, wherein the dose is administered in two fractions over the week, each fraction containing 500 micrograms. In some embodiments, the dose of a bispecific antibody or its antigen-binding fragment is divided into two or more fractions, wherein the fractions contain unequal amounts of antibody, for example, more or less than the first fraction. For example, a dose containing 1000 micrograms of anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment may be administered once weekly, wherein the dose is administered in two fractions over the week, wherein the first fraction contains 700 micrograms and the second fraction contains 300 micrograms. As another example, a dose containing 1000 micrograms of anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment may be administered once over two weeks, wherein the dose is administered in three portions over the two weeks, wherein the first portion contains 400 micrograms, the second portion contains 300 micrograms and the third portion contains 300 micrograms.

[0042] In some embodiments, this disclosure provides a method of treating a subject in need of a MUC16-expressing cancer (e.g., endometrial cancer, including cancers refractory to multiple rounds of prior therapy as discussed herein), comprising administering a bispecific antibody to the subject, the bispecific antibody comprising a first antigen-binding domain specifically binding to mucin 16 (MUC16) and a second antigen-binding domain specifically binding to human CD3, wherein the bispecific antibody is administered in a dosing regimen comprising: (i) administering 1 mg of the bispecific antibody in week 1, optionally wherein the dose is divided into a first portion of about 0.5 mg and a second portion of about 0.5 mg; (ii) administering 20 mg of the bispecific antibody in week 2, optionally wherein the dose is divided into a first portion of about 10 mg and a second portion of about 10 mg; and (iii) administering 250 mg of the bispecific antibody in week 3, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg. In some cases, the method further comprises administering the bispecific antibody once weekly at a dose of about 250 mg starting from week 4. In some cases, the method further includes administering the bispecific antibody every three weeks at a dose of approximately 250 mg, starting from week 4. In some cases, the method further includes administering the bispecific antibody every three weeks at a dose of approximately 800 mg, starting from week 4. In some cases, the method further includes administering the anti-PD-1 antibody to the subject every three weeks at a dose of 300 to 400 mg (e.g., 350 mg).

[0043] In some embodiments, this disclosure includes methods for inhibiting, delaying, or stopping tumor metastasis or tumor invasion into peripheral organs. According to this aspect, the method comprises administering to a subject in need a therapeutically effective amount of a single bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof, or a combination of said bispecific antibody or an antigen-binding fragment thereof with an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0044] In specific embodiments, this disclosure provides methods for increasing antitumor efficacy or increasing tumor suppression. According to this aspect of the disclosure, the method comprises administering a therapeutically effective amount of an antiPD-1 antibody or its antigen-binding fragment to a subject with endometrial cancer prior to the administration of a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof, wherein the antiPD-1 antibody or its antigen-binding fragment may be administered approximately 1 day, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more than 8 days prior to the bispecific antibody or its antigen-binding fragment. In some embodiments, the method provides increased tumor suppression, for example, an increase of approximately 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80%, compared to a subject who received the bispecific antibody or its antigen-binding fragment prior to the antiPD-1 antibody or its antigen-binding fragment.

[0045] In some embodiments, the method of this disclosure comprises administering a therapeutically effective amount of a single bispecific anti-CD3×MUC16 antibody or its antigen-binding fragment thereof, or a combination of said bispecific antibody or its antigen-binding fragment with an anti-PD-1 antibody or its antigen-binding fragment thereof, to a subject with endometrial cancer. In specific embodiments, the endometrial cancer is serous carcinoma. In other embodiments, the endometrial cancer is indolent or invasive. In some embodiments, the subject is unresponsive to prior therapy or has relapsed after prior therapy (e.g., platinum-based therapy). In some embodiments, the subject's CA-125 level is equal to or greater than twice the upper limit of normal (ULN) (e.g., equal to or greater than about 60 U / ml). In various embodiments, the subject's serum CA-125 level (before treatment) is 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 U / ml. In some embodiments, the method of this disclosure further includes administering an additional therapeutic agent to the subject.

[0046] In some embodiments, the method of this disclosure includes administering a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof to a subject with MUC16+ cancer. In a specific embodiment, the cancer is endometrial cancer. In other embodiments, the endometrial cancer is indolent or invasive. In some embodiments, the cancer is platinum-resistant endometrial cancer. In some embodiments, the cancer is paclitaxel-resistant endometrial cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is primary peritoneal cancer, optionally wherein the patient has elevated serum CA-125 levels (e.g., at least 2 × ULN). In some embodiments, the subject is unresponsive to prior therapy or has relapsed after prior therapy (e.g., chemotherapy).

[0047] In some embodiments, the method of this disclosure includes administering a combination of an anti-PD-1 antibody or its antigen-binding fragment thereof with a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof to a subject in need as "first-line" treatment (e.g., initial treatment). In other embodiments, administering a combination of an anti-PD-1 antibody or its antigen-binding fragment thereof with a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof is used as "second-line" treatment (e.g., after prior therapy). For example, administering a combination of an anti-PD-1 antibody or its antigen-binding fragment thereof with a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof to a subject who has relapsed after prior therapy such as chemotherapy (e.g., platinum-based chemotherapy) is used as "second-line" treatment.

[0048] In some embodiments, the methods of this disclosure are used to treat patients with MRD-positive disease. Minimal residual disease (MRD) refers to a small number of cancer cells remaining in a patient's body during or after treatment, where the patient may or may not exhibit symptoms or signs of disease. Such residual cancer cells often lead to disease relapse if not eliminated. This disclosure includes methods for inhibiting and / or eliminating residual cancer cells in a patient during an MRD test. MRD can be determined according to methods known in the art, such as MRD flow cytometry. According to this aspect of the disclosure, the method comprises administering a single bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof, or a combination of said bispecific antibody or an antigen-binding fragment thereof with an anti-PD-1 antibody or an antigen-binding fragment thereof, to a subject in need.

[0049] According to some embodiments, the method of this disclosure includes administering to a subject a therapeutically effective amount of a single bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, or a combination of said bispecific antibody or its antigen-binding fragment with an anti-PD-1 antibody or its antigen-binding fragment and an optional third therapeutic agent. The third therapeutic agent may be an agent selected from the group consisting of: for example, radiotherapy, chemotherapy, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), LAG3 inhibitors (e.g., anti-LAG3 antibodies), CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, Ang2 inhibitors, transforming growth factor β (TGF.β.) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, tumor-specific antigens. (e.g., antibodies against CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., BCG), granulocyte-macrophage colony-stimulating factors, cytotoxins, chemotherapeutic agents, IL-6R inhibitors (sarilumab), IL-4R inhibitors, IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, anti-inflammatory drugs such as corticosteroids and nonsteroidal anti-inflammatory drugs, and dietary supplements such as antioxidants. In some embodiments, the antibody may be administered in combination with therapies including chemotherapeutic agents (e.g., paclitaxel, carboplatin, doxorubicin, cyclophosphamide, cisplatin, gemcitabine, or docetaxel), radiation therapy, and surgery. As used herein, the phrase “in combination with” means administering the antibody to the subject concurrently with, before, or after administration of a third therapeutic agent. In some embodiments, the third therapeutic agent is administered as a co-regulation with the antibody.

[0050] In some embodiments, the method of this disclosure comprises administering to a subject in need a therapeutically effective amount of a single bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, or a combination of said bispecific antibody or its antigen-binding fragment with an anti-PD-1 antibody or its antigen-binding fragment. When the combination is administered, the administration of the antibody (or fragment) results in increased inhibition of tumor growth. In some embodiments, tumor growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, respectively, compared to untreated subjects or subjects receiving either antibody (or fragment) as monotherapy. In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment causes increased tumor regression, tumor shrinkage, and / or disappearance. In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment resulted in delayed tumor growth and development, for example, tumor growth could be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years, compared to treatment-naïve subjects or subjects receiving either antibody (or fragment) as monotherapy. In some embodiments, combined administration of an anti-PD-1 antibody or its antigen-binding fragment with a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment prevented tumor recurrence and / or increased the duration of survival in subjects, resulting in an increase in survival duration of more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months, compared to treatment-naïve subjects or subjects receiving either antibody (or fragment) as monotherapy. In some embodiments, combined administration of the antibodies increased development-free survival or overall survival. In some embodiments, combined administration of an anti-PD-1 antibody or its antigen-binding fragment with a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment increases the response and duration of response in subjects, for example, by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to untreated subjects or subjects receiving either antibody (or fragment) as monotherapy. In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment to a subject with endometrial cancer results in the complete disappearance of all evidence of tumor cells (“complete response”).In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof to a subject with endometrial cancer results in a reduction of tumor cells or tumor size by at least 30% or more (“partial response”). In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof to a subject with endometrial cancer results in the complete or partial disappearance of tumor cells / lesions (including new measurable lesions). Tumor reduction can be measured by any of the methods known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytological, histological, or molecular genetic analysis. In some embodiments, administration of an anti-PD-1 antibody or its antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof produces a synergistic antitumor effect that exceeds the combined effect of the two agents when administered alone.

[0051] In some embodiments, the combination of antibodies (or fragments) administered is safe and well tolerated by patients, wherein there is no increase in adverse side effects (e.g., increased cytokine release (cytokine storm) or increased T cell activation) compared to patients receiving bispecific antibodies (or fragments) administered as monotherapy.

[0052] In some cases, a subject's response to treatment is classified as a complete response (CR), partial response (PR), progressive disease (PD), or stable disease (SD). CR is defined as the disappearance of all target lesions and a reduction in the short axis of any pathological lymph node (whether target or non-target) to <10 mm (<1 cm). PR is defined as a reduction of at least 30% in the sum of the diameters of the target lesions, relative to the baseline sum. PD is defined as an increase of at least 20% in the sum of the diameters of the target lesions, relative to the minimum sum in the study (including the baseline sum if it is the minimum in the study). In addition to a relative increase of 20%, the sum must also indicate an absolute increase of at least 5 mm (0.5 cm). (Note: The appearance of one or more new lesions is also considered progression). SD is defined as a contraction that is neither sufficient to qualify as PR nor sufficient to qualify as PD, relative to the minimum sum of the diameters in the study.

[0053] Anti-PD-1 antibodies and their antigen-binding fragments

[0054] According to certain exemplary embodiments of this disclosure, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. The term “anti-PD-1 antibody or an antigen-binding fragment thereof” refers to the term “antibody or an antigen-binding fragment thereof that specifically binds to PD-1” and is used interchangeably with it. As used herein, the term “antibody” includes an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, and a polymer thereof (e.g., IgM). In a typical antibody, each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy-chain constant region contains three structural domains C. H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this article). L ) and the light chain constant region. The light chain constant region contains a structural domain (C L 1). V H District and V L The region can be further subdivided into highly variable regions called complementary determinant regions (CDRs), which are interspersed with more conservative regions called frame regions (FRs). Each V H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of the anti-IL-4R antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0055] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from, for example, whole antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by molecular biology techniques, for example, arranging one or more variable and / or constant domains into suitable conformations, or introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0056] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues of a hypervariable region of a mimic antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variant IgNAR domains, are also encompassed within the term "antigen-binding fragments" as used herein.

[0057] Antibody antigen-binding fragments typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will generally contain at least one CDR adjacent to or within one or more frame sequences. L V related to the structural domain H In the antigen-binding fragment of the domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0058] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody of this disclosure include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v)V H -C H 1-C H 2-CH 3; (vi)V H -C H 2-C H 3; (vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2; (x)V L -C H 3; (xi)V L -C H 1-C H 2; (xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or may be connected via full or partial hinge or connector regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which result in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise the domains listed above and / or one or more monomers V. H or V L Homodimers or heterodimers (or other polymers) of any variable and constant domain configurations in which the domains (e.g., via disulfide bonds) are non-covalently associated.

[0059] As used herein, the term "antibody" also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies typically comprise at least two distinct variable domains, each capable of specifically binding to a different epitope on a single antigen or a homogeneous antigen. Any multispecific antibody format can be adapted to the context of the antibodies or antigen-binding fragments of antibodies disclosed herein using conventional techniques available in the art. For example, this disclosure includes methods involving the use of bispecific antibodies, wherein one arm of an immunoglobulin is specific for PD-1 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or conjugated to a therapeutic portion. Exemplary bispecific formats that may be used in the context of this disclosure include, but are not limited to, scFv-based or bifunctional antibody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knocks-in-holes, common light chains (e.g., common light chains with knocks), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein for a review of the foregoing formats). Bispecific antibodies may also be constructed using peptide / nucleic acid conjugations, for example, where site-specific antibody-oligonucleotide conjugations are generated using non-natural amino acids with orthogonal chemical reactivity, and then self-assembled into multimeric complexes having defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [e-ed. December 4, 2012]).

[0060] The antibodies used in the methods of this disclosure may be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. However, human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in the CDR and specifically in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human scaffold sequence.

[0061] The antibodies used in the methods of this disclosure may be recombinant human antibodies. As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (described further below); antibodies isolated from recombinant, combined human antibody libraries (described further below); antibodies isolated from animals (e.g., mice) that are transgenic for the human immunoglobulin gene (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence into another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody is... H District and V L The amino acid sequence of the region is as follows: Although it is derived from human lineage V H Sequence and V L The sequence is associated with it, but it may not be naturally present in the human antibody germline library.

[0062] According to certain embodiments, the antibodies used in the methods of this disclosure specifically bind to PD-1. The term "specific binding," etc., means that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of this disclosure, antibodies that "specifically bind to PD-1" include antibodies that bind to PD-1 or a portion thereof at a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in a surface plasmon resonance assay. However, isolated antibodies that specifically bind to human PD-1 can be cross-reactive with other antigens, such as PD-1 molecules from other (non-human) species.

[0063] According to certain exemplary embodiments of this disclosure, an anti-PD-1 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR), wherein the HCVR, LCVR, and / or CDR comprises any amino acid sequence of an anti-PD-1 antibody as set forth in U.S. Patent No. 9,987,500. In certain exemplary embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof that may be used in the context of the methods of this disclosure comprises: a heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR), the HCVR comprising the amino acid sequence of SEQ ID NO: 33; and a light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR), the LCVR comprising the amino acid sequence of SEQ ID NO: 34. According to some embodiments, an anti-PD-1 antibody or its antigen-binding fragment comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 35; HCDR2 comprises the amino acid sequence of SEQ ID NO: 36; HCDR3 comprises the amino acid sequence of SEQ ID NO: 37; LCDR1 comprises the amino acid sequence of SEQ ID NO: 38; LCDR2 comprises the amino acid sequence of SEQ ID NO: 39; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 40. In yet other embodiments, an anti-PD-1 antibody or its antigen-binding fragment comprises HCVR and LCVR, wherein the HCVR comprises SEQ ID NO: 33 and the LCVR comprises SEQ ID NO: 34. In some embodiments, the method of this disclosure comprises using an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42. An exemplary antibody comprising an HCVR containing the amino acid sequence of SEQ ID NO: 33 and an LCVR containing the amino acid sequence of SEQ ID NO: 34 is a fully human anti-PD-1 antibody known as REGN2810 (also known as cimiprilmab; LIBTAYO®). According to certain exemplary embodiments, the methods of this disclosure involve the use of REGN2810 or a bioequivalence thereof. As used herein, the term "bioequivalence" refers to a pharmaceutical equivalent or substitute for an anti-PD-1 antibody or PD-1 binding protein or fragment thereof that, under similar experimental conditions, whether administered as a single or multiple dose at the same molar dose, does not show a significant difference in absorption rate and / or absorption extent compared to that of REGN2810.In the context of this disclosure, the term refers to an antigen-binding protein that binds to PD-1 and has no clinically significant differences from REGN2810 in terms of safety, purity, and / or potency.

[0064] Other anti-PD-1 antibodies that can be used in the context of the methods disclosed herein include, for example, antibodies known and referred to in the art as: nivolumab (US Patent No. 8,008,449), pembrolizumab (US Patent No. 8,354,509), MEDI0608 (US Patent No. 8,609,089), pidilizumab (US Patent No. 8,686,119), or any anti-PD-1 antibody as described in US Patent Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0065] Anti-PD-1 antibodies used in the context of the methods of this disclosure may have pH-dependent binding properties. For example, PD-1 antibodies used in the methods of this disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, anti-PD-1 antibodies of this disclosure may exhibit enhanced binding to their antigen at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values ​​less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or smaller. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0066] In some cases, "the binding of PD-1 is reduced at acidic pH compared to neutral pH" refers to the K+ binding of antibodies to PD-1 at acidic pH. D Value and K value of antibody binding to PD-1 at neutral pH D This is expressed in terms of the ratio of values ​​(or vice versa). For example, if the antibody or its antigen-binding fragment exhibits an acidic / neutral K value of approximately 3.0 or greater. D The ratio, for the purposes of this disclosure, can be considered as the antibody or its antigen-binding fragment exhibiting "reduced binding to PD-1 at acidic pH compared to neutral pH." In some exemplary embodiments, the acidic / neutral K of the antibody or antigen-binding fragment of this disclosure... DThe ratio can be approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

[0067] Antibodies exhibiting pH-dependent binding properties can be obtained, for example, by screening for antibody groups that show reduced (or enhanced) binding to specific antigens at acidic pH compared to neutral pH. Alternatively, modifications to the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For instance, by replacing one or more amino acids (e.g., within the CDR) of the antigen-binding domain with histidine residues, antibodies with reduced antigen binding at acidic pH relative to neutral pH can be obtained. As used herein, the term "acidic pH" refers to a pH of 6.0 or lower.

[0068] Bispecific anti-MUC16 / anti-CD3 antibody and its antigen-binding fragment

[0069] According to certain exemplary embodiments of this disclosure, the method comprises administering a therapeutically effective amount of a bispecific antibody that specifically binds to CD3 and MUC16, or an antigen-binding fragment thereof. Such antibodies and fragments may be referred to herein as, for example, “anti-MUC16 / anti-CD3” or “anti-MUC16×CD3” or “MUC16×CD3” bispecific antibody or an antigen-binding fragment thereof, or other similar terms.

[0070] As used herein, the term "bispecific antibody" refers to an immunoglobulin comprising at least one first antigen-binding domain and one second antigen-binding domain. In the context of this disclosure, the first antigen-binding domain specifically binds to a first antigen (e.g., MUC16), and the second antigen-binding domain specifically binds to a second, different antigen (e.g., CD3). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), and each domain contains three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix "A," and the CDRs of the second antigen-binding domain may be designated with the prefix "B." Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1, B-HCDR2, and B-HCDR3.

[0071] The first antigen-binding domain and the second antigen-binding domain are each linked to a separate multimerizing domain. As used herein, a "multimerizing domain" means any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to bind to a second multimerizing domain of the same or similar structure or composition. In the context of this disclosure, the multimerizing component is the Fc portion of an immunoglobulin (containing C... H2 -C H3 (domains), for example, Fc domains selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype of IgG within each isotype group.

[0072] The bispecific antibodies disclosed herein typically comprise two multimerizing domains, for example, two Fc domains that are each independently part of a separate antibody heavy chain. The first and second multimerizing domains can be the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerizing domains can be different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0073] The bispecific antigen-binding molecules of this disclosure can be prepared using any bispecific antibody form or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in the context of this disclosure include, but are not limited to, the following: for example, scFv-based or biantibody bispecific formats, IgG-scFv fusion, dual variable domain (DVD)-Ig, tetrahybridoma, mortise and tenon joint, common light chain (e.g., common light chain with mortise and tenon joint, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab2 bispecific formats (see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and the references cited therein for a review of the foregoing formats).

[0074] In the context of bispecific antibodies disclosed herein, the Fc domain may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring version of the Fc domain. For example, this disclosure includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain to produce a modified Fc domain having a modified binding interaction (e.g., enhanced or weakened) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule contains C H2 Zone or C H3 Modifications to the Fc domain that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in U.S. Patent Publication No. 20150266966, which is incorporated herein by reference in its entirety.

[0075] This disclosure also includes a bispecific antigen-binding molecule, said bispecific antigen-binding molecule comprising a first C H 3 structural domains and second Ig C H 3 structural domains, where the first Ig C H 3 structural domains and second Ig C H The three domains differ from each other by at least one amino acid, and wherein, compared to a bispecific antibody lacking amino acid differences, at least one amino acid difference reduces the binding of the bispecific antibody to protein A. In one embodiment, the first Ig C H The 3-domain binds to protein A, and the second IgC... H Domain 3 contains mutations that reduce or eliminate protein A binding, such as H95R modification (via IMGT exon numbering; H435R, via EU numbering). Second C H 3 may also include the Y96F modification (via IMGT number; Y436F, via EU number). See, for example, U.S. Patent No. 8,586,713. This may be included in the second C. HThe additional modifications found in 3 include: in the case of IgG1 antibody, D16E, L18M, N44S, K52N, V57M, and V82I (via IMGT number; D356E, L358M, N384S, K392N, V397M, and V422I, via EU number); in the case of IgG2 antibody, N44S, K52N, and V82I (via IMGT number; N384S, K392N, and V422I, via EU number); and in the case of IgG4 antibody, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (via IMGT number; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I, via EU number).

[0076] In some embodiments, the Fc domain may be chimeric, with combinations derived from Fc sequences of more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise sequences derived from human IgG1, human IgG2, or human IgG4 C. H C in area 2 H 2. Part or all of the sequence, and C derived from human IgG1, human IgG2, or human IgG4. H 3. Part or all of the sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise a combination of an “upper hinge” sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region and a “lower hinge” sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule described herein include, from the N-terminus to the C-terminus: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any antigen-binding molecule described herein comprises, from the N-terminus to the C-terminus: [IgG1 C H [1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any antigen-binding molecule of this disclosure are described in U.S. Patent Publication No. 20140243504, the entire contents of which are incorporated herein by reference. Chimeric Fc domains having these general structural arrangements and their variants may have altered Fc receptor binding, which in turn affects Fc effector function.

[0077] According to certain exemplary embodiments of the present disclosure, a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment comprises a heavy chain variable region (A-HCVR and B-HCVR), a light chain variable region (A-LCVR and B-LCVR), and / or a complementarity-determining region (CDR) comprising any amino acid sequence of a bispecific anti-MUC16 / anti-CD3 antibody as shown in U.S. Patent Publication No. 20180112001. In some exemplary embodiments, a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment that may be used in the context of the methods of this disclosure comprises: (a) a first antigen-binding arm comprising a heavy chain complementarity-determining region (A-HCDR1, A-HCDR2, and A-HCDR3) comprising a heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1 and a light chain complementarity-determining region (A-LCVR) comprising the amino acid sequence of SEQ ID NO: 2; and (b) a second antigen-binding arm comprising a heavy chain CDR (B-HCDR1, B-HCDR2, and B-HCDR3) comprising an HCVR (B-HCVR) containing the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 and a light chain complementarity-determining region (A-LCVR) comprising the amino acid sequence of SEQ ID NO: 2; ... The light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of the LCVR (B-LCVR) with amino acid sequence 2.According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 8; A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 9; A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 10; A-LCDR1 comprises the amino acid sequence of SEQ ID NO: 11; A-LCDR2 comprises the amino acid sequence of SEQ ID NO: 12; A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, or SEQ ID NO: 26; B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, or SEQ ID NO: 27; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 26. The amino acid sequence of SEQ ID NO: 18; and B-LCDR1 contains the amino acid sequence of SEQ ID NO: 11; B-LCDR2 contains the amino acid sequence of SEQ ID NO: 12; B-LCDR3 contains the amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment comprises: (a) a first antigen-binding arm comprising HCVR (A-HCVR) comprising SEQ ID NO: 1 and LCVR (A-LCVR) comprising SEQ ID NO: 2; and (b) a second antigen-binding arm comprising HCVR (B-HCVR) comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 and LCVR (B-LCVR) comprising SEQ ID NO: 2. In some exemplary embodiments, the bispecific anti-CD3×MUC16 antibody comprises a MUC16 binding arm and a CD3 binding arm, wherein the MUC16 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 30, and the CD3 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 30.In some exemplary embodiments, the bispecific anti-CD3×MUC16 antibody comprises a MUC16 binding arm and a CD3 binding arm, wherein the MUC16 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 30, and the CD3 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 30. An exemplary bispecific anti-MUC16 / anti-CD3 antibody that can be used in the methods of this disclosure and comprises A-HCVR of SEQ ID NO: 1, B-HCVR of SEQ ID NO: 3, and LCVR of SEQ ID NO: 2 is REGN4018 (urbatium).

[0078] Other bispecific anti-MUC16 / anti-CD3 antibodies that can be used in the context of the methods of this disclosure include, for example, any of the antibodies described in U.S. Patent Publication No. 20180112001.

[0079] Combination therapy

[0080] According to some embodiments, the methods of this disclosure include administering to a subject of need a combination of an anti-MUC16 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof with an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the methods of this disclosure include administering an antibody (fragment) having additional or synergistic activity to treat cancer, such as endometrial cancer. As used herein, the expression “in combination with” means administering the anti-MUC16 / anti-CD3 bispecific antibody or an antigen-binding fragment thereof before, after, or simultaneously with the anti-PD-1 antibody or an antigen-binding fragment thereof. The term “in combination with” also includes the sequential or concurrent administration of the anti-PD-1 antibody or an antigen-binding fragment thereof and the bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof. For example, when administered "before" the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment may be administered more than 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes prior to administration of the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment. When administered “following” the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment may be administered approximately 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more than 72 hours after administration of the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment. Concurrent administration with the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment means that the anti-PD-1 antibody or its antigen-binding fragment is administered to the subject in a single dosage form within 5 minutes (before, after, or simultaneously) of administration of the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, or as a single combined dose formulation comprising both the anti-PD-1 antibody or its antigen-binding fragment and the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment.

[0081] In some embodiments, the method of this disclosure includes administering a third therapeutic agent, wherein the third therapeutic agent is an anticancer drug. In some embodiments, the method of this disclosure includes administering an additional therapy, wherein the additional therapy is surgery. In some embodiments, the method of this disclosure includes administering a combination of an antiPD-1 antibody or its antigen-binding fragment and an antiMUC16 / antiCD3 bispecific antibody or its antigen-binding fragment with radiotherapy to produce a long-lasting antitumor response and / or prolong the survival of cancer patients.

[0082] In some embodiments, the methods disclosed herein include administering radiotherapy before, simultaneously with, or after administering an anti-PD-1 antibody or its antigen-binding fragment and a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment to a cancer patient. For example, one or more doses of radiotherapy may be administered to the tumor lesion after administering one or more doses of the antibody (or fragment). In some embodiments, after systemic administration of an anti-PD-1 antibody or its antigen-binding fragment and / or a bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, local radiotherapy may be administered to the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablation radiation).

[0083] In any of the various embodiments discussed herein, the methods of this disclosure may also include administration of a steroid (e.g., dexamethasone or an equivalent steroid) or an anti-IL-6 receptor antibody. In some cases, the anti-IL-6 receptor antibody is tocilizumab or thalidomide. In some cases, the steroid (e.g., dexamethasone) may be administered IV or PO at a dose of 1 mg to 20 mg (e.g., 5 mg to 10 mg). In some cases, these agents may be administered as a prodrug prior to the administration of the bispecific antibody.

[0084] Pharmaceutical composition and administration

[0085] This disclosure includes methods comprising administering to a subject a single bispecific anti-MUC16 / anti-CD3 antibody or an antigen-binding fragment thereof, or a combination of said bispecific antibody or an antigen-binding fragment thereof with an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein said one or more antibodies (or fragments) are contained in a single or combined (single) pharmaceutical composition. The pharmaceutical compositions of this disclosure can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and vesicle-containing lipids (cationic or anionic) (such as LIPOFECTIN). TMDNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbon wax emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See, for example, Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998). J Pharm Sci Technol 52:238-311.

[0086] Various delivery systems are known and can be used to administer the pharmaceutical compositions disclosed herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered co-administered with other bioactive agents.

[0087] The disclosed pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the disclosed pharmaceutical compositions. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0088] Many reusable pen-based delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen-type delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), KWIKPEN™ (Eli Lilly), and SURECLICK. TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM Pens (Abbott Labs, Abbott Park IL), to name just a few.

[0089] In some cases, drug compositions can be delivered in controlled-release systems. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the comments of Langer, 1990, Science 249:1527-1533.

[0090] Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying the aforementioned antibody or its salts in a sterile aqueous or oily medium conventionally used for injections. As an aqueous medium for injection, examples include physiological saline, isotonic solutions containing glucose and other excipients, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (an adduct of polyethylene oxide (50 mol) and hydrogenated castor oil)]. As an oily medium, examples include sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The resulting injection is preferably filled into a suitable ampoule.

[0091] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared into unit-dose dosage forms suitable for containing a specific dose of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0092] Application regimen and dosage

[0093] According to certain embodiments of this disclosure, multiple doses of antigen-binding molecules (e.g., bispecific antibodies that specifically bind to MUC16 and CD3, or antigen-binding fragments thereof, or anti-PD-1 antibodies, or antigen-binding fragments thereof) can be administered to a subject over a defined time period. Methods according to this aspect of the disclosure involve sequentially administering multiple doses of the antigen-binding molecules of the disclosure to a subject. As used herein, “sequentially administering” means that each dose of the antigen-binding molecule is administered to the subject at different time points, for example, on different dates spaced apart by predetermined intervals (e.g., hours, days, weeks, or months). The disclosure includes methods comprising sequentially administering a single initial dose of an antigen-binding molecule to a patient, followed by one or more second doses of the antigen-binding molecule, and optionally subsequently administering one or more third doses of the antigen-binding molecule.

[0094] The terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence in which the antigen-binding molecules of this disclosure (e.g., bispecific anti-MUC16×CD3 antibody or anti-PD-1 antibody) are administered. Thus, the “initial dose” is the dose administered at the start of a treatment regimen (also referred to as the “baseline dose”); the “second dose” is the dose administered after the initial dose; and the “third dose” is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of antigen-binding molecules, but may generally differ from each other in terms of frequency of administration. However, in some embodiments, the amounts of antigen-binding molecules contained in the initial, second, and / or third doses during treatment may differ from each other (e.g., appropriately increased or decreased). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the start of a treatment regimen as “starting doses,” followed by subsequent doses (e.g., “maintenance doses”) administered at a lower frequency. In any embodiment, the initial dose (e.g., a first weekly dose) may be divided into two doses administered on separate days (e.g., consecutive days) at intervals not exceeding three days. In any embodiment, the first nominal dose (i.e., the second dose) may be divided into two doses administered on different dates no more than three days apart (e.g., two consecutive days). For example, if the initial dose or the second dose is 6 mg, the dose may be divided into two 3 mg doses, administered, for example, on two consecutive days or on different dates no more than three days apart. In various embodiments, the dosage (e.g., a weekly dose, as a single dose, or as two separate portions of a dose) is or is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 ... mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76mg, 77mg、78 mg、79 mg、80 mg、81 mg、82 mg、83 mg、84 mg、85 mg、86 mg、87 mg、88 mg、89mg、90 mg、91 mg、92 mg、93 mg、94 mg、95 mg、96 mg、97 mg、98 mg、99 mg、100 mg、105 mg、110 mg、115 mg、120 mg、125 mg、130 mg、135 mg、140 mg、145 mg、150 mg、155 mg、160 mg、165 mg、170 mg、175 mg、180 mg、185 mg、190 mg、195 mg、200 mg、205 mg、210 mg、215 mg、220 mg、225 mg、230 mg、235 mg、240 mg、245 mg、250 mg、255 mg、260 mg、265 mg、270 mg、275 mg、280 mg、285 mg、290 mg、295 mg、300 mg、305 mg、310 mg、315 mg、320 mg、325 mg、330 mg、335 mg、340 mg、345 mg、350 mg、355 mg、360 mg、365 mg、370 mg、375 mg、380 mg、385 mg、390 mg、395 mg、400 mg、405 mg、410 mg、415 mg、420 mg、425 mg、430 mg、435 mg、440 mg、445 mg、450 mg、455 mg、460 mg、465 mg、470 mg、475 mg、480 mg、485 mg、490 mg、495 mg、500 mg、510 mg、520 mg、530 mg、540 mg、550 mg、560 mg、570 mg、580 mg、590 mr、600 mg、610 mg、620 mg、630 mg、640 mg、650 mg、660 mg、670 mg、680 mg、690 mg、700 mg、710 mg、720 mg、730 mg、740 mg、750 mg、760 mg、770 mg、780 mg、790 mg、800 mg、810 mg、820 mg、830 mg、840 mg、850 mg、860 mg、870 mg、880 mg、890 mg、900 mg、910 mg、920 mg、930mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, 10 g or more. Any of these amounts can be used to define the range of the initial, second or third dose discussed herein and is covered within the scope of this disclosure. In some embodiments, all doses are administered as a single dose (e.g., a single infusion), including doses administered during the first and second weeks of the dosing regimen. For example, an initial dose of 1 mg to 5 mg may be administered during the first week (as a single dose), a second dose of 3 mg to 400 mg may be administered during the second week (as a single dose), and a third dose of 50 mg to 800 mg may be administered during the third week and thereafter during the weekly dosing portion of the dosing regimen (as a single dose). In another example, an initial dose of 5 mg may be administered in the first week (as a single dose), a second dose of 25 mg may be administered in the second week (as a single dose), and a third dose of 50 mg to 800 mg may be administered in the third week and thereafter during the weekly dosing portion of the dosing regimen (as a single dose). In some cases, the subsequent dosing schedule (e.g., after 12 to 16 weeks) may include administration every two weeks, every three weeks, once a month, etc.

[0095] In one exemplary embodiment of this disclosure, each second and / or third dose is administered 1 to 26 weeks after the previous dose (e.g., 1, 1½, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½ or more). As used herein, the phrase “previous dose” means, in a sequence of multiple administrations, the dose of the antigen-binding molecule administered to the patient before the next dose in the sequence without an intermediate dose.

[0096] The dosage of the antibody (or fragment thereof) administered to a patient may vary depending on the patient's age and physical condition, route of administration, etc. A preferred dosage is typically calculated based on body weight or body surface area. When the antibody (or fragment thereof) of this disclosure is used to treat endometrial cancer in an adult patient, the treatment may include intravenous administration of the antibody of this disclosure in a single dose of about 0.01 to about 100 mg / kg body weight or more. In some cases, the dosage is about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg or more.

[0097] Depending on the severity of endometrial cancer, the dosage, frequency, and / or duration of treatment can be adjusted. Treatment options may be available at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, and 22 weeks. For a duration of 1 month, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, 60 months, or longer, administer 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the disclosed antibody weekly, every two weeks, or every three weeks. The dosing schedule may be modified, for example, from weekly to every two weeks, or vice versa, or otherwise adjusted as needed. Effective doses and schedules for antibody administration can be determined empirically; for example, patient progression can be monitored through regular assessments, and doses adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., Pharmaceut Res 8:1351 (1991)).

[0098] The method according to this aspect of the disclosure may comprise administering any number of second and / or third doses of an antigen-binding molecule (e.g., a bispecific anti-MUC16×CD3 antibody or an anti-PD-1 antibody) to a patient. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0099] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administration of the second and / or third doses to the patient may vary during the treatment regimen. The physician may also adjust the administration frequency during treatment, depending on the individual patient's needs after clinical examination.

[0100] This disclosure includes methods comprising administering to a subject a bispecific anti-MUC16×CD3 antibody or an antigen-binding fragment thereof and / or an anti-PD-1 antibody or an antigen-binding fragment thereof at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, or less (provided a therapeutic response is achieved).

[0101] According to certain embodiments of this disclosure, multiple doses of a single bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, or a combination of said bispecific antibody or its antigen-binding fragment with an anti-PD-1 antibody or its antigen-binding fragment, may be administered to a subject over a defined time period. According to this aspect of the disclosure, the method comprises sequentially administering one or more doses of a single bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, or a combination of said bispecific antibody or its antigen-binding fragment with one or more doses of an anti-PD-1 antibody or its antigen-binding fragment, to a subject in need. As used herein, sequential administration means administering each dose of the antibody or its antigen-binding fragment to the subject at different time points, for example, on different dates separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering a single initial dose of an antibody or its antigen-binding fragment to a patient, followed by administering one or more second doses of the antibody or its antigen-binding fragment, and optionally followed by administering one or more third doses of the antibody or its antigen-binding fragment.

[0102] The amount of bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof and optionally anti-PD-1 antibody or its antigen-binding fragment thereof administered to a subject according to the methods of this disclosure is generally a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means the amount of one or more of the following antibodies or their antigen-binding fragments (anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody) that produce: (a) a reduction in the severity or duration of symptoms of cancer (e.g., endometrial cancer); (b) inhibition of tumor growth, or increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay in tumor growth and development; (d) inhibition, delay, or cessation of tumor metastasis; (e) prevention of tumor growth recurrence; (f) increased survival in a subject with cancer (e.g., endometrial cancer); and / or (g) a reduction or need for the use of conventional anticancer therapy (e.g., reduction or elimination of the use of chemotherapeutic agents or cytotoxic agents) compared to a treatment-naïve subject or a subject administered either antibody (or fragment) as monotherapy.

[0103] In the case of bispecific anti-MUC16 / anti-CD3 antibodies or their antigen-binding fragments, the therapeutically effective dose can be from about 0.1 mg to about 1000 mg, for example, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 3 mg, about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, etc. Bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment, in doses of approximately 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. In some cases, the dose is approximately 1 mg. In some cases, the dose is approximately 2 mg. In some cases, the dose is approximately 20 mg. In some cases, the dose is approximately 25 mg. In some cases, the dose is approximately 250 mg. In some cases, the dose is approximately 800 mg. Any of these doses may be an initial dose, an intermediate or transitional dose, or a full dose.

[0104] In some cases, bispecific anti-MUC16 / anti-CD3 antibodies are administered at a dose of at least 1 mg or 1.5 mg or 2.0 mg or 2.5 mg or 3.0 mg or 3.5 mg or 4 mg or 5 mg or 6 mg or 7 mg or 8 mg or 9 mg or 10 mg or 15 mg or 20 mg or 25 mg or 30 mg or 35 mg or 40 mg or 45 mg or 50 mg or 55 mg or 60 mg or 65 mg or 70 mg or 70 mg or 70 mg or 8 mg or 9 mg or 10 mg or 15 mg or 20 mg or 25 mg or 30 mg or 35 mg or 40 mg or 45 mg or 50 mg or 55 mg or 60 mg or 65 mg or 70 mg or 70 mg or 80 mg or 65 mg or 70 mg or 80 mg or 70 mg or 80 mg or 70 mg or 80 mg or 70 mg or 80 mg or 70 mg or 80 mg or 80 mg or 70 mg or 80 mg or 80 mg or 80 mg or 70 mg or 80 mg or 80 mg or 80 mg or 80 mg or 85 ... The following doses were administered to the subject: 1 mg, at least 75 mg per week or every two weeks, at least 80 mg per week or every two weeks, at least 85 mg per week or every two weeks, at least 90 mg per week or every two weeks, at least 95 mg per week or every two weeks, at least 100 mg per week or every two weeks, at least 150 mg per week or every two weeks, at least 200 mg per week or every two weeks, at least 250 mg per week or every two weeks, at least 300 mg per week or every two weeks, at least 350 mg per week or every two weeks, at least 400 mg per week or every two weeks, at least 450 mg per week or every two weeks, at least 500 mg per week or every two weeks, at least 550 mg per week or every two weeks, at least 600 mg per week or every two weeks, at least 650 mg per week or every two weeks, at least 700 mg per week or every two weeks, at least 750 mg per week or every two weeks, at least 800 mg per week or every two weeks, at least 850 mg per week or every two weeks, or at least 900 mg per week or every two weeks.

[0105] In the case of anti-PD-1 antibodies or their antigen-binding fragments, the effective therapeutic dose can be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, etc. Anti-PD-1 antibody or its antigen-binding fragment, in doses of approximately 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, or 600 mg. In some embodiments, 350 mg of anti-PD-1 antibody or its antigen-binding fragment is administered.

[0106] In some cases, anti-PD-1 antibodies are administered at a dose of at least 1 mg or every two weeks, at least 1.5 mg or every two weeks, at least 2.0 mg or every two weeks, at least 2.5 mg or every two weeks, at least 3.0 mg or every two weeks, at least 3.5 mg or every two weeks, at least 4 mg or every two weeks, at least 5 mg or every two weeks, at least 6 mg or every two weeks, at least 7 mg or every two weeks, at least 8 mg or every two weeks, at least 9 mg or every two weeks, at least 10 mg or every two weeks, at least 15 mg or every two weeks, at least 20 mg or every two weeks, at least 25 mg or every two weeks, at least 30 mg or every two weeks, at least 35 mg or every two weeks, at least 40 mg or every two weeks, at least 45 mg or every two weeks, at least 50 mg or every two weeks, at least 55 mg or every two weeks, at least 60 mg or every two weeks, at least 65 mg or every two weeks, at least 70 mg or every two weeks, at least 75 mg or every two weeks. The following doses were administered to the subject: at least 80 mg per week or every two weeks, at least 85 mg per week or every two weeks, at least 90 mg per week or every two weeks, at least 95 mg per week or every two weeks, at least 100 mg per week or every two weeks, at least 150 mg per week or every two weeks, at least 200 mg per week or every two weeks, at least 250 mg per week or every two weeks, at least 300 mg per week or every two weeks, at least 350 mg per week or every two weeks, at least 400 mg per week or every two weeks, at least 450 mg per week or every two weeks, at least 500 mg per week or every two weeks, at least 550 mg per week or every two weeks, at least 600 mg per week or every two weeks, at least 650 mg per week or every two weeks, at least 700 mg per week or every two weeks, at least 750 mg per week or every two weeks, at least 800 mg per week or every two weeks, at least 850 mg per week or every two weeks, or at least 900 mg per week or every two weeks.

[0107] In any of the embodiments of the dosing regimen, the initial dose of the bispecific anti-MUC16 / anti-CD3 antibody and / or anti-PD-1 antibody is 1 mg to 5 mg. In any of the embodiments of the dosing regimen, the second dose is 3 mg to 400 mg. In any of the embodiments of the dosing regimen, the third dose is 3 mg to 800 mg. In some embodiments, the initial dose is 5 mg, the second dose is 25 mg, and the third dose is 50 mg to 800 mg. In some cases, the dosing regimen includes weekly administration of the third dose during the weekly dosing period of the dosing regimen for at least 12 weeks (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or longer). In some cases, the dosing regimen also includes administration of the third dose every two weeks during every two-week dosing period of the dosing regimen following the weekly cycle of the dosing regimen. In some cases, the dosing regimen also includes administration of the third dose every three weeks or every four weeks.In various embodiments, the dosage may be at least 1 mg per week or every two weeks, at least 1.5 mg per week or every two weeks, at least 2.0 mg per week or every two weeks, at least 2.5 mg per week or every two weeks, at least 3.0 mg per week or every two weeks, at least 3.5 mg per week or every two weeks, at least 4 mg per week or every two weeks, at least 5 mg per week or every two weeks, at least 6 mg per week or every two weeks, at least 7 mg per week or every two weeks, at least 8 mg per week or every two weeks, at least 9 mg per week or every two weeks, at least 10 mg per week or every two weeks, at least 15 mg per week or every two weeks, at least 20 mg per week or every two weeks, at least 25 mg per week or every two weeks, at least 30 mg per week or every two weeks, at least 35 mg per week or every two weeks, at least 40 mg per week or every two weeks, at least 45 mg per week or every two weeks, at least 50 mg per week or every two weeks, at least 55 mg per week or every two weeks, at least 60 mg per week or every two weeks, at least 65 mg per week or every two weeks, at least 70 mg per week or every two weeks, at least 75 mg per week or every two weeks, at least 80 mg per week or every two weeks. mg, at least 85 mg per week or every two weeks, at least 90 mg per week or every two weeks, at least 95 mg per week or every two weeks, at least 100 mg per week or every two weeks, at least 150 mg per week or every two weeks, at least 200 mg per week or every two weeks, at least 250 mg per week or every two weeks, at least 300 mg per week or every two weeks, at least 350 mg per week or every two weeks, at least 400 mg per week or every two weeks, at least 450 mg per week or every two weeks, at least 500 mg per week or every two weeks, at least 550 mg per week or every two weeks, at least 600 mg per week or every two weeks, at least 650 mg per week or every two weeks, at least 700 mg per week or every two weeks, at least 750 mg per week or every two weeks, at least 800 mg per week or every two weeks, at least 850 mg per week or every two weeks, or at least 900 mg per week or every two weeks.

[0108] The amount of bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof and optionally anti-PD-1 antibody or its antigen-binding fragment contained in an individual dose can be expressed in milligrams of antibody or its antigen-binding fragment per kilogram of subject body weight (i.e., mg / kg). In some embodiments, the bispecific anti-MUC16 / anti-CD3 antibody or its antigen-binding fragment thereof and optionally anti-PD-1 antibody or its antigen-binding fragment thereof used in the methods of this disclosure can be administered to the subject at a dose of about 0.0001 to about 100 mg / kg of subject body weight. For example, bispecific anti-MUC16 / anti-CD3 antibodies or their antigen-binding fragments can be available in dosages of approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.3 mg / kg, approximately 0.4 mg / kg, approximately 0.5 mg / kg, approximately 0.6 mg / kg, approximately 0.7 mg / kg, approximately 0.8 mg / kg, approximately 0.9 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, and approximately 20 mg / kg. The anti-PD-1 antibody or its antigen-binding fragment may be administered at a dose of approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.3 mg / kg, approximately 0.4 mg / kg, approximately 0.5 mg / kg, approximately 0.6 mg / kg, approximately 0.7 mg / kg, approximately 0.8 mg / kg, approximately 0.9 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, to approximately 20 mg / kg of the patient's body weight.

[0109] Table 1 below provides an overview of the sequences and their corresponding SEQ ID NOs cited in this article.

[0110] Table 1: Sequence Overview

[0111] Example

[0112] The following examples are provided to provide a complete disclosure and description of how to prepare and use the methods and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their disclosure. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0113] Example 1: Generation of bispecific antibodies binding to MUC16 and CD3

[0114] This disclosure provides a method of use of a bispecific antigen-binding molecule comprising binding to CD3 and MUC16; such bispecific antigen-binding molecules are also referred to herein as “anti-MUC16 / anti-CD3 or anti-MUC16×CD3 bispecific molecules.” The anti-MUC16 portion of the anti-MUC16 / anti-CD3 bispecific molecule can be used to target tumor cells expressing MUC16 (also referred to as CA-125), and the anti-CD3 portion of the bispecific molecule can be used to activate T cells. In some preferred embodiments, the bispecific antigen-binding molecule is a bispecific antibody. The simultaneous binding of MUC16 to CD3 on both tumor cells and T cells promotes targeted killing (cell lysis) of target tumor cells by activated T cells.

[0115] Bispecific antibodies comprising an anti-MUC16-specific binding domain and an anti-CD3-specific binding domain are constructed using standard methods, wherein each of the anti-MUC16 antigen-binding domain and the anti-CD3 antigen-binding domain contains a different unique HCVR that pairs with a common LCVR. In the illustrated bispecific antibody, the molecule is constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a common light chain from an anti-MUC16 antibody. In other cases, bispecific antibodies can be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody, or known antibody light chains that are mixed with or paired with various heavy chain arms.

[0116] As described in U.S. Patent Application Publication No. US20140243504A1, published August 28, 2014, illustrative bispecific antibodies having IgG1 Fc domains (BSMUC16 / CD3-001, -002, -003, and -004) or modified (chimeric) IgG4 Fc domains (BSMUC16 / CD3-005) have been prepared. An illustrative bispecific antibody containing BSMUC16 / CD3-001 for VR and CDR is REGN4018 (urbatium). REGN4018 comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 29, a second heavy chain containing the amino acid sequence of SEQ ID NO: 31, and a common light chain containing the amino acid sequence of SEQ ID NO: 30.

[0117] An overview of the components of the antigen-binding domains of the various anti-MUC16×CD3 bispecific antibodies constructed is shown in Table 2.

[0118] Table 2: Overview of the components of the anti-MUC16×CD3 bispecific antibody

[0119] Example 2: ImmunoPET imaging in engineered mice showed that anti-MUC16×CD3 bispecific antibodies were localized in T cell-rich organs.

[0120] The in vivo localization of BSMUC16 / CD3-001 and BSMUC16 / CD3-005 and the expression of MUC16 protein were evaluated using PET imaging in wild-type and genetically humanized mice. 89 The biodistribution of Zr-labeled anti-MUC16 antibodies (bivalent anti-MUC16 antibodies produced using the same anti-MUC16 heavy and light chains as bispecific antibodies, referred to herein as "parental") was similar in both wild-type and humanized mice, indicating low expression / availability of the humanized MUC16 protein for the antibody. In contrast, when mice were administered treatment-related doses of the antibody... 89 When Zr-labeled BSMUC16 / CD3-001 bispecific antibody was applied, the distribution in the spleen and lymph nodes was significant, attributed to recognition of CD3-positive T cells in these lymphoid organs (data not shown). In vitro biodistribution analysis of individual tissues confirmed antibody localization in lymph nodes and spleen (data not shown). Compared to BSMUC16 / CD3-001, the distribution in lymphoid tissues via… 89The uptake of Zr-labeled BSMUC16 / CD3-005 bispecific antibody was significantly reduced due to its low affinity for CD3. To assess whether BSMUC16 / CD3-001 and BSMUC16 / CD3-005 could accumulate in tumors expressing MUC16, mice carrying ID8-VEGF-huMUC16Δ tumors were administered [a specific treatment / treatment]. 89 Zr-marked BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005. Although BSMUC16 / CD3-001 showed high lymphoid uptake, there was no significant difference in tumor uptake between the bispecific antibodies (data not shown).

[0121] Preparation of immune conjugates and small animal PET: After deglycosylation of the antibody with peptidyl-N-glycosidase F (PNGase F), BSMUC16 / CD3-001 and the control antibody were transamided by microbial glutamine transaminase to bind DFO to the glutamine residue at position 295. Subsequently, the antibody binding to DFO was combined with zirconium-89 (… 89 Zr chelation. Mice received a final dose of 0.5 mg / kg of antibody via tail vein injection. PET imaging was then performed to assess the in vivo localization of the radioimmunoconjugate on day 6 post-administration, prior to in vitro biodistribution studies. For experiments in tumor-bearing mice, 10 × 10⁶ ions were subcutaneously implanted. 6 Individual ID8-VEGF-huMUC16Δ tumor cells. When the average tumor volume is 150 mm. 3 At that time, 20 days after implantation, tumor-bearing mice were administered drugs via... 89 Zr radiolabeled antibodies.

[0122] PET and CT images were acquired using a pre-calibrated Sofie Biosciences G8 PET / CT instrument (Sofie Biosciences (Culvercity, CA) and Perkin Elmer). The energy window ranged from 150 to 650 keV, with a reconstruction resolution of 1.4 mm at the center of the field of view. Mice were induced anesthetized with isoflurane on day 6 post-administration and maintained inhalation of isoflurane during a 10-minute static PET acquisition. CT images were acquired after the PET acquisition. The PET images were then reconstructed using pre-configured settings. Decay-corrected PET and CT data were processed into a pseudo-color co-registered PET-CT maximum intensity projection on a color scale calibrated to indicate the signal range of 0 to 30% per volume of the injected dose, expressed as %ID / g, using VivoQuant software (inviCRO Imaging Services). For in vitro biodistribution analysis, mice were euthanized after imaging on day 6 post-administration. Blood was collected into counting tubes via cardiac puncture. Normal tissue (groin and axillary lymph nodes, thymus, spleen, heart, lungs, stomach, small intestine, liver, kidneys, bones, and ovaries) was then removed and placed in counting tubes. Tumor tissue was similarly collected into counting tubes. All tubes were pre-weighed and then weighed again to determine the weight of blood and tissue. The gamma emission radioactivity of all samples was then counted on an automated gamma counter (Wizard 2470, Perkin Elmer), and the results were reported in counts per minute (cpm). The %ID of each sample was determined using the sample count relative to a dose standard count prepared from the original injection material. Subsequently, individual %ID / g values ​​were obtained by dividing the %ID value by the appropriate weight of the blood, tissue, or tumor sample.

[0123] through 89 Zr-marked BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005 exhibited specific localization in MUC16+ tumors and CD3+ lymphoid tissues, with lymphoid distribution correlated with relative CD3 affinity. Both MUC16×CD3 bispecific antibodies showed comparable tumor localization in the presence of CD3+ tissues.

[0124] Example 3: Toxicological studies in cynomolgus monkeys showed that the anti-MUC16×CD3 bispecific antibody had no significant toxicity.

[0125] BSMUC16 / CD3-001 cross-reacts with monkey MUC16 and CD3. To determine the safety and tolerability of the bispecific antibody and to characterize its pharmacokinetics, a multi-dose toxicity study was conducted in cynomolgus monkeys. Six monkeys / sex / groups received weekly administrations of BSMUC16 / CD3-001 at doses of 0.01, 0.1, or 1 mg / kg for a total of five doses. At the end of the dosing period, three animals / sex / groups were euthanized and their tissues examined for microscopic findings, while the remaining three animals / sex / groups underwent a 12-week treatment-free recovery period to assess the reversibility or persistence of any effects associated with BSMUC16 / CD3-001. BSMUC16 / CD3-001 was well tolerated, and all animals survived to the scheduled time for necropsy. Toxoplasmic analysis demonstrated dose-proportionate exposure and linear kinetics between the dose groups, with no sex differences observed (data not shown). Continuous exposure to BSMUC16 / CD3-001 was observed throughout the dosing period, and in 50% of animals in all (n=6) and in the 0.1 and 1.0 mg / kg groups, exposure persisted until the end of the recovery phase. Following the 8-week recovery period, BSMUC16 / CD3-001 was undetectable in the serum of any animal in the 0.01 mg / kg group. The elimination half-life of BSMUC16 / CD3-001 is approximately 10 days.

[0126] During administration or recovery, no clinical observations related to BSMUC16 / CD3-001 were observed; no changes were made to urinalysis parameters, peripheral blood immunophenotype, food consumption, or body weight. Importantly, BSMUC16 / CD3-001 administration did not result in any changes to respiratory, neurological, or cardiovascular safety pharmacological assessments, including no changes to ECG parameters. No BSMUC16 / CD3-001-related organ weight changes were observed, and no macroscopic changes were noted at terminal or post-mortem examinations. Dose-related and reversible increases in circulating inflammatory markers (C-reactive protein (CRP) and IL-6) were observed within 1 day following an initial dose of 1.0 or 0.1 mg / kg, but these increases were not significant after subsequent doses (data not shown). Consistent with minimal increases in serum cytokines, no T-cell redistribution was detected after BSMUC16 / CD3-001 administration (data not shown), contrary to descriptions of several CD3 bispecific molecules for hematologic malignancies.

[0127] The cynomolgus monkey study was conducted according to IACUC guidelines. Cynomolgus monkeys (6 animals / sex / group) were administered either a control product (diluted placebo) or BSMUC16 / CD3-001 (0.01, 0.1, or 1 mg / kg) weekly via 30-minute IV infusion. The control product was 10 mM histidine containing 10% sucrose and 0.05% polysorbate 20, pH 6, diluted with 0.9% sodium chloride for injection (USP, sterile saline). Blood or tissue samples were collected at different time points for clinicopathology and histopathology. BSMUC16 / CD3-001 concentrations were determined by ELISA, and toxicokinetics were performed using WinNonLin software. CRP was analyzed on a Roche Modular P 800 system. Cytokines were measured by MSD (Meso Scale Diagnostics, Rockville, MD). T cell counts were quantified using flow cytometry. In simple terms, blood is collected in EDTA potassium tubes, lysed, stained for CD3, CD4, and CD8 (BD Biosciences), and the relative values ​​for each phenotype are determined using FACS Canto II. These values ​​are then multiplied by the absolute lymphocyte count (via hematological analysis) to count the absolute cell count for each phenotype.

[0128] Immunohistochemical staining for MUC16 was observed in the expected tissues: pancreas (mesothelial, ductal epithelium), heart and ovary (data not shown), salivary glands (goblet cells), liver (mesothelial, bile ducts), lungs (mesothelial, bronchiolar / bronchial epithelium), small intestine (mesothelial), testes (mesothelial, reticulum testis / vas deferens), and tonsils (epithelium, mucous glands) (not shown). BSMUC16 / CD3-001-related microscopic changes assessed by hematoxylin and eosin (H&E) histological staining included inflammation (leukocyte infiltration) and increased mesothelial cell size and cellular structure, resulting in non-adverse thickening of the serosal layer and / or submesothelial connective tissue in multiple thoracic and peritoneal organs. These changes were generally focal or multifocal in nature and minimal to mild in severity, and were considered to be targeting BSMUC16 / CD3-001, induced by conjugation of MUC16 expressed on serosal epithelial (mesothelial) cells and activation of T cells. Importantly, the changes in the serous membrane had reversed or were showing a reversal trend by the end of the recovery period (data not shown).

[0129] Toxicological studies in cynomolgus monkeys showed that after administration of BSMUC16 / CD3-001, there was a minimal and transient increase in serum cytokine and C-reactive protein levels, with no significant toxicity.

[0130] Example 4: Effects of MUC16 expression and anti-MUC16×CD3 bispecific antibody on MUC16-positive tissues in humanized mice

[0131] To investigate the antitumor efficacy of BSMUC16 / CD3-001 in mice with a fully intact immune system, mice were genetically engineered to express human CD3 on T cells and the MUC16 region covering the antibody-binding region at an endogenous murine locus (knock-in mice). To validate these mice, MUC16 expression was examined by both RT-PCR and IHC. RNA expression was detected in the trachea, and low levels were detected in the lungs, heart, ovaries, pancreas, and bladder (data not shown), consistent with publicly available data on murine MUC16 expression. To assess MUC16 protein expression, selected tissues were subjected to IHC using an anti-human MUC16 antibody that recognizes the proximal membrane region of MUC16. MUC16 protein expression was confirmed in the surface epithelium of the ovaries and stomach of these mice. MUC16 was also observed in the tracheal lining / epithelium and submucosal glands, consistent with descriptions in human studies (data not shown).

[0132] Histology of mouse tissueTissues were collected from humanized or WT mice and stained with anti-MUC16 antibody binding to the proximal membrane domain of MUC16 via IHC using Ventana DiscoveryXT (Ventana; Tucson, AZ). 5 μm paraffin sections were cut, placed on Superfrost PLUS slides, and baked at 60°C for one hour. Immunohistochemical staining was performed on the Discovery XT automated IHC staining system using the Ventana DAB Map Detection Kit. Dewaxing was performed at 75°C for 8 minutes using EZPrep solution. Mild antigen retrieval was performed using Tris-EDTA buffer pH 9 (CC1) from Ventana (95°C, 8 minutes, then 100°C, 24 minutes). This was followed by multiple blocking steps. Tissue sections were incubated at room temperature for 8 hours with anti-MUC16 antibody (2 μg / ml). An isotype control antibody recognizing an irrelevant non-binding antibody was used as a negative control. Primary antibody and negative control were manually applied. Biotinylated goat anti-human IgG (Jackson ImmunoResearch) was used as a secondary antibody (1 μg / ml), and samples were incubated at room temperature for one hour. Colorimetric signals were developed using the Ventana DAB MAP kit. Slides were manually counterstained with hematoxylin (2 min), dehydrated, and covered with coverslips. Images were acquired on an Aperio AT 2 slide scanner (Leica Biosystems; Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs; Corrales, NM). H&E staining was performed by Histoserv (Germantown, MD, USA).

[0133] As assessed by T-cell receptor (TCR) Vβ gene accession, T cells in these mice were polyclonal, expressed human CD3, and present in numbers similar to those in wild-type mice (data not shown). To determine whether BSMUC16 / CD3-001 induced any T-cell activation or had an effect on normal tissues in these animals, non-tumor-bearing mice were injected with a high dose of BSMUC16 / CD3-001 (10 mg / kg), and blood T-cell counts, serum cytokines, and histopathology were subsequently examined. Measured by peripheral migration of T cells in the blood and increased serum cytokine levels (data not shown), BSMUC16 / CD3-001 did not induce any such effects, although T cells can be activated by an anti-human CD3 antibody (OKT3), suggesting limited accessibility to the MUC16 target (data not shown). To determine whether BSMUC16 / CD3-001 induced any microscopic changes in MUC16-expressing tissues, MUC16 and CD3 humanized mice were administered BSMUC16 / CD3-001 at two doses of 10 mg / kg on days 0 and 3. On day 5, several MUC16-expressing tissues (trachea, stomach, and ovary) were examined, and no cell infiltration or necrosis was observed in these tissues after BSMUC16 / CD3-001 administration (data not shown).

[0134] Histopathological examination revealed that, at the time of examination, no inflammation or infiltration was observed in the MUC16-expressing tissues of mice after administration of BSMUC16 / CD3-001.

[0135] This study, along with the cynomolgus monkey study discussed in Example 5, demonstrates the safety profile of BSMUC16 / CD3-001. BSMUC16 / CD3-001 induced minimal serum cytokines, and although focal induction of inflammation and thickening of the serous membrane lining were observed in MUC16 expression (indicating targeting activity), these effects subsided by the end of the recovery period and were consistent with results indicating repaired inflammation and increased cellular structure. The observed serous membrane changes were independent of any clinical observations, clinicopathological findings (excluding inflammatory responses), or microscopic changes in the underlying parenchyma. Therefore, studies in genetically humanized mice and cynomolgus monkeys demonstrate the good tolerability of BSMUC16 / CD3-001.

[0136] Example 5: Treatment of endometrial cancer with a single anti-MUC16×anti-CD3 bispecific antibody or a combination thereof with an anti-PD-1 antibody.

[0137] A phase 1 / 2 study is underway to investigate the safety, pharmacokinetics (PK), and preliminary antitumor activity of REGN4018 (anti-MUC16 × anti-CD3 bispecific antibody; urbutumab alone) or its combination with cimiprimab (anti-PD-1 antibody) in patients with advanced endometrial cancer. Selected patients have previously received anti-PD1 therapy and platinum-based therapy, and their tumors are confirmed to express MUC16.

[0138] Patients with advanced endometrial cancer that has relapsed after 1-2 lines of prior therapy (including prior anti-PD-1 therapy and prior platinum-based chemotherapy) will be included. Patients with tumors that may express MUC16 will also be included. Only patients with elevated serum CA-125 levels (≥2×ULN) will be included, as these levels may reflect the presence of MUC16 on tumor cells.

[0139] Target

[0140] Main objectives The primary objective is to evaluate the initial efficacy of urbutuzumab as monotherapy or in combination with cimiprimab for the treatment of endometrial cancer. During the dose escalation phase, the primary objective is to assess safety and pharmacokinetics (PK) to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of REGN4018 as monotherapy and in combination with cimiprimab. During the dose extension phase, the primary objective is to evaluate the initial efficacy of REGN4018, as determined by the objective response rate (ORR) according to the Responsive Clinical Trial of Solid Tumors (RECIST) 1.1.

[0141] Secondary objectivesThis includes: (1) assessing the preliminary efficacy of REGN4018 as monotherapy and in combination with cimiprimab during the dose escalation phase, as determined according to RECIST 1.1; (2) characterizing the safety profile in each expansion cohort during the dose extension phase, characterizing the pharmacokinetic (PK) profile of REGN4018 as monotherapy and in combination with cimiprimab, and assessing the impact of REGN4018 as monotherapy and in combination with cimiprimab on patient-reported outcomes (PROs), including health-related quality of life (HRQoL), function, and symptoms; and (3) assessing the preliminary efficacy of REGN4018 as monotherapy and in combination with cimiprimab (separately by cohort) during both the dose escalation and dose extension phases, as measured by ORR based on RECIST (iRESIST), based on RECIST. 1.1 The best overall response (BOR), duration of response (DOR), disease control rate, CR rate, and progression-free survival (PFS) of REGN4018 as monotherapy and in combination with cimiprimab were evaluated, as measured by CA-125 levels, and the immunogenicity of REGN4018 and cimiprimab were assessed.

[0142] Research Exploratory goals yes: (1) Evaluate the preliminary efficacy of REGN4018 as monotherapy and in combination with cimiprimab (separately by group), such as by measuring ORR using the combined assessment of RECIST and CA-125 using the Gynecologic Cancer International Group (GCIG) criteria; (2) Evaluate biomarkers that may be related to the mechanism of action, a deeper understanding of the disease / target, observed toxicity, and potential antitumor activity, including but not limited to: - Circulating proteins - Circulating immune cells - Changes in gene expression in peripheral blood and tumors - Expression levels of proteins such as MUC16 and programmed cell death ligand 1 (PD-L1) in tumors (3) Assess tumor mutation burden and circulating tumor DNA; (4) Where possible, assess the relationship between exposure and efficacy and safety endpoints; and (5) Total lifespan (OS).

[0143] Research Design

[0144] Patients with histologically confirmed endometrial cancer (e.g., ≥25% MUC16+ tumor cells as assessed by immunohistochemistry [IHC]) that has progressed or recurred after prior anti-PD-1 therapy (≥60 days since the last administration) and prior platinum-based chemotherapy will be recruited. The first 20 patients recruited in the endometrial cohort will receive urbutuzumab 250 mg IV Q3W after an initial escalation dose (1 mg in week 1 and 20 mg in week 2, which may be in fractionated doses). If there are fewer than three objective responses, another 20 patients may be recruited to receive urbutuzumab 800 mg IV Q3W. If evidence of monotherapy activity is demonstrated in the first 20 patients, an additional treatment group may be opened, receiving urbutuzumab 250 mg IV Q3W + cimiprimab 250 mg or 350 mg IV Q3W, concurrent with the monotherapy cohort. To limit the risk of cytokine release syndrome, all arms will include escalating weekly dosing of urbutanol prior to Q3W dosing. The cohorts will use the Simon Phase 2 study design, with interim analyses following the first 20 patients. Any treatment arm with ≥3 objective responses will be expanded to 50 patients.

[0145] The primary endpoint will be the objective response rate (RECIST v1.1) for each arm. Secondary endpoints include assessing duration of response and progression-free survival, safety, pharmacokinetic (PK), and changes in quality of life and physical function compared to baseline. Exploratory endpoints include assessing baseline tumor MUC16 IHC expression and other biomarkers as predictors of response.

[0146] In addition, dosing will be evaluated in a 3-arm randomized phase 2 cohort that evaluates three doses: REGN4018 250 mg IV Q3W, REGN4018 800 mg IV Q3W as monotherapy, and REGN4018 250 mg (or the highest tolerable dose in combination with cimiprimab if 250 mg QW is not tolerated) IV Q3W in combination with cimiprimab 350 mg IV Q3W.

[0147] Study Duration: All REGN4018 monotherapy cycles will be approximately 6 weeks (42 days). In combination therapy, cycle 1 will be approximately 28 or 35 days, depending on when patients can tolerate the full dose of REGN4018 without CRS. In the randomized phase 2 cohort, cycle 1 in all arms will be approximately 42–49 days, depending on when patients can tolerate the full dose of REGN4018 without CRS. Subsequent cycles in all cohorts will be approximately 6 weeks (42 days). Treatment will continue until disease progression, intolerable adverse events, withdrawal of consent, or other criteria for treatment discontinuation. For patients who do not withdraw consent, follow-up after treatment will be approximately 90 or 168 days, depending on the reason for treatment discontinuation.

[0148] Study cohort: Up to approximately 100 patients with advanced endometrial cancer will be recruited. The initial cohort will evaluate the efficacy of REGN4018 250 mg IV every 3 weeks following an initial escalation dose. The treatment regimen will be the same as the first arm of the randomized phase 2 cohort. This cohort will follow the Simon phase 2 design. In phase 1, 20 patients will be recruited. An additional 30 phase 2 patients will be recruited only if 3 or more patients in Simon phase 1 have an unconfirmed partial response or better that meets RECIST 1.1 criteria. The study will recruit patients with recurrent or advanced endometrial cancer that has progressed or recurred after prior anti-PD-1 therapy and platinum-based chemotherapy.

[0149] If there is evidence of monotherapy activity (defined as 3 or more patients having an unconfirmed partial response or better that meets RECIST 1.1 criteria) among the first 20 patients treated with REGN4018 250 mg IV Q3 weeks, an additional Simon Phase 2 cohort (the third arm of the randomized Phase 2 cohort) of REGN4018 250 mg IV in combination with cimiprimab 350 mg IV Q3 weeks can be initiated in parallel with the monotherapy cohort.

[0150] Inclusion criteria -Patients must meet the following criteria to be eligible for inclusion in the study: 1. Women aged 18 and over 2. Willing and able to comply with clinical visit and research procedures. Note: Patients must provide newly obtained biopsy (unless medically inappropriate and discussed with a medical monitor, in which case a newly obtained biopsy must be provided at screening) or archived tumor tissue.

[0151] 3. Expanded group only: There must be imaging records showing disease progression after previous therapy and there must be at least one measurable lesion (not previously treated with radiotherapy) that can be accurately measured by the Responsive Evaluation Criteria in Solid Tumors (RECIST).

[0152] 4. Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1.

[0153] 5. Organ and bone marrow functions are normal, as detailed below: a. Hemoglobin ≥ 8.5 g / dL b. Absolute neutrophil count ≥1.5×10 9 / L c. Platelet count ≥75×10 9 / L d. Serum creatinine ≤1.5×ULN, or estimated glomerular filtration rate >50 mL / min / 1.73m 2 (Dose escalation group), or estimated glomerular filtration rate >30 mL / min / 1.73m 2 (Dose extension group) e. Total bilirubin ≤ 1.5 × ULN f. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤3×ULN, or ≤5×ULN if liver cancer metastasis is present. g. Alkaline phosphatase ≤2.5×ULN (or ≤5.0×ULN if liver or bone cancer metastases are present) 6. Life expectancy is at least 3 months.

[0154] 7. Provide informed consent forms signed by the study patient or their legal representative.

[0155] 8. Histologically confirmed endometrial cancer that has progressed or recurred after prior anti-PD1 therapy and platinum-based chemotherapy.

[0156] a. MUC16 positivity in tumor cells is confirmed by immunohistochemical (IHC) assay of ≥25% MUC16. This assay will be performed at a Ventana Clinical Laboratory Improvement Amendments-certified and College of American Pathologists-accredited laboratory in Tucson, Arizona, USA. Samples may be biopsy samples obtained during screening or archived tissue blocks from within the past two years.

[0157] b. Previous treatment with anti-PD-1 therapy and platinum-based chemotherapy

[0158] c. Previous treatment with 1-2 lines of systemic therapy, excluding systemic adjuvant therapy administered >12 months prior to the first treatment in cases of relapse.

[0159] d. 60 days have passed since the last administration of anti-PD-1 (approximately 3 half-lives).

[0160] Exclusion criteria - Patients meeting any of the following criteria will be excluded from the study: 1. Currently receiving treatment in another therapeutic study or already participating in and receiving treatment with the investigational agent, or using the investigational device within 4 weeks of the first dose of the investigational therapy, or receiving approved systemic therapy within 3 weeks of the first dose of the investigational therapy, or receiving any prior systemic therapy within 5 half-lives of the first dose of the investigational therapy (whichever is longer). Patients who have already received or participated in studies involving treatment with the minimum dose of the investigational immuno-PET reagent are not excluded. Patients who have previously received bevacizumab treatment, without a history of bevacizumab-related intestinal perforation or wound complications, and whose last dose was >30 days after the first REGN4018 dose, are eligible for enrollment after discussion with the sponsor; in addition, patients who have used other non-investigational, non-immunomodulatory antibodies with a half-life of more than 7 days, and whose last treatment was at least 3 half-lives, are also eligible for enrollment after discussion with the sponsor.

[0161] 2. Previous anti-cancer immunotherapies are as follows: a. Those who have received anti-PD-1 / PD-L1 therapy within 60 days of the first dose (approximately 3 half-lives). b. Received CAR-T cell therapy within 30 days of the first dose of the investigational drug. 3. Previously treated with therapies targeting MUC16.

[0162] 4. Expanded Group Only: Other malignant tumors that are progressive or require active treatment, except for non-melanoma skin cancer, cervical cancer in situ, or any other tumors that have been effectively treated with definite local control (whether or not adjuvant hormone therapy has been continued) for at least 2 years prior to recruitment.

[0163] 5. Patients who have received corticosteroid treatment (>10 mg prednisone / day or equivalent) within 1 week prior to their first dose of the study drug. Patients requiring only a short course of corticosteroid treatment (maximum 2 days within the week prior to recruitment) will not be excluded.

[0164] 6. Having treatment-related immune-mediated adverse events caused by immunomodulatory agents (including but not limited to anti-PD-1 / PD-L1 or anti-CTLA-4 monoclonal antibodies or PI3Kδ inhibitors) that have not recovered to baseline levels for at least 30 days prior to starting the study therapy.

[0165] Note: Endocrine-immune dysregulation controlled (but not completely resolved) by hormones or other non-immunosuppressive therapies. An event, or a Grade 1 imAE that affects any organ system and has subsided prior to recruitment, is eligible for enrollment.

[0166] 7. Other malignant tumors that are progressive or require active treatment, except for non-melanoma skin cancer, cervical cancer in situ, or any other tumors that have been effectively treated with definite local control (whether or not adjuvant hormone therapy has been continued) for at least 2 years prior to recruitment.

[0167] 8. Untreated or active primary brain tumors, CNS metastases, or spinal cord compression. Patients who have previously received treatment for central nervous system metastases or spinal cord compression should be eligible if their condition is stable (i.e., no signs of progression on imaging at least 4 weeks prior to the first dose of study therapy, and any neurological symptoms have returned to baseline), and there are no signs of new or enlarging central nervous system metastases. Furthermore, patients should not be using any systemic corticosteroids to control central nervous system metastases or spinal cord compression within 2 weeks prior to the first dose of study therapy.

[0168] 9. Has experienced encephalitis, meningitis, or uncontrolled epilepsy within one year prior to signing the informed consent form.

[0169] 10. The investigator determines that there are clinically significant abnormal ECG readings, and / or the following criteria are met: a. QTc (Friedericia) interval > 470 ms. In cases of asymptomatic QTc interval prolongation (> 470 ms), ECG can be repeated up to 2 times. If the subsequent QTc interval is < 470 ms, the patient can only be recruited after review and approval by a cardiologist.

[0170] b. Evidence of second-degree AV block type II (Mobitz type) or AV block type III (complete heart conduction block).

[0171] 11. Left ventricular ejection fraction (LVEF) less than 50% as measured by echocardiography at baseline. Patients may be recruited after review and approval by a cardiologist in the absence of clinical symptoms and with an LVEF of 45-50%.

[0172] 12. A clinically significant history of heart disease within 6 months prior to screening, including but not limited to the following conditions: - Myocardial infarction - Unstable angina - Stroke or transient ischemic attack - Peripheral artery disease events - Heart failure (NYHA Class III and IV or ACC / AHA Heart Failure Classification C or D) 13. A history of any clinically significant arrhythmia, including paroxysmal atrial fibrillation requiring intervention at any time, or a history of implantation of a pacemaker or defibrillator.

[0173] 14. Has any history of myocarditis.

[0174] 15. Signs or symptoms of active angina, arrhythmia, or heart failure.

[0175] 16. Any moderate to severe valvular abnormality (stenosis or regurgitation) and / or clinically significant valvular heart disease that has not yet been treated surgically.

[0176] 17. If measured by echocardiography at baseline, moderate to large pericardial effusion (e.g., >100 mL).

[0177] 18. Patients who require two or more therapeutic punctures in the month prior to screening.

[0178] 19. Baseline serum troponin is above the upper limit of institutional normal. Patients may be recruited in the absence of clinical symptoms and with minimally elevated troponin levels, and with the approval of a cardiologist.

[0179] 20. Evidence of persistent or recent (within 5 years) severe autoimmune disease, and a history of systemic immunosuppressive therapy, may indicate a risk of imAE (imAE). The following conditions cannot be excluded: vitiligo, remission of childhood asthma, hypothyroidism requiring only hormone replacement therapy, type 1 diabetes, or psoriasis not requiring systemic treatment.

[0180] 21. A history of interstitial lung disease or any evidence of active non-infectious pneumonia (within the past 5 years).

[0181] 22. Patients with moderate to large pleural effusions, where thoracentesis may be necessary within the next 4 weeks due to the size or rate of increase of the effusion. Pre-placed chest tubes are acceptable if the patient meets all other inclusion / exclusion criteria.

[0182] 23. Having an uncontrolled infection with human immunodeficiency virus, hepatitis B, or hepatitis C; or being diagnosed with immunodeficiency.

[0183] Note: - HIV patients with controlled infection (whether naturally or through a stable antiretroviral therapy regimen, with undetectable viral load and CD4 count above 350) are permitted to participate.

[0184] Patients who are positive for hepatitis B surface antigen (HepBsAg+) and whose infection is under control (serum hepatitis B virus DNA PCR test result is below the detection limit and who are receiving antiviral treatment for hepatitis B) are allowed to participate.

[0185] Participants who are HBsAg negative but positive for total HBV core antibody (HBc Ab+) are permitted to participate if: If serum HBV DNA PCR is above the detection limit at screening, antiviral therapy for HBV must be started before enrollment in the study; if serum HBV DNA PCR is below the detection limit, regular monitoring of HBsAg must be performed.

[0186] 24. Active infections requiring systemic treatment, including: a. Infections requiring hospitalization or IV antibiotic treatment within 2 weeks of the start of the study therapy. b. Known active tuberculosis or a history of incompletely treated active or latent tuberculosis. Acceptable treatment for latent tuberculosis would be 9 months of isoniazid 300 mg orally daily or an equivalent proven regimen according to local guidelines.

[0187] c. History of invasive opportunistic infections, including but not limited to histoplasmosis, coccidioidomycosis, pneumocystic jirovecii, aspergillosis, or John Cunningham virus (progressive multifocal leukoencephalopathy).

[0188] 25. Receive the live vaccine within 30 days of the planned start of drug treatment research.

[0189] 26. Underwent major surgery, open biopsy, or major traumatic injury within 2 weeks prior to recruitment.

[0190] 27. Has previously received allogeneic stem cell transplantation.

[0191] 28. If you have experienced intestinal obstruction, perforation, or severe diverticulosis within the past 3 months, or (as the investigator believes) you are at high risk of intestinal obstruction or currently require parenteral nutrition.

[0192] 29. Any medical condition that the researchers believe could prevent patients from participating in this study from being in the best interests of the patients.

[0193] 30. Documented allergic or acute hypersensitivity reactions attributed to antibody therapy.

[0194] 31. Known to have an allergic or hypersensitivity reaction to a component of the study drug.

[0195] 32. Known mental illness or substance abuse disorder may interfere with participation in the study.

[0196] 33. A member of the clinical field research team or his / her immediate family member.

[0197] 34. Women who are pregnant or breastfeeding.

[0198] 35. Severe and / or uncontrolled hypertension at the time of screening. Patients taking antihypertensive medication must follow a stable antihypertensive regimen.

[0199] Example 6: Assessment of MUC16 expression by immunohistochemistry

[0200] MUC16 expression in clinically annotated hysterectomy samples from patients with endometrial cancer was assessed by immunohistochemistry (IHC) (using Abnova Clone X325). Assessment included apical expression as well as combined membrane and cytoplasmic expression. MUC16 expression was quantified by total protein score (TPS, number of MUC16+ cancer cells) and H score incorporating staining intensity. Clinical annotation included available demographic data, highest serum CA125, next-generation sequencing (NGS), p53 IHC, and histology. Clinical relevance was assessed using the Kruskal-Wallis test, Spearman analysis, and the Wilcoxon test.

[0201] 147 cases were evaluated. Patient demographics: 64% Black (n=94), 26% White (n=39), and 10% of other ethnicities (n=14). Histology: 67 cases (46%) were serous carcinoma, 42 cases (29%) were carcinosarcoma, 20 cases (13%) were grade 3 endometrioid carcinoma, and 18 cases (12%) had mixed histology. Based on p53 IHC (n=72, 71% positive) and NGS sequencing (n=36), 14 cases (39%) had p53 mutations without microsatellite instability. MUC16 analysis showed 97% (143 / 147) positive cases, with a median overall H score of 145 (interquartile range (IQR) 70, 205) and a median TPS of 70%. 79% (117 / 147) of cases had a TPS ≥ 25%, the threshold for inclusion in the study discussed in Example 5. H scores varied across subtypes (Kruskal-Wallis test, p = 0.00022): serous: median 175 (IQR 90, 205); carcinosarcoma: median 105 (IQR 12, 161); grade 3 endometrial carcinoma: median 110 (IQR 16, 203); mixed serous and endometrioid: median 200 (IQR 120, 214); mixed serous and clear cell: median 115 (IQR 10, 210); and the H score for mixed clear cell and grade 3 endometrial carcinoma cases was 155. Heterogeneous MUC16 expression was observed in mixed histological cases, particularly carcinosarcoma, where MUC16 typically stained only in the epithelial component. No difference in MUC16 H score was observed with p53 IHC status. Among the 106 patients with available CA125 data, 48% had CA125 levels >70 u / mL; 25% had CA125 levels >500 u / mL. In this study, CA125 was not correlated with MUC16 H score.

[0202] MUC16 is widely expressed in well-differentiated endometrial cancer.

[0203] The scope of this disclosure is not limited to the specific embodiments described herein. In fact, various modifications to this disclosure, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0204] sequence

[0205] SEQ ID NO: 1

[0206] QVQLVESGGGLVKPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWISYISGRGSTIFYADSVKGRITISRDNAKNSLFLQMNSLRAEDTAVYFCVKDRGGYSPYWGQGTLVTVSS

[0207] SEQ ID NO: 2

[0208] DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0209] SEQ ID NO: 3

[0210] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKYGSGYGKFYHYGLDVWGQGTTVTVSS

[0211] SEQ ID NO: 4

[0212] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDGSGYGKFYYYGMDVWGQGTTVTVSS

[0213] SEQ ID NO: 5

[0214] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDGSGYGKFYHYGLDVWGQGTTVTVSS

[0215] SEQ ID NO: 6

[0216] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKYGSGYGKFYYYGLDVWGQGTTVTVSS

[0217] SEQ ID NO: 7

[0218] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKYGSGYGKFYYYGMDVWGQGTTVTVSS

[0219] SEQ ID NO: 8

[0220] GFTFSNYY

[0221] SEQ ID NO: 9

[0222] ISGRGSTI

[0223] SEQ ID NO: 10

[0224] VKDRGGYSPY

[0225] SEQ ID NO: 11

[0226] QSISTY

[0227] SEQ ID NO: 12

[0228] TAS

[0229] SEQ ID NO: 13

[0230] QQSYSTPPIT

[0231] SEQ ID NO: 14

[0232] GFTFDDYS

[0233] SEQ ID NO: 15

[0234] ISWNSGSK

[0235] SEQ ID NO: 16

[0236] AKYGSGYGKFYHYGLDV

[0237] SEQ ID NO: 17

[0238] GFTFDDYS

[0239] SEQ ID NO: 18

[0240] ISWNSGSI

[0241] SEQ ID NO: 19

[0242] AKDGSGYGKFYYYGMDV

[0243] SEQ ID NO: 20

[0244] GFTFDDYS

[0245] SEQ ID NO: 21

[0246] ISWNSGSK

[0247] SEQ ID NO: 22

[0248] AKDGSGYGKFYHYGLDV

[0249] SEQ ID NO: 23

[0250] GFTFDDYS

[0251] SEQ ID NO: 24

[0252] ISWNSGSK

[0253] SEQ ID NO: 25

[0254] AKYGSGYGKFYYYGLDV

[0255] SEQ ID NO: 26

[0256] GFTFDDYS

[0257] SEQ ID NO: 27

[0258] ISWNSGSI

[0259] SEQ ID NO: 28

[0260] AKYGSGYGKFYYYGMDV

[0261] SEQ ID NO: 29

[0262] QVQLVESGGGLVKPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWISYISGRGSTIFYADSVKGRITISRDNAKNSLFLQMNSLRAEDTAVYFCVKDRGGYSPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0263] SEQ ID NO: 30

[0264] DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0265] SEQ ID NO: 31

[0266] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYCAKYGGYGKFYHYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPPAPPVAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLPGS

[0267] SEQ ID NO: 32

[0268] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYCAKYGGYGKFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPPAPPVAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLPGS

[0269] SEQ ID NO: 33

[0270] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSS

[0271] SEQ ID NO: 34

[0272] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFR

[0273] SEQ ID NO: 35

[0274] GFTFSNFG

[0275] SEQ ID NO: 36

[0276] ISGGGRDT

[0277] SEQ ID NO: 37

[0278] VKWGNIYFDY

[0279] SEQ ID NO: 38

[0280] LSINTF

[0281] SEQ ID NO: 39

[0282] AAS

[0283] SEQ ID NO: 40

[0284] QQSSNTPFT

[0285] SEQ ID NO: 41

[0286] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0287] SEQ ID NO: 42

[0288] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A method of treating endometrial cancer in a subject in need, comprising administering a bispecific antibody to the subject, the bispecific antibody comprising a first antigen-binding domain specifically binding to mucin 16 (MUC16) and a second antigen-binding domain specifically binding to human CD3, wherein the bispecific antibody is administered to the subject at a dose of at least 1 mg.

2. The method of claim 1, wherein the subject has previously been treated with platinum-based chemotherapy.

3. The method of claim 1 or 2, wherein the subject has previously been treated with an antibody that binds to human programmed death receptor-1 (PD-1).

4. The method according to any one of claims 1 to 3, wherein the subject has previously received at least two lines of therapy to treat the endometrial cancer.

5. The method according to any one of claims 1 to 4, wherein the endometrial cancer is advanced or recurrent endometrial cancer.

6. The method according to any one of claims 1 to 5, wherein the endometrial cancer is a cancer expressing MUC16.

7. The method according to any one of claims 1 to 6, wherein the first antigen-binding domain comprises: (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), said three heavy chain complementarity-determining regions being contained within a heavy chain variable region (HCVR), said HCVR containing the amino acid sequence of SEQ ID NO: 1; and (b) Three light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:

2.

8. The method according to claim 7, wherein the first antigen-binding domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence of SEQ ID NO:

10.

9. The method according to claim 7 or 8, wherein the first antigen-binding domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

13.

10. The method according to any one of claims 7 to 9, wherein the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO:

2.

11. The method according to any one of claims 1 to 10, wherein the second antigen-binding domain comprises: (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), said three heavy chain complementarity-determining regions being contained within a heavy chain variable region (HCVR), said HCVR containing the amino acid sequence of SEQ ID NO: 3; and (b) Three light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:

2.

12. The method of claim 11, wherein the second antigen-binding domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprising the amino acid sequence of SEQ ID NO:

16.

13. The method according to claim 11 or 12, wherein the second antigen-binding domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

13.

14. The method according to any one of claims 11 to 13, wherein the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 3 and an LCVR comprising the amino acid sequence of SEQ ID NO:

2.

15. The method according to any one of claims 1 to 14, wherein the bispecific antibody comprises a constant region of the human IgG heavy chain.

16. The method of claim 15, wherein the human IgG heavy chain constant region is isotype IgG1.

17. The method of claim 15, wherein the constant region of the human IgG heavy chain is isotype IgG4.

18. The method of claim 16 or 17, wherein the bispecific antibody comprises a chimeric hinge that reduces Fc relative to a wild-type hinge of the same isotype. Receptor binding.

19. The method according to any one of claims 15 to 18, wherein the first heavy chain or the second heavy chain comprises a CH3 domain, but not both simultaneously comprise a CH3 domain, the CH3 domain comprising H435R (EU designation) modification and Y436F (EU designation) modification.

20. The method according to any one of claims 1 to 14, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:

29.

21. The method according to any one of claims 1 to 14, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:

31.

22. The method according to any one of claims 1 to 14, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31, and a common light chain comprising the amino acid sequence of SEQ ID NO:

30.

23. The method according to any one of claims 1 to 22, wherein the subject has a serum CA-125 level equal to or greater than 60 U / ml.

24. The method according to any one of claims 1 to 23, wherein the method further comprises administering a second therapeutic agent or treatment regimen.

25. The method of claim 24, wherein the second therapeutic agent or treatment regimen comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

26. The method of claim 25, wherein the anti-PD-1 antibody or antigen-binding fragment comprises: (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), said three heavy chain complementarity-determining regions being contained within a heavy chain variable region (HCVR), said HCVR containing the amino acid sequence of SEQ ID NO: 33; and (b) Three light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:

34.

27. The method of claim 26, wherein the anti-PD-1 antibody or antigen-binding fragment comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:

37.

28. The method according to claim 26 or 27, wherein the anti-PD-1 antibody or antigen-binding fragment comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

40.

29. The method according to any one of claims 26 to 28, wherein the anti-PD-1 antibody or antigen-binding fragment comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 33 and an LCVR comprising the amino acid sequence of SEQ ID NO:

34.

30. The method of claim 29, wherein the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 41 and the light chain comprises the amino acid sequence of SEQ ID NO:

42.

31. The method according to any one of claims 25 to 30, wherein the anti-PD-1 antibody is cimipril.

32. The method according to any one of claims 1 to 31, wherein the bispecific antibody is administered in a dosing regimen comprising a fractionated initial dose.

33. The method according to any one of claims 1 to 32, wherein the bispecific antibody is administered to the subject at a dose of 10 mg to 1000 mg per week.

34. The method of claim 33, wherein the bispecific antibody is administered to the subject at a dose of about 250 mg per week, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg.

35. The method of claim 33, wherein the bispecific antibody is administered to the subject at a dose of about 800 mg per week, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 750 mg.

36. The method according to any one of claims 1 to 32, wherein the bispecific antibody is administered to the subject at a dose of 10 mg to 1000 mg every three weeks.

37. The method of claim 36, wherein the bispecific antibody is administered to the subject at a dose of about 250 mg every three weeks, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg.

38. The method of claim 36, wherein the bispecific antibody is administered to the subject at a dose of about 800 mg every three weeks, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 750 mg.

39. The method of claim 33, wherein the bispecific antibody is administered in a dosing regimen comprising: (i) administering 1 mg of the bispecific antibody in week 1, optionally wherein the dose is divided into a first portion of about 0.5 mg and a second portion of about 0.5 mg; (ii) administering 20 mg of the bispecific antibody in week 2, optionally wherein the dose is divided into a first portion of about 10 mg and a second portion of about 10 mg; and (iii) administering 250 mg of the bispecific antibody in week 3, optionally wherein the dose is divided into a first portion of about 50 mg and a second portion of about 200 mg.

40. The method of claim 39, wherein the method further comprises administering the bispecific antibody once weekly at a dose of about 250 mg, starting from week 4.

41. The method of claim 39, wherein the method further comprises administering the bispecific antibody every three weeks at a dose of about 250 mg, starting from week 4.

42. The method of claim 39, wherein the method further comprises administering the bispecific antibody every three weeks at a dose of about 800 mg, starting from week 4.

43. The method according to any one of claims 25 to 42, wherein the anti-PD-1 antibody is administered to the subject at a dose of 300 to 400 mg every three weeks.

44. The method of claim 43, wherein the anti-PD-1 antibody is administered to the subject at a dose of 350 mg every three weeks.

45. The method according to any one of claims 1 to 44, wherein the subject has a stable disease, a partial response, or a complete response at least one week after administration of the bispecific antibody at a dose of 1-800 mg.

46. ​​The method according to any one of claims 1 to 44, wherein the subject has a stable disease, a partial response, or a complete response at least one week after administration of the bispecific antibody at a dose of 20-800 mg.

47. The method according to any one of claims 1 to 32, wherein the bispecific antibody is administered to the subject at a dose sufficient to achieve a serum concentration of at least 4 mg / L.

48. The method according to any one of claims 1 to 47, wherein MUC16 is expressed in large quantities in ≥25% of tumor cells in the subject, as determined by immunohistochemical staining.

49. The method according to any one of claims 1 to 47, wherein the subject: ■ The baseline MUC16 immunohistochemical staining score in tumors expressing MUC16 was 2; or ■ Baseline MUC16 immunohistochemical staining score of 2+ in tumors expressing MUC16; or ■ The baseline MUC16 immunohistochemical staining score in tumors expressing MUC16 was 3; or ■ Baseline MUC16 immunohistochemical staining score of 3+ in tumors expressing MUC16; or ■ The baseline MUC16 immunohistochemical staining score in tumors expressing MUC16 was 4; or ■ Baseline MUC16 immunohistochemical staining score of 4+ in tumors expressing MUC16; or ■ The baseline MUC16 immunohistochemical staining score in tumors expressing MUC16 was 5; or ■ Tumors with ≥ 25% of tumor cells expressing MUC16; or ■ Tumors with ≥ 30% of tumor cells expressing MUC16; or ■ Tumors with ≥ 35% of tumor cells expressing MUC16; or ■ Tumors with ≥ 40% of tumor cells expressing MUC16; or ■ Tumors with ≥ 45% of tumor cells expressing MUC16; or ■ Tumors with ≥ 50% of tumor cells expressing MUC16; or ■ Tumors with ≥ 55% of tumor cells expressing MUC16; or ■ Tumors with ≥ 60% of tumor cells expressing MUC16; or ■ Tumors with ≥ 65% of tumor cells expressing MUC16; or ■ Tumors with ≥ 70% of tumor cells expressing MUC16; or ■ Tumors with ≥ 75% of tumor cells expressing MUC16.

50. The method according to any one of claims 1 to 47, wherein the subject has: ■ H score ≥100; or ■ H score ≥105; or ■ H score ≥110; or ■ H score ≥115; or ■ H score ≥120; or ■ H score ≥125; or ■ H score ≥130; or ■ H score ≥135; or ■ H score ≥140; or ■ H score ≥145; or ■ H score ≥150; or ■ H score ≥155; or ■ H score ≥160; or ■ H score ≥165; or ■ H score ≥170; or ■ H score ≥175; or ■ H score ≥180; or ■ H score ≥185; or ■ H score ≥190; or ■ H score ≥195; or ■ H score ≥200 For example, it can be determined by immunohistochemical staining.

51. The method according to any one of claims 1 to 50, wherein the bispecific antibody is administered intravenously.

52. The method according to any one of claims 1 to 50, wherein the bispecific antibody is administered subcutaneously.

53. The method according to any one of claims 25 to 52, wherein the anti-PD-1 antibody or antigen-binding fragment is administered intravenously.

54. The method according to any one of claims 1 to 53, wherein the bispecific antibody is REGN4018 (ubamatamab).

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