Methods for treating recurrent ovarian cancer with a bispecific anti-MUC16 x anti-CD3 antibody alone or in combination with an anti-PD-1 antibody

Abstract

The present invention provides methods for treating, reducing the severity of, or inhibiting the growth of cancer (e.g., recurrent ovarian cancer). The methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that specifically binds mucin 16 (MUC16) and CD3, alone or in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds programmed death 1 (PD-1).

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 11048WO01_Sequence, created on January 6, 2023, and containing 54,660 bytes.

[0002] The present invention relates to methods of treating cancer using a bispecific antibody that binds mucin 16 (MUC16) and CD3, alone or in combination with an anti-PD-1 antibody. [Background technology]

[0003] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a single transmembrane domain, highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer. MUC16 is composed of three major domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with sea urchin sperm, enterokinase, and agrin (SEA) domains, and a carboxyl-terminal domain that contains a segment of the transmembrane region and a short cytoplasmic tail. Proteolytic cleavage releases much of the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass-forming type, adenocarcinoma of the cervix, and adenocarcinoma of the gastric tract, as well as diseases and conditions including inflammatory bowel disease, liver cirrhosis, heart failure, peritoneal infection, and abdominal surgery. (Haridas, D. et al., 2014, FASEB J., 28:4183-4199). Expression on cancer cells has been shown to protect tumor cells from the immune system. (Felder, M. et al., 2014, Molecular Cancer, 13:129) Methods for treating ovarian cancer using antibodies against MUC16 are being investigated. Oregovomab and abgovomab are anti-MUC16 antibodies with limited success. (Felder, Das, S. and Batra, SK 2015, Cancer Res. 75:4660-4674, supra.)

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta, and gamma. CD3 dimeric configurations include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby triggering T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes, including T cell activation. Furthermore, bispecific antibodies capable of binding CD3 and a target antigen have been proposed for therapeutic uses, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0005] Programmed death receptor-1 (PD-1) signaling in the tumor microenvironment plays a key role in enabling tumor cells to escape immunosurveillance by the host immune system. Blockade of the PD-1 signaling pathway has demonstrated clinical activity in patients with multiple tumor types, and antibody therapeutics that block PD-1 (e.g., nivolumab and pembrolizumab) have been approved for the treatment of, for example, metastatic melanoma and metastatic squamous non-small cell lung cancer. Recent data have demonstrated clinical activity of PD-1 blockade 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).

[0006] Ovarian cancer is the most lethal of gynecologic malignancies. Although the estimated number of new cases of ovarian cancer in U.S. women is much lower than certain other cancers, the mortality-to-morbidity ratio of ovarian cancer is quite high (Siegal et al., CA Cancer J Clin 66:7-30, 2016). Ovarian cancer is often diagnosed at an advanced stage, which contributes to its lethality. The current standard of care for ovarian cancer is surgery followed by chemotherapy, i.e., a combination of platinum-based drugs and taxanes. While the majority of patients respond to initial treatment, most patients experience disease recurrence, resulting in cycles of repeated surgery and additional rounds of chemotherapy. Although recurrent ovarian cancers can respond to further treatments, nearly all of them eventually become resistant to currently available therapies. Despite recent advances in therapy, such as PARP inhibitors for patients with BRCA or other homologous recombination defect (HRD) mutations, advanced ovarian cancer remains a disease of high unmet need.

[0007] Evidence suggests that ovarian cancer may be amenable to some immunotherapy (Kandalaft et al., J. Clin. Oncol., 29:925-933, 2011). For example, tumors expressing intraepithelial CD8 + Ovarian cancer patients who were positive for T lymphocyte infiltration had intraepithelial CD8 +Patients with T-lymphocyte infiltration had significantly better overall survival and progression-free survival than those without T-lymphocyte infiltration (Hamanishi et al., PNAS, 104:3360-65, 2007, and Zhang et al., N. Engl. J. Med., 348:203-213, 2003). In addition, some patients demonstrate an innate immune response to the tumor, as demonstrated by the detection of tumor-reactive T cells and antibodies in the blood, tumor, or ascites of patients with advanced disease (Schliengar et al., Clin Cancer Res, 9:1517-1527, 2003). Blockade of the PD-1 / PD-L1 checkpoint pathway has shown some benefit in ovarian cancer, with PD-1 blockade monotherapy resulting in an overall response rate (ORR) of approximately 10-15% in early clinical trials (Hamanishi et al., supra). However, blockade of this pathway alone is clearly not sufficient.

[0008] Given the high unmet need, additional therapies targeting ovarian cancer are needed. Summary of the Invention

[0009] In one aspect, the disclosure includes a method of treating cancer (e.g., a MUC16-expressing cancer) in a subject in need thereof, comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to mucin 16 (MUC16) on a target tumor cell and a second antigen-binding domain that specifically binds to human CD3 on a T cell. 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 cancer (e.g., MUC16-expressing cancer) is ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. In some cases, the cancer (e.g., MUC16-expressing cancer) is resistant to platinum-based chemotherapy. In some cases, the subject has previously been treated with platinum-based chemotherapy.

[0011] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain comprising (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1, and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2. In some cases, the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:8, an HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:10. In some cases, the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:11, an LCDR2 comprising the amino acid sequence of SEQ ID NO:12, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:13. In some cases, 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.

[0012] In some embodiments, including those in which the first antigen-binding domain is as described above, the bispecific antibody or antigen-binding fragment thereof comprises a second antigen-binding domain comprising (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 3, and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2. In some cases, the second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16. In some cases, the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some instances, the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO:3, and a LCVR comprising the amino acid sequence of SEQ ID NO:2.

[0013] In some embodiments, the bispecific antibody comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region is of isotype IgG1. Optionally, the human IgG heavy chain constant region is of isotype IgG4.

[0014] In some embodiments, the bispecific antibody comprises a chimeric hinge that has reduced Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0015] In some embodiments, the first heavy chain or the second heavy chain, but not both, of the bispecific antibody comprises a CH3 domain that comprises an H435R (EU numbering) modification and a Y436F (EU numbering) modification.

[0016] In some embodiments, 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.

[0017] In some embodiments, the subject has an elevated serum CA-125 level. In some embodiments, the subject has a serum CA-125 level that is at least twice the upper limit of normal. In some embodiments, the subject has a CA-125 level greater than 92 U / ml.

[0018] In some embodiments, the method further comprises administering a second therapeutic agent or treatment regimen. In some cases, the second therapeutic agent or treatment regimen comprises an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 antibody is cemiplimab.

[0019] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising 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) comprising the amino acid sequence of SEQ ID NO: 34. In some cases, the anti-PD-1 antibody or antigen-binding fragment comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some cases, the anti-PD-1 antibody or antigen-binding fragment comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some instances, the anti-PD-1 antibodies and antigen-binding fragments comprise a HCVR comprising the amino acid sequence of SEQ ID NO: 33, and a LCVR comprising the amino acid sequence of SEQ ID NO: 34. In some instances, the anti-PD-1 antibodies and antigen-binding fragments are anti-PD-1 antibodies that comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0020] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered in a dosing regimen that includes a split initial dose. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject in a weekly dose of 1 mg to 1000 mg. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered to the subject in a weekly dose of 2 mg to 1000 mg. In some embodiments, the initial dose (e.g., 1 mg or 2 mg) is split into two fractions of equal or unequal amounts. For example, in some cases, the initial dose is split in half (e.g., an initial dose of 1 mg is split into two fractions of 0.5 mg each, which are administered on separate days, e.g., on consecutive days). Alternatively, the initial dose may be split into unequal fractions that are administered on separate days, e.g., on consecutive days. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to the subject in a weekly dose of about 250 mg. In some cases, the 250 mg dose is split into two fractions comprising 50 mg and 200 mg, respectively. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a dose of about 800 mg every week. In some cases, the 800 mg dose is divided into two fractions containing 50 mg and 750 mg, respectively. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to the subject at a frequency of once every three weeks (e.g., once every three weeks, about 250 mg or about 800 mg, or 10 mg to 1000 mg). In some cases, the bispecific antibody or antigen-binding fragment is administered at a dose of about 250 mg every three weeks. In some cases, the 250 mg dose is divided into two fractions containing 50 mg and 200 mg, respectively. In some embodiments, the bispecific antibody is administered to the subject at a dose sufficient to achieve a serum concentration of at least 4 mg / L.In some embodiments, the bispecific antibody is administered in a dosing regimen comprising: (i) administering 1 mg of the bispecific antibody in week 1, optionally split into a first fraction of about 0.5 mg and a second fraction of about 0.5 mg; (ii) administering 20 mg of the bispecific antibody in week 2, optionally split into a first fraction of about 10 mg and a second fraction of about 10 mg; and (iii) administering 250 mg of the bispecific antibody in week 3, optionally split into a first fraction of about 50 mg and a second fraction of about 200 mg. In some cases, the dosing regimen further comprises administering the bispecific antibody at a dose of about 250 mg once a week from week 4 onwards. In some cases, the dosing regimen further comprises administering the bispecific antibody at a dose of about 250 mg once every three weeks from week 4 onwards. In some embodiments, the bispecific antibody is administered in a dosing regimen comprising: (i) administering 1 mg of the bispecific antibody in week 1; (ii) administering 20 mg of the bispecific antibody in week 2, optionally with the dose split into a first fraction of about 10 mg and a second fraction of about 10 mg; and (iii) administering 800 mg of the bispecific antibody in week 3, optionally with the dose split into a first fraction of about 50 mg and a second fraction of about 750 mg. In some cases, the dosing regimen further comprises administering the bispecific antibody at a dose of about 800 mg once a week from week 4 onwards. In some cases, the dosing regimen further comprises administering the bispecific antibody at a dose of about 800 mg once every three weeks from week 4 onwards. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to the subject via intravenous administration. In some cases, the bispecific antibody or antigen-binding fragment thereof is administered to the subject via subcutaneous administration. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 300 mg to 400 mg once every three weeks. In some cases, the anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 350 mg once every three weeks. In some cases, the anti-PD-1 antibody or antigen-binding fragment thereof is administered intravenously.

[0021] In some embodiments of the method, the subject has stable disease, a partial response, or a complete response after at least one week of administration of the bispecific antibody at a dose of 1-800 mg. In some embodiments of the method, the subject has stable disease, a partial response, or a complete response after at least one week of administration of the bispecific antibody at a dose of 20-800 mg.

[0022] In some embodiments of the methods, the bispecific antibody is administered to the subject at a dose sufficient to achieve a serum concentration of at least 4 mg / L.

[0023] In some embodiments of the method, MIC16 is highly expressed in > 75% of tumor cells in the subject as determined by immunohistochemical staining. In some embodiments of the method, the subject has a baseline MUC16 immunohistochemical staining score of 2 in MUC16-expressing tumors, or a baseline MUC16 immunohistochemical staining score of 2+ in MUC16-expressing tumors, or a baseline MUC16 immunohistochemical staining score of 3 in MUC16-expressing tumors, or a baseline MUC16 immunohistochemical staining score of 3+ in MUC16-expressing tumors, or a baseline MUC16 immunohistochemical staining score of 4 in MUC16-expressing tumors, or a baseline MUC16 immunohistochemical staining score of 5 in MUC16-expressing tumors. C16 immunohistochemistry staining score of 4+, or a baseline MUC16 immunohistochemistry staining score of 5 in MUC16-expressing tumors, or tumors with MUC16 expression in ≧50% of tumor cells, or tumors with MUC16 expression in ≧55% of tumor cells, or tumors with MUC16 expression in ≧60% of tumor cells, or tumors with MUC16 expression in ≧65% of tumor cells, or tumors with MUC16 expression in ≧70% of tumor cells, or tumors with MUC16 expression in ≧75% of tumor cells.

[0024] In some embodiments of the method, the bispecific antibody is administered intravenously. In some embodiments, the bispecific antibody is administered subcutaneously. In some embodiments of the method, the anti-PD-1 antibody or antigen-binding fragment is administered intravenously.

[0025] Other embodiments of the invention will become apparent from consideration of the detailed description that follows. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows the binding of various concentrations of anti-MUC16 clone 3A5 and BSMUC16 / CD3-001 to CA125 as determined by ELISA (described in Example 2 herein). BSMUC16 / CD3-001 and its MUC16 parent antibody showed significantly reduced binding signals at all concentrations tested compared to anti-MUC16 clone 3A5, which binds to the repeat region of MUC16. [Diagram 2] Figure 2 shows the mean tumor growth curves for groups of mice (5 per group) treated with CD3 binding control + isotype control (△), BSMUC16 / CD3-005 + isotype control (□), CD3 binding control + anti-PD-1 (▲), and BSMUC16 / CD3-005 + anti-PD-1 (■) (as described in Example 3 herein). The combination of anti-PD-1 antibody and anti-CD3xMUC16 bispecific antibody synergistically inhibited tumor growth. [Diagram 3] FIG. 3 shows the effect of incubation of T cells with BSMUC16 / CD3-001 on the percentage of PD-1 positive T cells. [Figure 4] FIG. 4 shows a waterfall plot of monotherapy REGN4018 dose escalation (20-800 mg) illustrating best response in various patients. [Diagram 5] FIG. 5 shows one embodiment of a monotherapy dosing regimen for intravenous administration of REGN4018. [Figure 6] FIG. 6 shows one embodiment of a combination therapy dosing regimen for intravenous administration of REGN4018 in combination with cemiplimab. [Figure 7]Figure 7 shows one embodiment of a monotherapy dosing regimen with subcutaneous loading dose and transition dose of REGN4018. *If the first 3 patients tolerate the second transition IV dose and all subsequent IV doses of REGN4018 without significant CRS, the second transition IV dose may be omitted for the remainder of the cohort. [Figure 8] Figure 8 shows one embodiment of a combination therapy dosing regimen with subcutaneous initial dose and transition doses of REGN4018 in combination with cemiplimab. *If the first three patients tolerate the second transition IV dose and all subsequent IV doses of REGN4018 without significant CRS, the second transition IV dose may be omitted for the remainder of the cohort. [Figure 9] FIG. 9 shows one embodiment of a combination therapy dosing regimen of REGN4018 in combination with cemiplimab administered intravenously Q3W. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are used for the purpose of describing only specific embodiments, and are not intended to be limiting, since the scope of the present invention is limited only by the scope of the appended claims. Any embodiment or feature of the embodiment can be combined with each other, and such combinations are expressly included within the scope of the present invention. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are included within the scope of the present disclosure.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value may vary by 1% or less from the referenced value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0029] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0030] Methods for Treating or Inhibiting Cancer The present invention includes methods for treating, ameliorating, or reducing the severity of at least one symptom or sign of cancer or inhibiting the growth of cancer (e.g., recurrent ovarian cancer) in a subject. Methods according to aspects of the invention include administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof against MUC16 and CD3, alone or in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1. As used herein, the terms "treat," "treating," and the like, refer to alleviating a symptom, eliminating the cause of a symptom, either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, and / or increasing the survival of a subject.

[0031] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer, and / or a human or non-human mammal diagnosed with cancer, including ovarian cancer, and in need of 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 with one or more symptoms or signs, including, but not limited to, enlarged lymph nodes, abdominal bloating, chest pain / tightness, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, and itching. This phrase includes a subject with a primary or established ovarian tumor. In specific embodiments, this phrase includes a human subject having ovarian cancer or another tumor that expresses MUC16, such as, for example, endometrial cancer, and in need of treatment thereof. In other specific embodiments, this phrase includes a subject having a MUC16+ tumor (e.g., a tumor with MUC16 expression as determined by flow cytometry or immunohistochemistry). In certain embodiments, expression includes human subjects having tumors that exhibit high expression of MUC16 in >50%, >55%, >60%, >65%, >70%, or >75% of tumor cells. MUC16 expression can be determined and assessed by any method known in the art (see, e.g., Shimizu et al 2012, Cancer Sci. 103:739-746). In certain embodiments, expression includes human subjects with a baseline MUC16 immunohistochemical staining score of 2+ (e.g., 2, 3, 4, or 5) in MUC16-expressing tumors. In certain embodiments, expression includes human subjects with a baseline MUC16 immunohistochemical staining score of 2, 2+, 3, 3+, 4, 4+, or 5 in MUC16-expressing tumors. Immunohistochemical staining scores, in this context, incorporate the percentage of cells, as well as the intensity and pattern of staining according to the following criteria: 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 certain embodiments, the term "subject in need thereof" includes patients with ovarian cancer that is resistant or refractory to previous therapy (e.g., treatment with conventional anti-cancer drugs) or not adequately controlled by previous therapy. For example, the term includes subjects treated with chemotherapy, such as platinum-based chemotherapy agents (e.g., cisplatin) or taxol compounds (e.g., docetaxel). The term also includes subjects with ovarian tumors for which conventional anti-cancer therapy is not advisable, for example, due to toxic side effects. For example, the term includes patients who have undergone one or more cycles of chemotherapy with toxic side effects. In certain embodiments, the term "subject in need thereof" includes patients with ovarian cancer that have been treated but have subsequently recurred or metastasized. For example, patients with ovarian cancer who have been treated with one or more anti-cancer drugs and may have undergone tumor regression, but who subsequently recur with cancer that is resistant to one or more anti-cancer drugs (e.g., chemotherapy-resistant cancer), are treated with the methods of the present invention.

[0032] The term "subject in need thereof" also includes subjects at risk of developing ovarian cancer, such as those with a family history of ovarian cancer, those with a past medical history of an infection associated with ovarian cancer, those with a mutation in the BRCA1 / 2 gene, or those with a compromised immune system due to HIV infection or immunosuppressant medications.

[0033] In certain embodiments, the methods of the invention can be used to treat patients exhibiting elevated levels of one or more cancer-associated biomarkers (e.g., programmed death-ligand 1 (PD-L1), MUC16, CA125 human epididymis protein 4 (HE4), and / or carcinoembryonic antigen (CEA)). For example, the methods of the invention include administering a therapeutically effective amount of an anti-PD-1 antibody, or antigen-binding fragment thereof, in combination with a bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, to a patient with elevated levels of MUC16 and / or CA125. Methods for determining MUC16 and / or CA125 expression are well known in the art. In certain embodiments, expression of MUC16 in tumor tissue is determined by immunohistochemistry (IHC) assay (see, e.g., Bast et al 1981, J. Clin. Invest. 68:1331-1337). MUC16 expression may be assessed by any method known in the art (e.g., Shimizu et al 2012, Cancer Sci. 103:739-746). In certain embodiments, MUC16 expression is determined by imaging with a labeled anti-MUC16 antibody, such as, for example, by immunopositron emission tomography or iPET (described elsewhere herein).

[0034] In certain embodiments, the methods of the present invention are used in subjects with ovarian cancer. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "ovarian cancer" refers to tumors of the ovaries and fallopian tubes, and includes serous carcinoma, endometrioid carcinoma, clear cell carcinoma, and mucinous carcinoma.

[0035] According to certain embodiments, the invention includes methods for treating a tumor or slowing or inhibiting the growth of a tumor. In certain embodiments, the invention includes methods for promoting tumor regression. In certain embodiments, the invention includes methods for reducing tumor cell burden or reducing tumor burden. In certain embodiments, the invention includes methods for preventing tumor recurrence. The method according to this aspect of the invention includes administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, wherein each antibody is administered to the subject in multiple doses, e.g., as part of a particular therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of an anti-MUC16xCD3 antibody or antigen-binding fragment thereof to a subject approximately once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less frequently. In certain embodiments, one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof are administered in combination with one or more doses of a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, and the one or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof are administered to a subject about once per day, once every two days, once every three days, once every four days, once every five days, once every six days, once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every two months, once every three months, once every four months, or less frequently.

[0036] In certain embodiments, each dose of the anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof is administered in more than one fraction, e.g., 2-5 fractions, within a given administration period ("split doses"). The anti-MUC16 / anti-CD3 bispecific antibody or antigen-binding fragment thereof may be administered in split doses to reduce or eliminate the cytokine "spike" induced in response to administration of the antibody. Cytokine spike refers to the clinical symptoms of cytokine release syndrome ("cytokine storm") and infusion-related reactions. In certain embodiments, the methods of the invention comprise administering to a subject in need thereof one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the dose of the bispecific antibody or antigen-binding fragment thereof is administered as a split dose or in more than one fraction, e.g., 2, 3, 4, or 5 fractions, within a given administration period. In certain embodiments, the dose of the bispecific antibody or antigen-binding fragment thereof is divided into two or more fractions, each fraction containing an equal amount of the antibody or antigen-binding fragment thereof as the other fractions. For example, a dose of the anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof containing 1000 micrograms may be administered once a week, the dose being administered in two fractions within the week, each fraction containing 500 micrograms. In certain embodiments, the dose of the bispecific antibody or antigen-binding fragment thereof is divided into two or more fractions, the fractions containing unequal amounts of the antibody, such as more or less than the first fraction. For example, a dose of the anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof containing 1000 micrograms may be administered once a week, the dose being administered in two fractions within the week, the first fraction containing 700 micrograms and the second fraction containing 300 micrograms. As another example, a dose of anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof containing 1000 micrograms may be administered once every two weeks, the dose being administered in three fractions within two weeks, with the first fraction containing 400 micrograms, the second fraction containing 300 micrograms, and the third fraction containing 300 micrograms.

[0037] In certain embodiments, the invention provides a method of treating a MUC16-expressing cancer (e.g., ovarian, fallopian tube, or primary peritoneal cancer, including cancers that are refractory to multiple prior therapies described herein) in a subject in need of such treatment, comprising administering to the subject a bispecific antibody comprising a first antigen-binding domain that specifically binds mucin 16 (MUC16) on a target tumor cell and a second antigen-binding domain that specifically binds human CD3 on a T cell, the bispecific antibody comprising: The bispecific antibody is administered in a regimen of: (i) administering 1 mg of the bispecific antibody in week 1, optionally divided into a first fraction of about 0.5 mg and a second fraction of about 0.5 mg; (ii) administering 20 mg of the bispecific antibody in week 2, optionally divided into a first fraction of about 10 mg and a second fraction of about 10 mg; (iii) administering 250 mg of the bispecific antibody in week 3, optionally divided into a first fraction of about 50 mg and a second fraction of about 200 mg. In some cases, the method further comprises administering the bispecific antibody at a dose of about 250 mg once a week from week 4 onwards. In some cases, the method further comprises administering the bispecific antibody at a dose of about 250 mg once every three weeks from week 4 onwards. In some cases, the method further comprises administering the bispecific antibody at a dose of about 800 mg once every three weeks starting after week 4. In some cases, the method further comprises administering to the subject an anti-PD-1 antibody at a dose of 300-400 mg (e.g., 350 mg) once every three weeks.

[0038] In certain embodiments, the invention includes a method of inhibiting, slowing, or stopping tumor metastasis or tumor invasion to peripheral organs. The method according to this aspect includes administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody, or antigen-binding fragment thereof.

[0039] In a specific embodiment, the invention provides a method for increasing anti-tumor efficacy or increasing tumor inhibition. The method according to this aspect of the invention comprises administering to a subject having ovarian cancer a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof prior to administering a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, where the anti-PD-1 antibody or antigen-binding fragment thereof can be administered about 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 antigen-binding fragment thereof. In a specific embodiment, the method provides an increase in tumor inhibition of, e.g., about 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 administered the bispecific antibody or antigen-binding fragment thereof prior to the anti-PD-1 antibody or antigen-binding fragment thereof.

[0040] In certain embodiments, the methods of the invention comprise administering to a subject with ovarian cancer a therapeutically effective amount of a bispecific anti-CD3xMUC16 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In specific embodiments, the ovarian cancer is a serous carcinoma. In further embodiments, the ovarian cancer is indolent or aggressive. In certain embodiments, the subject is unresponsive to a previous therapy or has relapsed after a previous therapy (e.g., a platinum-based therapy). In some embodiments, the subject has a CA-125 level that is greater than or equal to twice the upper limit of normal (ULN) (e.g., greater than or equal to about 60 U / ml). In various embodiments, the subject's serum CA-125 level (pre-treatment) is greater than or equal to 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 certain embodiments, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

[0041] In certain embodiments, the methods of the invention comprise administering a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject with a MUC16+ cancer. In specific embodiments, the cancer is ovarian cancer. In further embodiments, the ovarian cancer is indolent or aggressive. In some embodiments, the cancer is platinum-resistant ovarian cancer. In some embodiments, the cancer is taxol-resistant ovarian cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is primary peritoneal cancer, and optionally, the patient has elevated serum CA-125 levels (at least 2x the ULN). In certain embodiments, the subject is unresponsive to or has relapsed after previous therapy (e.g., chemotherapy).

[0042] In certain embodiments, the methods of the invention include administering an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject in need thereof as a "first line" treatment (e.g., initial treatment). In other embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof is administered as a "second line" treatment (e.g., after prior therapy). For example, an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof is administered as a "second line" treatment to a subject who has relapsed after prior therapy, e.g., with chemotherapy (e.g., platinum-based chemotherapy).

[0043] In certain embodiments, the methods of the invention 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 during or after treatment, who may or may not show symptoms or signs of disease. Such residual cancer cells, if not removed, often lead to disease recurrence. The invention includes methods of inhibiting and / or removing residual cancer cells in a patient upon MRD testing. MRD can be assayed according to methods known in the art (e.g., MRD flow cytometry). The method according to this aspect of the invention includes administering a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, to a subject in need thereof.

[0044] Methods of the invention according to certain embodiments comprise administering to a subject a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, and optionally, a third therapeutic agent. The third therapeutic agent can be, for example, radiation, chemotherapy, surgery, a cancer vaccine, a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a LAG3 inhibitor (e.g., an anti-LAG3 antibody), a CTLA-4 inhibitor (anti-CTLA-4 antibody), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGF.beta.) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen (e.g., 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), a vaccine (e.g., Bacillus Calmette-Guerin), granulocyte macrophage colony stimulating factor, cytotoxins, chemotherapeutic agents, cytokines such as IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, and IL-15, anti-inflammatory agents such as corticosteroids and nonsteroidal anti-inflammatory drugs, and dietary supplements such as antioxidants. In certain embodiments, the antibody may be administered in combination with a therapy including a chemotherapeutic agent (e.g., paclitaxel, carboplatin, doxorubicin, cyclophosphamide, cisplatin, gemcitabine, or docetaxel), radiation, and surgery. As used herein, the phrase "in combination with" means that the antibody is administered to the subject simultaneously with, immediately before, or immediately after administration of a third therapeutic agent. In certain embodiments, the third therapeutic agent is administered as a co-formulation with the antibody.

[0045] In certain embodiments, the methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. When the combination is administered, administration of the antibody (or fragment) results in increased inhibition of tumor growth. In certain 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% compared to an untreated subject or a subject administered either antibody (or fragment) as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof leads to increased tumor regression, tumor shrinkage and / or elimination. In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof leads to a delay in tumor growth and progression, e.g., tumor growth may 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 an untreated subject or a subject treated with either antibody (or fragment) as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof prevents tumor recurrence and / or increases the survival of the subject, e.g., increases the duration of survival by 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 an untreated subject or a subject treated with either antibody (or fragment) as monotherapy. In certain embodiments, administration of a combination of antibodies increases progression-free survival or overall survival.In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof increases the response and duration of response in a subject by, for example, 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% than an untreated subject or a subject receiving either antibody (or fragment) as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject with ovarian cancer leads to the complete disappearance of all traces of tumor cells (a "complete response"). In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject with ovarian cancer leads to at least a 30% or greater reduction in tumor cells or tumor size ("partial response"). In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject with ovarian cancer leads to a 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, for example, X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis. In certain embodiments, administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a subject with ovarian cancer results in a synergistic anti-tumor effect that exceeds the combined effect of the two agents when administered alone.

[0046] In certain embodiments, the administered antibody (or fragment) combination is safe and well tolerated by patients, without an increase in adverse side effects (e.g., increased cytokine release ("cytokine storm") or increased T cell activation) compared to patients receiving the bispecific antibody (or fragment) as monotherapy.

[0047] In certain cases, a subject's response to therapy is classified as 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 nodes (target or non-target) to <10 mm (<1 cm). PR is defined as at least a 30% reduction in the sum of the diameters of the target lesions, based on the baseline sum of the diameters. PD is defined as at least a 20% increase in the sum of the diameters of the target lesions, based on the minimum sum on study (including the baseline sum, if it is the minimum on study). In addition to the 20% relative increase, the sum must also show 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 neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, based on the minimum sum of the diameters on study.

[0048] Anti-PD-1 antibodies and antigen-binding fragments thereof According to certain exemplary embodiments of the invention, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (referred to herein as HCVR or VL). H The heavy chain constant region is made up of three domains, C H 1. C H 2, and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V LThe regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), separated by regions that are relatively conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-IL-4R antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the aligned analysis of two or more CDRs.

[0049] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. 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 may be derived from complete antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding antibody variable regions and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0050] Non-limiting examples of antibody 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) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0051] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V bound to the domain H In the case of an antigen-binding fragment having a V domain, H Domains and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer and may have a V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H Or V L It may contain domains.

[0052] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the invention include: (i) a VH -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may be in non-covalent association with each other and / or with one or more monomeric V H Or V LIt may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (e.g., via disulfide bonds) of the domains. The term "antibody" as used herein also includes multispecific (eg, bispecific) antibodies.

[0053] Multispecific antibodies or antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format can be adapted for use in connection with the antibodies or antigen-binding fragments of antibodies of the invention using routine techniques available in the art. For example, the invention includes methods involving the use of bispecific antibodies, in which one arm of the 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 is conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (such as common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2.sup.2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6,1-11, and references cited therein, for a discussion of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0054] The antibody used in the method of the present invention may be a human antibody. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the human antibody of the present invention may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) that are not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0055] The antibodies used in the methods of the invention may be recombinant human antibodies. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (discussed in more detail below), antibodies isolated from recombinant, combinatorial human antibody libraries (discussed in more detail below), antibodies isolated from animals (e.g., mice) that have been transgenic for human immunoglobulins (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when human Ig-sequence transgenic animals are used), thereby improving the V H Area and V L The amino acid sequence of the region is H Array and V LWhile the sequences are derived from and related to the sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0056] According to certain embodiments, the antibody used in the methods of the invention specifically binds to PD-1. The term "specifically binds" and the like means that the antibody or antigen-binding fragment thereof 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, and the like. For example, as used in the context of the present invention, an antibody that "specifically binds" to PD-1 has a K 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 by a surface plasmon resonance assay. D The term "antibody" includes antibodies that specifically bind to PD-1, or portions thereof, in a human PD-1 context. However, an isolated antibody that specifically binds to human PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0057] According to certain exemplary embodiments of the present invention, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity determining region (CDR) comprising any of the amino acid sequences of the anti-PD-1 antibodies described in U.S. Patent No. 9,987,500. In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof that may be used in connection with the methods of the present invention comprises a heavy chain complementarity determining region (HCDR) of the heavy chain variable region (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) comprising the amino acid sequence of SEQ ID NO:34. According to certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), where 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, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 33 and a LCVR comprising SEQ ID NO: 34. In certain embodiments, the methods of the invention comprise the use of an anti-PD-1 antibody, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42. An exemplary antibody comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 33 and a LCVR comprising the amino acid sequence of SEQ ID NO: 34 is the fully human anti-PD-1 antibody known as REGN2810 (also known as cemiplimab, LIBTAYO®). According to certain exemplary embodiments, the methods of the invention comprise the use of REGN2810, or a biological equivalent thereof.As used herein, the term "bioequivalent" refers to an anti-PD-1 antibody or PD-1 binding protein or fragment thereof that is a pharmaceutical equivalent or pharmaceutical substitute that exhibits no significant difference in rate and / or extent of absorption from REGN2810 when administered in either single or multiple doses at the same molar dose under similar experimental conditions. In the context of the present invention, the term refers to an antigen binding protein that binds to PD-1 that has no clinically meaningful differences in safety, purity and / or potency from REREGN2810.

[0058] Other anti-PD-1 antibodies that may be used in the context of the methods of the invention include antibodies referred to and known in the art as, for example, nivolumab (U.S. Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (U.S. Pat. No. 8,609,089), pidilizumab (U.S. Pat. No. 8,686,119), or any of the anti-PD-1 antibodies described in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0059] The anti-PD-1 antibodies used in connection with the methods of the invention may have pH-dependent binding properties. For example, an anti-PD-1 antibody for use in the methods of the invention may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, an anti-PD-1 antibody of the invention may exhibit enhanced binding to its antigen at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, e.g., 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 less. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0060] In certain instances, "reduced binding to PD-1 at acidic pH compared to neutral pH" refers to the K D K value of antibody binding to PD-1 at neutral pH D For example, an antibody or antigen-binding fragment thereof may have an acidic / neutral K value of about 3.0 or greater. D For purposes of the present invention, an antibody or antigen-binding fragment of the present invention may be considered to exhibit "reduced binding to PD-1 at acidic pH compared to neutral pH." In certain exemplary embodiments, an antibody or antigen-binding fragment of the present invention may exhibit an acidic / neutral K D The ratio can be about 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 more. Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced binding (or increased binding) to a particular antigen at acidic pH compared to neutral pH.

[0061] In addition, modifications of the antigen-binding domain in amino acid concentration can produce antibodies with pH-dependent properties. For example, by replacing one or more amino acids in the antigen-binding domain (e.g., in the CDRs) with histidine residues, an antibody can be obtained that has reduced antigen binding at acidic pH compared to neutral pH. As used herein, the term "acidic pH" refers to a pH of 6.0 or less.

[0062] Bispecific anti-MUC16 / anti-CD3 antibodies and antigen-binding fragments thereof According to certain exemplary embodiments of the invention, the methods include administering a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds CD3 and MUC16. Such antibodies and fragments may be referred to herein, for example, as "anti-MUC16 / anti-CD3," or "anti-MUC16xCD3," or "MUC16xCD3" bispecific antibodies or antigen-binding fragments thereof, or other similar terms.

[0063] As used herein, the expression "bispecific antibody" refers to an immunoglobulin protein comprising at least a first antigen-binding domain and a second antigen-binding domain. In the context of the present invention, 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 distinct 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), each comprising 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.

[0064] The first antigen-binding domain and the second antigen-binding domain are each connected to a separate multimerizing domain. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or composition. In the context of the present invention, a multimerizing component is a multimerizing component having a structure such as (C H2 -C H3The Fc portion of an immunoglobulin (including its Fc domain), such as the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0065] A bispecific antigen-binding molecule of the invention typically comprises two multimerization domains, e.g., two Fc domains, each of which is part of an individual, separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0066] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be operatively linked (e.g., by chemical conjugation, genetic fusion, or non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to generate a bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in connection with the present invention include, but are not limited to, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (such as common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zipper, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6,1-11, and references cited therein for a discussion of the foregoing formats).

[0067] In the context of the bispecific antigen-binding molecules of the invention, the Fc domain may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a naturally occurring version of the wild-type Fc domain. For example, the invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain with an altered binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H2 Area or C H3 The FcRn domain may comprise modifications in regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes at a pH ranging from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in U.S. Patent Publication No. 2015 / 0266966, which is incorporated herein in its entirety.

[0068] The present invention also relates to a first C H 3 domain and second Ig C H The bispecific antigen-binding molecule comprises a first and a second IgC domain. H In one embodiment, the first Ig C domain is a 2-amino acid ... H The 3 domain binds to protein A and the second Ig C H The third domain contains a mutation that reduces or eliminates Protein A binding, for example the H95R modification (according to the IMGT exon numbering; H435R according to the EU numbering). H 3 may further include a Y96F modification (Y436F according to IMGT and according to the EU). See, e.g., U.S. Patent No. 8,586,713. HFurther modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0069] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4. H The chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A particular example of a chimeric Fc domain that may be included in any of the antigen binding molecules described herein is, from the N-terminus to the C-terminus, [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from the N-terminus to the C-terminus, [IgG1 C HIgG1 CH3]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen binding molecules of the invention are described in U.S. Patent Publication No. 2014 / 0243504, which is incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding and therefore affect Fc effector function.

[0070] According to certain exemplary embodiments of the invention, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof 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 of the amino acid sequences of the bispecific anti-MUC16 / anti-CD3 antibodies described in U.S. Patent Publication No. 2018 / 0112001. In certain exemplary embodiments, bispecific anti-MUC16 / anti-CD3 antibodies or antigen-binding fragments thereof that may be used in connection with the methods of the invention include (a) a first antigen-binding arm comprising heavy chain complementarity determining regions (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO:1 and light chain complementarity determining regions (A-LCDR1, A-LCDR2, and A-LCDR3) of a light chain variable region (A-LCVR) comprising the amino acid sequence of SEQ ID NO:2, and (b) a second antigen-binding arm comprising heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) comprising 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 light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a LCVR (B-LCVR) comprising the amino acid sequence of SEQ ID NO: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:28; and B-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; B-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; and B-LCDR3 comprises the amino acid sequence of SEQ ID NO:13.In yet other embodiments, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof comprises (a) a first antigen-binding arm comprising an HCVR comprising SEQ ID NO:1 (A-HCVR) and a LCVR comprising SEQ ID NO:2, and (b) a second antigen-binding arm comprising an HCDR comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 (B-HCDR) and a LCVR comprising SEQ ID NO:2 (B-LCVR). In certain exemplary embodiments, the bispecific anti-CD3xMUC16 antibody comprises a MUC16-binding arm comprising 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 a CD3-binding arm comprising 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 certain exemplary embodiments, the bispecific anti-CD3xMUC16 antibody comprises a MUC16-binding arm comprising 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 a CD3-binding arm comprising 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.

[0071] In certain embodiments, the anti-tumor activity of the bispecific anti-CD3xMUC16 antibodies or antigen-binding fragments thereof of the invention is not substantially inhibited by the presence of high levels (e.g., up to 10,000 U / ml) of circulating CA125. Serum levels of CA125 are elevated in the serum of most ovarian cancer patients (median published levels are about 656 U / ml). As shown in Example 2 below, high levels of CA125 in serum or ascites do not significantly interfere with the anti-tumor profile of the bispecific antibodies of the invention.

[0072] Other bispecific anti-MUC16 / anti-CD3 antibodies that may be used in connection with the methods of the invention include, for example, any of the antibodies described in U.S. Patent Application Publication No. 20180112001.

[0073] Combination therapy According to certain embodiments, the methods of the invention include administering to a subject an anti-MUC16 / anti-CD3 bispecific antibody or antigen-binding fragment thereof in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the methods of the invention include administering the antibodies (or fragments) for additive or synergistic activity to treat cancer, preferably ovarian cancer. As used herein, the term "in combination with" means that the anti-MUC16 / anti-CD3 bispecific antibody or antigen-binding fragment thereof is administered before, after, or simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof. The term "in combination with" also includes sequential or simultaneous administration of the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof. For example, when administered "prior to" the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, the anti-PD-1 antibody, or antigen-binding fragment thereof, 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 before administration of the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof. For example, when administered "after" the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, the anti-PD-1 antibody, or antigen-binding fragment thereof, may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than about 72 hours after administration of the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof."Concurrent administration" with the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, means that the anti-PD-1 antibody, or antigen-binding fragment thereof, is administered to the subject in a separate dosage form within less than five minutes of (before, after, or simultaneously with) administration of the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof, or is administered to the subject as a single, combined dosage formulation that includes both the anti-PD-1 antibody, or antigen-binding fragment thereof, and the bispecific anti-MUC16 / anti-CD3 antibody, or antigen-binding fragment thereof.

[0074] In certain embodiments, the methods of the invention include administration of a third therapeutic agent, where the third therapeutic agent is an anti-cancer agent. In certain embodiments, the methods of the invention include administering an anti-PD-1 antibody, or antigen-binding fragment thereof, and an anti-MUC16 / anti-CD3 bispecific antibody, or antigen-binding fragment thereof, in combination with radiation therapy to generate a long-lasting anti-tumor response and / or enhance survival of patients with cancer.

[0075] In some embodiments, the methods of the invention include administering radiation therapy before, simultaneously with, or after administering the anti-PD-1 antibody or antigen-binding fragment thereof and the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to a cancer patient. For example, radiation therapy may be administered in one or more doses to a tumor lesion after administration of one or more doses of the antibody (or fragment). In some embodiments, radiation therapy may be administered locally to a tumor lesion after systemic administration of an anti-PD-1 antibody or antigen-binding fragment thereof and / or a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof to enhance local immunogenicity of the patient's tumor (adjuvinating radiation) and / or kill tumor cells (ablative radiation).

[0076] Pharmaceutical Compositions and Administration The present invention includes methods comprising administering to a subject a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, the antibody or antibody (or fragment) being contained within a separate or combined (single) pharmaceutical composition. The pharmaceutical compositions of the present invention may be formulated with suitable carriers, excipients, and other agents that provide suitable transport, delivery, tolerance, and the like. Numerous suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311. Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, and the like (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).

[0077] Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents.

[0078] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily applied in the delivery of the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is sold in a pre-filled state with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0079] A number of reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 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 ... Examples of disposable pen delivery devices having application in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR Pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK (trademark) Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (trademark) Pen (Abbott Labs, Abbott Park IL).

[0080] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.

[0081] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. The aqueous medium for injection may be, for example, physiological saline, an isotonic solution containing glucose, and other auxiliary agents, which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, and the like may be employed, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0082] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient.Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0083] Dosing regimen The invention includes methods comprising administering to a subject a bispecific anti-MUC16xCD3 antibody, or antigen-binding fragment thereof, and / or an anti-PD-1 antibody, or antigen-binding fragment thereof, at a dosing frequency of about four times per week, twice per week, once per 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 twelve weeks, or less frequently, so long as a therapeutic response is achieved.

[0084] According to certain embodiments of the invention, multiple doses of the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, may be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering to a subject one or more doses of the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. As used herein, "sequentially administering" means that each dose of the antibody or antigen-binding fragment thereof is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient a single initial dose of the antibody or antigen-binding fragment thereof, followed by one or more secondary doses of the antibody or antigen-binding fragment thereof, and optionally followed by one or more tertiary doses of the antibody or antigen-binding fragment thereof.

[0085] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of an antibody or antigen-binding fragment thereof (anti-PD-1 antibody or bispecific antibody). However, in certain embodiments, the amounts contained in the initial dose, secondary dose, and / or tertiary dose differ from each other (e.g., adjusted up or down) during the course of treatment. In certain embodiments, one or more doses (e.g., 1, 2, 3, 4, or 5) are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis. For example, an anti-PD-1 antibody or antigen-binding fragment thereof may be administered to a patient with ovarian cancer at a loading dose of about 1 to 3 mg / kg, followed by one or more maintenance doses of about 0.1 to about 20 mg / kg of the patient's body weight. In an exemplary embodiment of the invention, each secondary dose and / or tertiary dose is administered ½ to 14 weeks after the immediately preceding dose (e.g., ½ week, 1 week, 1 ½ weeks, 2 weeks, 2 ½ weeks, 3 weeks, 3 ½ weeks, 4 weeks, 4 ½ weeks, 5 weeks, 5 ½ weeks, 6 weeks, 6 ½ weeks, 7 weeks, 7 ½ weeks, 8 weeks, 8 ½ weeks, 9 weeks, 9 ½ weeks, 10 weeks, 10 ½ weeks, 11 weeks, 11 ½ weeks, 12 weeks, 12 ½ weeks, 13 weeks, 13 ½ weeks, 14 weeks, 14 ½ weeks, or more).

[0086] As used herein, the phrase "the immediately preceding dose" refers to a dose of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof (and / or an anti-PD-1 antibody or antigen-binding fragment thereof) administered to a patient prior to administration of the immediately succeeding dose in a series of multiple doses, with no intervening doses between.

[0087] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof (and / or an anti-PD-1 antibody or antigen-binding fragment thereof) to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0088] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding administration. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding administration. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0089] In certain embodiments, one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof (e.g., and anti-PD-1 antibody or antigen-binding fragment thereof) are administered more frequently (twice weekly, once weekly, or once every two weeks) at the start of a treatment regimen as an "induction dose," followed by subsequent doses ("consolidation doses" or "maintenance doses") administered less frequently (e.g., once every 4-12 weeks).

[0090] The present invention includes methods comprising sequentially administering to a patient a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, alone or in combination with an anti-PD-1 antibody or antigen-binding fragment thereof, to treat ovarian cancer (e.g., serous cancer). In some embodiments, the method comprises administering one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, optionally followed by one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the method comprises administering a single dose of an anti-PD-1 antibody or antigen-binding fragment thereof, followed by one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof. For example, to inhibit tumor growth and / or prevent tumor recurrence in a subject with ovarian cancer, one or more doses of about 0.1 mg / kg to about 20 mg / kg of an anti-PD-1 antibody or antigen-binding fragment thereof can be administered, followed by one or more doses of about 0.1 mg / kg to about 20 mg / kg of a bispecific antibody or antigen-binding fragment thereof. In some embodiments, administration of one or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof, followed by administration of one or more doses of a bispecific antibody or antigen-binding fragment thereof, results in an increased anti-tumor effect (e.g., greater inhibition of tumor growth, improved prevention of tumor recurrence compared to untreated subjects or subjects administered either the antibody or antigen-binding fragment thereof as monotherapy). Alternative embodiments of the invention relate to co-administration of an anti-PD-1 antibody or antigen-binding fragment thereof and a bispecific antibody or antigen-binding fragment thereof, administered at a similar or different frequency in separate doses compared to the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is administered before, after, or simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof, hi certain embodiments, the bispecific antibody or antigen-binding fragment thereof is administered together with the anti-PD-1 antibody or antigen-binding fragment thereof in a single dose formulation.

[0091] Dosage The amount of bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, and optionally anti-PD-1 antibody or antigen-binding fragment thereof, administered to a subject according to the methods of the invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of an antibody or antigen-binding fragment thereof (anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody) that results in one or more of the following, compared to an untreated subject or a subject administered either antibody (or fragment) as a monotherapy: (a) a reduction in the severity of cancer (e.g., ovarian cancer) or the duration of cancer symptoms; (b) inhibition of tumor growth or an increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) a delay in tumor growth and development; (d) inhibition, delay, or cessation of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) an increase in survival of a subject with cancer (e.g., ovarian cancer); and / or (g) a reduction in the use or need for conventional anti-cancer therapy (e.g., a reduction or elimination of the use of chemotherapeutic or cytotoxic agents).

[0092] For a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, a therapeutically effective amount may be from about 0.1 milligrams (mg) to about 1000 mg, e.g., 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 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, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 400 mg, about 400 mg, about 450 mg, about 50 mg, about 600 mg, about 650 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 250 mg, about The bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof may be about 1 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, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg. In some cases, the dose is about 1 mg. In some cases, the dose is about 2 mg. In some cases, the dose is about 20 mg. In some cases, the dose is about 25 mg. In some cases, the dose is about 250 mg. In some cases, the dose is about 800 mg. Any of these doses can be an initial dose, an intermediate or transitional dose, or a full dose.

[0093] In the case of an anti-PD-1 antibody or an antigen-binding fragment thereof, the therapeutically effective amount is 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, or about 280 mg. The amount of the anti-PD-1 antibody or antigen-binding fragment thereof may be about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. In a specific embodiment, 350 mg of the anti-PD-1 antibody or antigen-binding fragment thereof is administered.

[0094] The amount of bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, and optionally anti-PD-1 antibody or antigen-binding fragment thereof, contained within an individual dose may be expressed in terms of milligrams of antibody or antigen-binding fragment thereof per kilogram of subject's body weight (i.e., mg / kg). In certain embodiments, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof, and optionally anti-PD-1 antibody or antigen-binding fragment thereof used in the methods of the invention may be administered to a subject at a dose of about 0.0001 to about 100 mg / kg of the subject's body weight. For example, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof may be administered at a dose of about 0.1 mg / kg to about 20 mg / kg of the patient's body weight, and the optional anti-PD-1 antibody or antigen-binding fragment thereof may be administered at a dose of about 0.1 mg / kg to about 20 mg / kg of the patient's body weight.

[0095] A summary of the sequences referred to herein and their corresponding SEQ ID NOs is provided in Table 1 below. [Table 1] EXAMPLES

[0096] The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure. Example 1: Generation of a bispecific antibody that binds ovarian cell-specific (MUC16) and CD3 The present invention also provides bispecific antigen-binding molecules that bind to CD3 and MUC16, such bispecific antigen-binding molecules are also referred to herein as "anti-MUC16 / anti-CD3 or anti-MUC16xCD3 bispecific molecules."

[0097] The anti-MUC16 portion of the anti-MUC16 / anti-CD3 bispecific molecule is useful for targeting tumor cells expressing MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of MUC16 on tumor cells and CD3 on T cells promotes direct killing (cytolysis) of the targeted tumor cells by the activated T cells.

[0098] Bispecific antibodies comprising an anti-MUC16 specific binding domain and an anti-CD3 specific binding domain were constructed using standard methodology, with the anti-MUC16 antigen binding domain and the anti-CD3 antigen binding domain each comprising a distinct HCVR paired with a common LCVR. In the exemplified bispecific antibody, the molecule was constructed utilizing 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 examples, bispecific antibodies may be constructed utilizing 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 an antibody light chain that is known to be promiscuous or that is known to pair effectively with a variety of heavy chain arms.

[0099] Exemplary bispecific antibodies were generated with an IgG1 Fc domain (BSMUC16 / CD3-001, -002, -003, and -004) or an engineered (chimeric) IgG4 Fc domain (BSMUC16 / CD3-005) as described in U.S. Patent Application Publication No. US2014 / 0243504A1, published August 28, 2014.

[0100] An overview of the antigen-binding domain components of the various anti-MUC16xCD3 bispecific antibodies constructed is provided in Table 2. [Table 2]

[0101] Example 2: CA-125 does not interfere with anti-MUC16xCD3 antibody activity in vitro The effect of soluble CA-125 (a shed form of MUC16) on the activity of BSMUC16 / CD3-001 was assessed using FACS binding and cytotoxicity assays in the presence of high levels of CA-125 purified from ascites of ovarian cancer patients. CA-125 levels are increased in the serum of most ovarian cancer patients, and circulating levels may impact any MUC16-targeted therapy by acting as an antigen sink. The levels of CA-125 used in the assay (10,000 U / ml) significantly exceeded the published median level of 656.6 U / mL in ovarian cancer patients. The ability of BSMUC16 / CD3-001 to kill MUC16-expressing OVCAR-3 cells in the presence of soluble CA-125 enriched from human ascites (creative Biomart, NY, USA) or a membrane proximal construct expressing the five carboxy-terminal SEA domains and the juxtamembrane region of MUC16 (MUC16Δ) was performed at a fixed concentration of BSMUC16 / CD3-001 or CD3-binding control antibody (100 pM) and serial dilutions of either MUC16-1H or MUC16Δ for 72 h at 37 °C at an effector / target ratio of 4:1. To monitor the specific killing of MUC16-bearing target cells, OVCAR-3 cells were labeled with 1 uM Violet Cell tracker. After labeling, cells were cultured overnight at 37 °C. Separately, human PBMCs were cultured at 1 × 10 6PBMCs were cultured in RPMI medium supplemented with 1000 ng / mL of IgG and incubated overnight at 37°C to enrich for lymphocytes by depleting adherent cells. The next day, target cells were co-incubated with naïve PBMCs depleted of adherent cells (effector / target cell ratio 4:1) and serial dilutions of either BSMUC16 / CD3-001 or CD3-binding control for 72 h at 37°C. Cells were removed from the culture plate using trypsin and analyzed by FACS. For FACS analysis, cells were stained with a dead / live far-red cell tracker (Invitrogen). To assess the specificity of killing, cells were gated on the population labeled with a violet cell tracker. To calculate adjusted viability, the percentage of live target cells was reported as follows: adjusted viability = (R1 / R2) * 100, where R1 = percentage of live target cells in the presence of antibody, and R2 = percentage of live target cells in the absence of test antibody. T cell activation was assessed by incubating cells with directly conjugated antibodies against CD2, CD69, and CD25 and reporting the percentage of activated (CD69+) or (CD25+) T cells among total T cells (CD2+).

[0102] Binding of BSMUC16 / CD3-001 to an antibody known to bind CA-125 (clone 3A5) from human ascites fluid was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, soluble CA-125 (Creative Biomart, NY, USA) was passively adsorbed to 96-well microtiter plates at a concentration of 4000 units / mL in PBS overnight at 4°C. Plates were then washed with PBST and blocked with 0.5% BSA in PBS for 1 h. Biotinylated BSMUC16 / CD3-001, MUC16 parental antibody, a-MUC16 3A5, and non-binding controls (BSMUC16 / CD3-001 isotype control and a-MUC16 3A5 isotype control) were added to the plates at concentrations of 10, 1, 0.3, or 0.1 nM in 0.5% BSA in PBS for 1 h, followed by washing with PBST. Streptavidin conjugated with horseradish peroxidase (SA-HRP) (ThermoFisher Scientific, Waltham, MA, USA) at a 1:10000 dilution of a 1.0 mg / mL stock solution was added to the wells and incubated for 1 h to detect plate-bound biotinylated antibodies. Plates were washed and developed with 3-3',5-5'-tetramethylbenzidine (BD Biosciences, Franklin Lakes, NJ, USA) substrate according to the manufacturer's instructions. The absorbance at 450 nm was recorded for each well on a Victor Multilabel Plate Reader (Perkin Elmer, Melville, NY). Data were analyzed using GraphPad Prism software.

[0103] Excess CA-125 had minimal effect on BSMUC16 / CD3-0001 binding to OVCAR-3 cells, suggesting minimal binding to CA-125 (Figure 1). In contrast, CA-125 significantly inhibited the ability of a comparison antibody that likely binds to the repeat region of MUC16 (in-house version of antibody clone 3A) (Figure 1). Furthermore, a soluble MUC16 construct containing the membrane proximal region up to the fifth SEA domain of MUC16 (MUC16Δ) dramatically inhibited BSMUC16 / CD3-001 binding, indicating that BSMUC16 / CD3-001 binds to the membrane proximal region, as discussed in more detail in WO 2018 / 067331, which is incorporated herein by reference. Consistent with the binding studies, BSMUC16 / CD3-001 could also induce T cell-mediated killing in the presence of CA-125, but not in the presence of high concentrations of MUC16Δ (data not shown). Thus, BSMUC16 / CD3-001 can bind MUC16 and induce T cell redirected killing even in the presence of high concentrations of CA-125.

[0104] Example 3: PD-1 blockade enhances the antitumor activity of anti-MUC16 x CD3 bispecific antibodies in xenogeneic and syngeneic tumor models The in vivo efficacy of anti-MUC16 / anti-CD3 bispecific antibodies in combination with PD-1 blockade was evaluated in xenogeneic and syngeneic tumor models.

[0105] A. Heterogeneous model - OVCAR-3 / Luc In the xenogeneic model, OVCAR-3 / Luc cell lines previously passaged in vivo (day 0) were injected intraperitoneally (IP) into immunodeficient NSG mice thirteen days after engraftment with human PBMCs. Mice were treated IP with 12.5ug / mouse BSMUC16 / CD3-001 or 12.5ug of CD3 binding control was administered on days 5 and 8, alone or in combination with 100ug of REGN2810. Tumor burden was assessed by BLI on days 4, 8, 12, 15, 20, and 25 after tumor engraftment. Treatment with 12.5ug of BSMUC16 / CD3-001 resulted in significant anti-tumor effects as determined by BLI measurements on day 25, and combination with REGN2810 (anti-PD-1) further enhanced the anti-tumor effects. All groups had similar tumor burdens assessed by BLI prior to dosing. There were no significant differences in tumor burden between the groups.

[0106] BSMUC16 / CD3-001 significantly reduces tumor burden at 12.5ug and the addition of anti-PD-1 enhances the anti-tumor effect more than BSMUC16 / CD3-001 alone. Human T cell engrafted NSG mice were implanted with human OVCAR-3 / Luc cells. Mice were treated with 12.5ug BSMUC16 / CD3-001 IV or CD3 binding or non-binding controls (12.5ug IV) on days 5 and 8. Data shown in Table 3 below is tumor burden assessed by BLI 25 days after tumor implantation. Statistical significance was determined using an unpaired non-parametric Mann-Whitney t-test. Treatment with BSMUC16 / CD3-001+ / -REGN2810 was compared to CD3 binding control (*p<0.05 for BSMUC16 / CD3-001, **p<0.01 for BSMUC16 / CD3-001 and REGN2810) and treatment with BSMUC16 / CD3-001 alone was compared to the combination with REGN2810 (#p<0.05). [Table 3]

[0107] B. Syngeneic model - ID8-VEGF / huMUC16 To test efficacy in immune-competent models, the mouse CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with a human sequence, resulting in mice whose T cells express human CD3 and a chimeric MUC16 molecule that contains the portion of human MUC16 bound by the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies.

[0108] For this first syngeneic tumor model, we used the ID8-VEGF cell line, which was engineered to express a portion of human MUC16. Mice were implanted with ID8-VEGF / huMUC16 cells IP and treated three days after implantation with 5mg / kg BSMUC16 / CD3-001, or a CD3 binding control with an isotype control, or in combination with anti-PD-1 (5mg / kg IV). Treatment with BSMUC16 / CD3-001 extended median survival compared to the group that received the CD3 binding control, but the addition of anti-PD-1 blockade also resulted in 50% survival of the mice.

[0109] BSMUC16 / CD3-001 significantly increases median survival in the ID8-VEGF ascites model, and the addition of PD-1 (EGN2810) blockade allows survival in some mice. Mice expressing human CD3 in place of mouse CD3 and the chimeric MUC16 molecule were implanted with a mouse ovarian tumor line expressing a portion of human MUC16. Mice were administered BSMUC16 / CD3-001 (5 mg / kg IV) or CD3 binding control (5 mg / kg IV) along with isotype control or anti-PD-1 on day 3 after implantation. Mice were treated on days 3, 7, 10, 14, and 17 after tumor implantation. Data shown are median survival times. Mice were sacrificed when body weight increased by 20% or more due to abdominal distension induced by ascites. Statistical significance was determined using the Mantel-Cox method. Both BSMUC16 / CD3-001 and BSMUC16 / CD3-001+anti-PD-1 treatments resulted in increased median survival, with the combination of BSMUC16 / CD3-001+anti-PD-1 resulting in a 50% survival, demonstrating synergy between the MUC16xCD3 bispecific antibody and the anti-PD-1 antibody. The results are shown in Table 4 below. [Table 4]

[0110] Similar results were observed when BSMUC16 / CD3-001 was administered at 1 mg / kg in combination with an anti-PD-1 antibody.

[0111] C. Similar model - MC38 / huMUC16 As described above, the mice used in this experiment were genetically engineered to replace the mouse CD3 gene with human CD3 and to replace a portion of the mouse MUC16 gene with human sequences, resulting in mice whose T cells express human CD3 and a chimeric MUC16 molecule that contains the portion of human MUC16 that is bound by the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies.

[0112] For this second syngeneic tumor model, the MC38 cell line engineered to express a portion of human MUC16 was used. MC38 / huMUC16 cells SC were implanted into mice and treated 7 days after tumor implantation with BSMUC16 / CD3-005, or with CD3 binding control with isotype control (1 mg / kg IV), or in combination with anti-PD-1 (5 mg / kg IV). The anti-PD-1 antibody used in this experiment was a commercially available mouse antibody (clone RMP1-14, BioXCell). The combination of BSMUC16 / CD3-005 and anti-PD-1 showed synergistic anti-tumor effects.

[0113] The combination of BSMUC16 / CD3-005 and anti-PD-1 blockade resulted in better antitumor efficacy than BSMUC16 / CD3-005 alone in the MC38 SC model. Mice expressing human CD3 in place of mouse CD3 and the chimeric MUC16 molecule were implanted with the murine tumor line MC38, which expresses a portion of human MUC16. Mice were administered BSMUC16 / CD3-005 or CD3 binding control (1 mg / kg IV) along with isotype control or anti-PD-1 antibody (5 mg / kg IV) on day 7 post-implantation. Mice were treated on days 7, 11, and 14 post-tumor implantation. Results are shown in Figure 2. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparison test. BSMUC16 / CD3-005 plus anti-PD-1 significantly and synergistically inhibited tumor growth more than the CD3 binding control.

[0114] Example 4: ImmunoPET imaging in genetically engineered mice showed localization of anti-MUC16xCD3 bispecific antibodies to T cell-rich organs In vivo localization of BSMUC16 / CD3-001 and BSMUC16 / CD3-005, as well as MUC16 protein expression, was assessed in wild-type and genetically humanized mice using PET imaging. 89The biodistribution of Zr-labeled anti-MUC16 antibodies (herein referred to as "parental", bivalent anti-MUC16 antibodies generated using the same anti-MUC16 heavy and light chains as the bispecific) was similar in both wild-type and humanized mice, suggesting low expression / availability of the humanized MUC16 protein to the antibody. In contrast, in mice 89 When therapeutically relevant doses of the Zr-labeled BSMUC16 / CD3-001 bispecific antibody were administered, distribution to the spleen and lymph nodes was evident due to recognition of CD3-positive T cells in these lymphoid organs (data not shown). Ex vivo biodistribution analysis in individual tissues confirmed localization to lymph nodes and spleen (data not shown). 89 The uptake of the Zr-labeled BSMUC16 / CD3-005 bispecific antibody was significantly reduced compared to BSMUC16 / CD3-001 due to its lower affinity for CD3. To evaluate whether BSMUC16 / CD3-001 and BSMUC16 / CD3-005 could accumulate in MUC16-expressing tumors, 89 Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005 was administered to mice bearing ID8-VEGF-huMUC16Δ tumors. Tumor uptake between the bispecific antibodies was not significantly different, despite higher lymphocyte uptake of BSMUC16 / CD3-001 (data not shown).

[0115] Preparation of immunoconjugates and small animal PET: BSMUC16 / CD3-001 and control antibodies were conjugated with DFO at the glutamine residue at position 295 via transamidation with microbial transglutaminase after deglycosylation of the antibodies with PNGase F. The DFO-conjugated antibodies were then transferred to Zirconium-89 ( 89 The antibodies were chelated with 10×10 Zn (Zr). Mice received the antibodies at a final dose of 0.5 mg / kg via tail vein injection. PET imaging was then performed to assess the in vivo localization of the radioimmunoconjugates 6 days after administration prior to ex vivo biodistribution studies. For experiments in tumor-bearing mice, mice were injected with 10×10 6ID8-VEGF-huMUC16Δ tumor cells were implanted subcutaneously. Tumor-bearing mice had tumors with an average size of 150 mm 3 If so, 20 days after transplantation 89 Zr radiolabeled antibody was administered.

[0116] PET / CT images were taken using a pre-calibrated Sofie Biosciences G8 PET / CT instrument (Sofie Biosciences, Culver city, CA and Perkin Elmer). The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. On day 6 post-injection, mice were induced anesthetized using isoflurane and placed under a continuous flow of isoflurane for 10 min of static PET imaging. CT images were acquired after PET acquisition. PET images were then reconstructed using pre-set settings. Attenuation-corrected PET and CT data were processed into false-colored co-registered PET-CT maximum intensity projection images using VivoQuant software (invicro Imaging Services) with a color scale calibrated to indicate a signal range of 0–30% of the injected dose per volume, expressed as %ID / g. For ex vivo biodistribution analysis, mice were euthanized after imaging on day 6 post-injection. Blood was collected into counting tubes via cardiac puncture. Normal tissues (inguinal and axillary lymph nodes, thymus, spleen, heart, lungs, stomach, small intestine, liver, kidneys, bones, and ovaries) were then excised and placed into counting tubes. Tumors were similarly collected into counting tubes. All tubes were pre-weighed and then re-weighed to determine blood and tissue weights. All samples were then counted for gamma emission radioactivity in an automatic gamma counter (Wizard 2470, Perkin Elmer) and results were reported in counts per minute (cpm). The %ID of each sample was determined using the number of samples relative to the number of dose standards prepared from the original injected material. Individual %ID / g values ​​were then derived by dividing the %ID by the respective weights of the appropriate blood, tissue, or tumor sample.

[0117] 89Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005 showed specific localization to MUC16+ tumors and CD3+ lymphoid tissues, with lymphocyte distribution correlating with relative CD3 affinity. Both MUC16xCD3 bispecifics showed comparable tumor localization in the presence of CD3+ tissue.

[0118] Example 5: Toxicity studies in cynomolgus monkeys showed no obvious toxicity for anti-MUC16xCD3 bispecific antibodies BSMUC16 / CD3-001 cross-reacts with monkey MUC16 and CD3. A multiple-dose toxicity study was conducted in cynomolgus monkeys to determine the safety and tolerability of the bispecific antibody and to characterize its pharmacokinetics. Six monkeys / sex / group were administered BSMUC16 / CD3-001 weekly for a total of five doses at 0.01, 0.1, or 1 mg / kg. Upon completion of the dosing period, three animals / sex / group were euthanized and tissues examined for microscopic findings, while the remaining three animals / sex / group were allowed 12 weeks of treatment-free recovery to assess the reversibility or persistence of any BSMUC16 / CD3-001-associated effects. BSMUC16 / CD3-001 was well tolerated and all animals survived to the time of scheduled necropsy. Toxicokinetic analysis demonstrated dose-proportional exposure and linear kinetics across dose groups, with no gender differences observed (data not shown). Continuous exposure to BSMUC16 / CD3-001 was observed throughout the dosing phase, with BSMUC16 / CD3-001 exposure maintained until the end of the recovery phase in all (n=6) and 50% of animals in the 0.1 mg / kg and 1.0 mg / kg groups, respectively. BSMUC16 / CD3-001 was not detectable in serum in any animals in the 0.01 mg / kg group after week 8 of recovery. The elimination half-life of BSMUC16 / CD3-001 was approximately 10 days.

[0119] There were no BSMUC16 / CD3-001-related clinical findings, and no changes in urinalysis parameters, peripheral blood immunophenotyping, food consumption, or body weight during the treatment or recovery periods. Importantly, BSMUC16 / CD3-001 treatment did not result in any changes in respiratory, neurological, or cardiovascular safety pharmacological assessments, including no changes in ECG parameters. No BSMUC16 / CD3-001-related changes in organ weights were found, and no gross changes were observed at either terminal or recovery necropsies. Dose-related, reversible elevations in circulating inflammatory markers (C-reactive protein (CRP) and IL-6) were observed within 1 day of the first dose of 1.0 or 0.1 mg / kg, but these elevations were not evident after subsequent doses (data not shown). In accordance with the minimal increase in serum cytokines, in contrast to what has been described for some CD3 bispecific molecules for hematological tumors, no T-cell redistribution was detected after BSMUC16 / CD3-001 treatment (data not shown).

[0120] Cynomolgus monkey studies were performed in accordance with IACUC guidelines. Cynomolgus monkeys (6 / sex / group) received control substance (diluted placebo) or BSMUC16 / CD3-001 (0.01, 0.1, or 1 mg / kg) via 30-minute IV infusion once weekly. Control substance was 10 mM histidine with 10% sucrose and 0.05% polysorbate 20, pH 6, diluted in 0.9% Sodium Chloride for Injection, USP (sterile saline). Blood samples or tissues were collected at various time points for clinical pathology and histopathology. BSMUC16 / CD3-001 concentrations were determined by ELISA, and toxicokinetic analysis was performed using WinNonLin software. CRP was analyzed on a RocheRoche P 800 system. Cytokines were measured by MSD (Meso Scale Diagnostics, Rockville, MD). T cells were quantified using flow cytometry. Briefly, blood is collected into potassium EDTA tubes, lysed, and stained for CD3, CD4, and CD8 (BD Biosciences), and the relative values ​​of each phenotype are determined using a FACS Canto II. These values ​​are then multiplied by the absolute lymphocyte values ​​(via hematology analysis) to count the absolute cell numbers of each phenotype.

[0121] Immunohistochemical staining for MUC16 was present in the expected tissues, pancreas (mesothelium, ductal epithelium), heart and ovaries (data not shown), as well as salivary glands (goblet cells), liver (mesothelium, bile duct), lung (mesothelium, bronchioles / bronchial epithelium), small intestine (mesothelium), testis (mesothelium, rete testis / efferent ducts), and tonsils (epithelium, mucous glands) (not shown). BSMUC16 / CD3-001-associated microscopic changes, assessed by hematoxylin and eosin (H&E) histological staining, included inflammation (infiltration of leukocytes), and increased size and cellularity of mesothelial cells leading to non-nociceptive thickening of the serosal lining and / or submesothelial connective tissue in multiple thoracic and peritoneal organs. These changes were generally focal or multifocal, minimal to mild in severity, and resulted from engagement of MUC16 expressed on serosal epithelial (mesothelial) cells and activation of T cells, considered to be the target of BSMUC16 / CD3-001. Importantly, the serosal changes had reversed or trended toward reversal at the end of the recovery period (data not shown).

[0122] Toxicity studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein following BSMUC16 / CD3-001 administration, with no overt toxicity.

[0123] Example 6: Evaluation of serum cytokine induction in tumor-bearing mice Because cytokine release syndrome (CRS) is a frequent and severe side effect of CD3 bispecific and CAR T cell therapy, we performed studies to monitor serum cytokines in relevant models following treatment with BSMUC16 / CD3-001.In tumor-free genetically humanized MUC16 / CD3 mice, no serum cytokine response was evident upon BSMUC16 / CD3-001 administration.

[0124] To assess in vivo T cell activation by BSMUC16 / CD3-001, serum cytokine levels from tumor-bearing mice were measured. Serum samples were collected 4 hours after the first antibody dose in the 0.5 mg / kg BSMUC16 / CD3-001, CD3-binding control, and non-binding control groups. Treatment with BSMUC16 / CD3-001 activated T cells as determined by induction of IFNγ, TNFα, IL-2, IL-6, IL-8, and IL-10 compared to non-binding and CD3-binding controls (data not shown). BSMUC16 / CD3-001-induced cytokine responses required the presence of T cells as well as OVCAR-3 / Luc cells; mice with only OVCAR-3 / Luc cells had no detectable human IFNγ in serum, and mice without tumor cells providing MUC16 for cross-linking did not show an increase in serum IFNγ in response to BSMUC16 / CD3-001 (data not shown).

[0125] Measurement of serum cytokine levels: T cell activation in response to treatment with BSMUC16 / CD3-001 was assessed by measuring serum concentrations of interferon gamma (IFN γ), tumor necrosis factor alpha (TNFα), interleukin-2 (IL-2), IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and IL-1B four hours after the first 0.5 mg / kg dose. Cytokine levels were analyzed using a V-plex Human ProInflammatory-10 Plex kit (Meso Scale Diagnostics, Rockville, MA) according to the manufacturer's instructions. Cytokines were measured in two separate studies with 4-6 mice per group.

[0126] Example 7: MUC16 Expression in Humanized Mice and the Effect of Anti-MUC16xCD3 Bispecific Antibody on MUC16-Positive Tissues To investigate the antitumor effects of BSMUC16 / CD3-001 in mice with a completely intact immune system, mice were genetically engineered to express human CD3 on T cells and the region of MUC16 covering the antibody binding region at the endogenous mouse locus (knock-in mice). To validate these mice, MUC16 expression was examined by both RT-PCR and IHC. Similar to published data on mouse MUC16 expression, RNA expression was detected at low levels in the trachea as well as in the lung, heart, ovaries, pancreas, and bladder (data not shown). To assess MUC16 protein expression, IHC was performed on selected tissues using an anti-human MUC16 antibody that recognizes the membrane proximal 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 as well as submucosal glands as described in humans (data not shown).

[0127] Histology of Mouse Tissues:Tissues from humanized or WT mice were harvested and stained with anti-MUC16 antibody, which binds to the membrane proximal domain of MUC16, by IHC using a Ventana Discovery XT (Ventana; Tucson, AZ). 5 μm paraffin sections were cut and mounted on Superfrost PLUS slides and baked at 60°C for 1 h. Immunohistochemistry was performed on a Discovery XT automated IHC staining system using the Ventana DAB Map detection kit. Deparaffinization was performed at 75°C for 8 min using EZ Prep solution. Mild antigen retrieval (95°C, 8 min, followed by 100°C, 24 min) was performed using Ventana Tris-EDTA buffer pH 9 (CC1). This was followed by multiple blocking steps. Tissue sections were incubated with anti-MUC16 antibody (2 μg / ml) for 8 h at room temperature. An isotype control antibody that recognizes an irrelevant non-binding antibody was used as a negative control. Primary antibodies and negative controls were applied manually. Biotinylated goat anti-human IgG (Jackson ImmunoResearch) was used as the secondary antibody (1 μg / ml) and samples were incubated for 1 h at RT. Color signals were developed using the Ventana DAB MAP kit. Slides were manually counterstained with hematoxylin (2 min), dehydrated and coverslipped. Images were acquired with 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, Inc (Germantown, MD, USA).

[0128] T cells in these mice were polyclonal as assessed by T cell receptor (TCR) Vβ usage, expressed human CD3, and were present in numbers similar to wild-type mice (data not shown). To determine whether BSMUC16 / CD3-001 induced any T cell activation or effects on normal tissues in these animals, non-tumor-bearing mice were injected with a high dose of BSMUC16 / CD3-001 (10 mg / kg) and then examined T cell numbers in the blood, serum cytokines, and histopathology. Although T cells could be activated by an anti-human CD3 antibody (OKT3), as measured by margination of T cells from the blood and increased levels of serum cytokines (data not shown), BSMUC16 / CD3-001 did not induce such effects, suggesting limited access to MUC16 targets (data not shown). To determine whether BSMUC16 / CD3-001 induced any microscopic changes in MUC16-expressing tissues, MUC16 and CD3 humanized mice were administered two doses of 10 mg / kg BSMUC16 / CD3-001 on days 0 and 3. On day 5, several MUC16-expressing tissues (trachea, stomach, and ovary) were examined and no cellular infiltration or necrosis was observed in these tissues after BSMUC16 / CD3-001 administration (data not shown). Histopathological examination revealed no inflammation or infiltration of MUC16-expressing tissues at any time point examined following BSMUC16 / CD3-001 administration in mice.

[0129] The results of this study, as well as the cynomolgus monkey study described in Example 5, demonstrate the safety profile of BSMUC16 / CD3-001. BSMUC16 / CD3-001 induced only minimal serum cytokines, and there was focal induction of inflammation in MUC16 expression and thickening of the serosal lining, suggesting target activity, but these effects resolved by the end of the recovery period, consistent with increased inflammation and cellularity indicative of repair. The observed serosal changes did not correlate with any clinical findings, clinical pathology (except for inflammatory responses), or microscopic changes in the underlying parenchyma. Thus, studies in both genetically humanized mice and cynomolgus monkeys indicate that BSMUC16 / CD3-001 is well tolerated.

[0130] Example 8: Monitoring PD-1 expression in a FACS-based cytotoxicity assay using naive human effector cells To monitor specific killing of Muc16-bearing target cells by flow cytometry, the ovarian cell line OVCAR-3 was labeled with 1 uM Violet Cell Tracker. After labeling, cells were cultured overnight at 37°C. Separately, human PBMCs were cultured at 1 × 10 6 BSMUC16 / CD3-001 cells / mL in RPMI medium supplemented with 100% PBS and incubated overnight at 37°C to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and localized monocytes. The next day, target cells were co-incubated with naïve PBMCs depleted of adherent cells (effector / target cell ratio 4:1) and serial dilutions of either BSMUC16 / CD3-001 or CD3-binding control for 72 h at 37°C. Cells were removed from the culture plate using trypsin and analyzed by FACS. For FACS analysis, cells were stained with dead / live far-red cell tracker (Invitrogen). To assess the specificity of killing, cells were gated on the population labeled with violet cell tracker.

[0131] PD-1 expression was assessed by incubating cells with antibodies directly conjugated to CD2, CD4, CD8, and PD-1, and reporting the percentage of PD-1 / CD4 positive T cells or PD-1 / CD8 positive T cells among total T cells (CD2+). Incubation with BSMUC16 / CD3-001 increased the percentage of PD-1+ T cells by more than 10-fold (CD4+ T cells) or more than 3-fold (CD8+ T cells) compared to controls. The results are shown in Figure 3.

[0132] Example 9: Methods of Treating Ovarian Cancer with Anti-MUC16xAnti-CD3 Bispecific Antibodies Alone or in Combination with Anti-PD-1 Antibodies A phase 1 / 2 study of the safety, tolerability, preliminary antitumor activity, and pharmacokinetics (PK) of REGN4018 (an anti-MUC16 × anti-CD3 bispecific antibody) in patients with recurrent ovarian cancer who have exhausted all treatment options, including platinum-containing therapy, has been conducted and has shown meaningful clinical benefit.

[0133] Patients with difficult-to-treat, advanced, platinum-experienced, and / or intolerant epithelial ovarian cancer (excluding carcinosarcoma), primary peritoneal cancer, or fallopian tube cancer are being studied as part of the REGN4018 study.

[0134] In clinical trials for ovarian cancer, treatment evaluation is based on tumor response. Tumor response evaluation is based on the levels of the tumor marker CA-125 in patients with and without measurable disease. In addition, biomarker analysis will include peripheral T cell phenotyping, as REGN4018 treatment is expected to transiently reduce the population of peripheral CD3 T cells. Further analysis may include biomarkers such as tumor expression of proteins such as MUC16 and PD-L1, and ctDNA, tumor (RNA and somatic DNA sequencing) genetic analysis for variations that affect the clinical course of the underlying disease or modulate treatment side effects.

[0135] Objectives: To explore both primary and secondary endpoints.

[0136] This exam The main objective of teeth: (1) During the dose escalation phase of the study: To evaluate 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 cemiplimab in patients with recurrent ovarian cancer who have exhausted all treatment options expected to provide meaningful clinical benefit. The determination of the RP2D will be based on review of nonclinical and all clinical data, including those related to safety, pharmacokinetics (PK), and PK / PD (pharmacokinetic / pharmacodynamic) relationships. (2) To evaluate the preliminary efficacy of REGN4018 as monotherapy and in combination with cemiplimab (by cohort) as determined by objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST 1.1) during the dose expansion phase of the study.

[0137] Exam Secondary Objectives (both in the dose escalation and dose expansion phases), (1) To evaluate the preliminary efficacy of REGN4018 as monotherapy and in combination with cemiplimab, as measured by ORR, best overall response (BOR), duration of response (DOR), progression-free survival (PFS), disease control rate, and CR rate; (2) to evaluate the efficacy of REGN4018 as monotherapy and in combination with cemiplimab, as measured by CA-125 levels; (3) to characterize the immunogenicity of REGN4018 and cemiplimab; (4) In the dose escalation phase only: to evaluate the preliminary efficacy of REGN4018 as monotherapy and in combination with cemiplimab, as measured by ORR; and (5) In the dose expansion phase only: To evaluate the safety, PK, and tolerability of REGN4018 as monotherapy and in combination with cemiplimab.

[0138] This test exploratory purpose is as follows: (1) To evaluate the preliminary efficacy of REGN4018 as monotherapy and in combination with cemiplimab (separately for each cohort), as measured by ORR based on a combined assessment of RECIST and CA-125 using Gynecologic Cancer Intergroup (GCIG) criteria; (2) To evaluate the mechanism of action, improved understanding of the disease / target, observed toxicities, and biomarkers that may correlate with potential anti-tumor activity, including but not limited to: - Circulating Proteins - circulating immune cells - Changes in gene expression in peripheral blood and tumors - Tumor expression levels of proteins such as MUC16 and programmed cell death ligand 1 (PD-L1) (3) assessment of both tumor mutation burden and circulating tumor DNA; (4) assessing the relationship between exposure and efficacy and safety endpoints, when possible; and (5) Overall survival (OS).

[0139] Study Design: Utilizes a modified 3+3 dose escalation design ("4+3") with accelerated escalation (n=1 patient per cohort) at the lowest two dose levels (DL) of REGN4018 monotherapy. Dose escalation of REGN4018 monotherapy will proceed until the maximum tolerated dose (MTD) is achieved or a dose is selected for expansion based on sufficient evidence of tolerability and activity (RP2D).

[0140] A series of DLs of REGN4018 will be investigated as monotherapy. Dose escalation will begin at DL1 with a week 1 dose of 0.1 mg intravenous (IV) and a week 2 dose of 0.3 mg IV (if week 1 is tolerated). DL2 will use a week 1 dose of 0.3 mg IV and a week 2 dose of 1 mg IV (if week 1 is tolerated). Dose escalation will then progress through DL3, DL4, DL4a, and subsequent DLs if no dose-limiting toxicity (DLT), serious infusion-related reaction (IRR), or CRS is identified.

[0141] Thus, each REGN4018 DL consists of a loading dose and (if the loading dose is tolerated) a higher full dose. To minimize the risk of CRS, the loading dose is set to a maximum of 1 mg for all patients assigned to a DL from DL4 / DL4a and above, and from DL5a, a mandatory transition dose of 20 mg is set between the loading dose and the full dose. Intermediate dose levels may be added as needed for patient safety.

[0142] REGN4018 and cemiplimab combination therapy was initiated at DLC2, which was deemed to be a tolerable and pharmacologically active REGN4018 monotherapy dose. For REGN4018 and cemiplimab combination therapy, enrolled patients were dose-escalated according to a 4+3 dose-escalation design, similar to monotherapy patients. To more effectively identify the estimated RP2D / MTD and minimize the possibility of exposing patients to subtherapeutic and / or excessively toxic doses, continuous dose-escalation (DL) of dose levels follows a BOIN design.

[0143] The combination therapy will not be titrated beyond the monotherapy MTD. Study implementation of the combination therapy cohort will be conducted in a similar manner to the monotherapy cohort, including a monotherapy lead-in cycle in which REGN4018 will be gradually introduced.

[0144] Following DLC4, the selected transition dose and subsequent dose escalation route will be in accordance with the transition dose of the monotherapy. For example, if a transition dose of 20 mg is used in the monotherapy cohort, a transition dose of 20 mg will be used in the combination therapy cohort. Thus, dose escalation will continue to DLC4a (provided safety data from DLC4a are deemed adequate), followed by DLC5a-DLC9a.

[0145] A single full dose of REGN4018 must be tolerated without CRS in order for patients to begin cycle 2 of the cemiplimab combination. Combination cycle 1 continues for 4 to 5 weeks until patients have received at least one full dose of REGN4018 without developing CRS.

[0146] The monotherapy expansion cohort will be enrolled after identification of the REGN4018 MTD and / or RP2D, and the second combination expansion cohort will be enrolled after identification of the MTD and / or RP2D of REGN4018 in combination with cemiplimab. The dose levels of REGN4018 may differ between the monotherapy expansion cohort and the combination expansion cohort. Additionally, doses will be evaluated via a three-arm, randomized second cohort evaluating three doses: REGN4018 250mg IV Q3W; REGN4018 800mg IV Q3W as monotherapy; and REGN4018 250mg combined with cemiplimab 350mg IV Q3W (or maximum tolerated dose in combination with cemiplimab if 250mg QW is not tolerated) IV Q3W.

[0147] The DLT observation period to determine the safety of dose escalation is defined differently for the monotherapy and combination therapy cohorts. The purpose of the DLT observation period for the monotherapy cohort is to monitor the safety and tolerability of at least two full doses of REGN4018 during monotherapy dose escalation. For DL1-DL4, the DLT window is 28 days since no transition dose is used. For DL5a / DLC5a and all higher DLs, the DLT observation period is 28-35 days (depending on when the second full dose is administered) starting from day 1 of cycle 1.

[0148] The combination DLT observation period is defined as 21 days of combination therapy starting on Day 1 of Cycle 2 for the purpose of monitoring safety and tolerability during the first 3 weeks of REGN4018 and cemiplimab combination therapy. The DLT observation period to determine the safety of dose escalation in the combination is not necessary to evaluate REGN4018 monotherapy because, at each combination DL, the REGN4018 dose was previously deemed to be tolerable during REGN4018 monotherapy dose escalation.

[0149] In the first phase of the study, additional cohorts will explore subcutaneous (SC) administration of the loading and transition doses of REGN4018 solely to evaluate whether the SC route of administration is associated with reduced acute toxicities such as CRS. The total dose of REGN4018 (either as monotherapy or in combination with cemiplimab) for this cohort will be selected for this cohort based on a review of the safety and efficacy data demonstrated to date.

[0150] Study Duration: The screening period will be a maximum of 28 days for all patients. For REGN4018 monotherapy, each cycle will be 6 weeks (42 days) long. For combination therapy with REGN4018 and cemiplimab, the first cycle will be 28 or 35 days long, based on if the patient tolerates the full dose of REGN4018 without CRS. Subsequent cycles of combination therapy will be 6 weeks (42 days) long. Treatment will continue until disease progression, intolerable adverse events, withdrawal of consent, or any other treatment discontinuation criteria is met. Post-treatment follow-up will be approximately 90 days (core follow-up) or 168 days (surveillance follow-up), based on the reason for treatment discontinuation.

[0151] Study Population: We expect to enroll up to 554 patients (292 in the dose escalation phase, 12 in the exploratory SC cohort, and 250 in the expansion cohort). The actual number of patients enrolled will depend on DLTs observed during the monotherapy and combination therapy dose escalation cohorts, the number of patients added, whether a second stage (of the Simon two-stage design) is enrolled in each expansion cohort, and whether a trigger event occurs during dose expansion. The study will enroll patients with platinum-based therapy-experienced and / or intolerant ovarian, fallopian tube, or primary peritoneal cancer with elevated serum CA-125 levels (≥2x upper limit of normal).

[0152] Selection Criteria - Patients must meet the following criteria to be eligible for inclusion in this study: 1. Women aged 18 or older 2. Patients with a histologically or cytologically confirmed diagnosis of advanced epithelial ovarian cancer (excluding carcinosarcoma), primary peritoneal cancer, or fallopian tube cancer and all of the following: Serum CA-125 level ≥ 2x upper limit of normal (ULN) (at screening), b. Having received at least one platinum-containing therapy or being platinum intolerant; c. Documented recurrence or progression on or after the most recent line of treatment, and d. Absence of standard treatment options that may provide clinical benefit 3. Willing and able to comply with clinic visit and study related procedures Note: a. In the escalation cohort, patients must provide either a newly obtained biopsy (a freshly obtained biopsy is required at screening unless medically appropriate and discussed with the medical monitor) or archived tumor tissue. b. In the expansion cohort, patients must provide a fresh tumor biopsy at screening. 4. Expansion cohort only: Must have radiologically documented progression on prior therapy and must have at least one measurable lesion (not previously irradiated) that can be accurately measured by Response Evaluation Criteria in Solid Tumors (RECIST). Eastern Cooperative Oncology Group (ECOG) performance status of 5.0 or 1 6. Adequate organ and bone marrow function of: Hemoglobin ≥ 9.0 g / dL B. Absolute neutrophil count ≥ 1.5 × 10 9 / L c. Platelet count ≧75×10 9 / L d. Serum creatinine ≤ 1.5 x ULN OR estimated glomerular filtration rate > 50 mL / min / 1.73 m2 (dose escalation cohort) OR estimated glomerular filtration rate > 30 mL / min / 1.73 m2 (dose expansion cohort) e. Total bilirubin ≦ 1.5 × ULN f. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3 x ULN or ≤ 5 x ULN in the case of liver metastases g. Alkaline phosphatase ≤ 2.5 x ULN (or ≤ 5.0 x ULN in the case of liver or bone metastases) 7. Estimated life expectancy of at least 3 months. 8. Provision of informed consent signed by the study patient or legally acceptable representative 9. Randomized Phase 2 expansion cohort only: a. Platinum-resistant ovarian cancer meeting one of the following criteria: Patients who have received only one prior platinum-based therapy must have received at least four prior platinum-based therapies, must have experienced a response (complete response / remission [CR] or partial response / remission [PR]), and must have subsequently progressed between 0 and 6 months from the date of their last platinum dose. Patients who have received two or three prior platinum-based therapies must have progressed within six months of the date of their last platinum dose. b. Prior treatment with a PARP inhibitor for patients with a BRCA mutation or known homologous recombination repair (HRD) deficiency, or patients ineligible to receive a PARP inhibitor c. Previous treatment with bevacizumab or ineligible for bevacizumab

[0153] Exclusion criteria -Patients who meet any of the following criteria will be excluded from the study: 1. Currently receiving treatment in another therapeutic study or participating in a study of an investigational drug, have been treated or have used an investigational device within 4 weeks of the first dose of an investigational therapy, or have been treated with an approved systemic therapy within 3 weeks of the first dose of an investigational therapy, or have received any previous systemic therapy within 5 half-lives of the first dose of an investigational therapy (whichever is longer). Patients who have received or enrolled in a study that includes treatment with a minimum dose of an investigational immunoPET agent are not excluded. Patients previously treated with bevacizumab are permitted after consultation with the sponsor if they have no history of intestinal perforation or wound complications from bevacizumab and the last dose of REGN4018 is >30 days from the first dose, and other non-investigational non-immunomodulatory antibodies with half-lives longer than 7 days are permitted after consultation with the sponsor if at least 3 half-lives have elapsed since the last treatment. 2. Previous anti-cancer immunotherapy as listed below: Prior treatment with anti-PD-1 / PD-L1 therapy within 5 half-lives of first dose Note: The combination therapy cohort and the randomized phase 2 cohort also exclude patients who previously discontinued anti-PD-1 / PD-L1 therapy due to toxicity. b. History of CAR-T cell therapy within 30 days of first administration of study drug 3. Previous treatment with MUC16-targeted therapy. 4. Expansion cohort only: 4 or more prior cytotoxic chemotherapy regimens. NOTE: In dose escalation cohorts, there is no upper limit on the number of prior treatments required to be eligible. 5. Corticosteroid therapy (>10 mg prednisone / day or equivalent) within 1 week prior to the first dose of study medication. Patients requiring short-term steroid treatment (maximum 2 days in the week prior to enrollment) will not be excluded. 6. Treatment-related immune-mediated AEs with immunomodulatory agents (including but not limited to anti-PD-1 / PD-L1 or anti-CTLA-4 monoclonal antibodies or PI3Kdelta inhibitors) that have not resolved to baseline at least 30 days prior to initiating treatment with study therapy. Note: Endocrine immune-mediated AEs managed with hormonal therapy or other non-immunosuppressive therapy (without resolution), or Grade 1 irAEs in any organ system with resolution prior to enrollment. 7. Expansion cohort only: Another malignancy that is progressing or requiring active treatment, except for non-melanoma skin cancer or in situ cervical cancer that has undergone potentially curative therapy, or any other tumor deemed to have been effectively treated with definitive local control (with or without ongoing adjuvant hormonal therapy) for at least 2 years prior to enrollment. 8. Untreated or active primary brain tumor, CNS metastasis, or spinal cord compression. Patients with previously treated CNS metastases or spinal cord compression may participate as long as they are stable (i.e., no evidence of progression and return of neurological symptoms to baseline by imaging for at least 4 weeks prior to the first dose of study therapy), there is no evidence of new or enlarging CNS metastases, and the patient has not required any systemic corticosteroids for management of CNS metastases or spinal cord compression within 2 weeks prior to the first dose of study therapy. 9. Encephalitis, meningitis, or uncontrollable seizures in the year prior to informed consent. 10. Have clinically significant abnormal ECG measurements as determined by the investigator and / or meet the following criteria: a. QTc (Fridericia) interval >470 ms. In case of asymptomatic prolonged QTc interval (>470 ms), the ECG may be repeated up to two times. If the subsequent QTc interval is <470 ms, the patient may be enrolled only after review and approval by a cardiologist. b. Evidence of second-degree AV block type II (Mobitz type II) or AV block type III (complete heart block). 11. Left ventricular ejection fraction (LVEF) <50% as measured by baseline echocardiogram. Patients may be enrolled if LVEF 45-50% without clinical symptoms after review and clearance by a cardiologist. 12. History of clinically significant cardiac disease within 6 months prior to screening, including but not limited to: - Myocardial infarction - Unstable angina - Stroke or transient ischemic attack - Peripheral arterial disease events - Heart failure (NYHA class III and IV or ACC / AHA heart failure classification C or D) 13. History of any clinically significant arrhythmia, including paroxysmal pericardial fibrillation, or implantation of a pacemaker or defibrillator. 14. History of myocarditis. 15. Signs or symptoms of active angina, arrhythmia, or heart failure. 16. Any moderate to severe valvular abnormality (stenosis or regurgitation) and / or clinically significant valvular heart disease that has not already been managed surgically. 17. Moderate to large pericardial fluid (e.g., >≈100 mL) as measured by baseline echocardiogram. 18. Patients requiring 2 or more therapeutic paracentesis in the month prior to screening. 19. Baseline serum troponin above the institutional upper limit of normal. If troponin is minimally elevated in the absence of clinical symptoms, after clearance by a cardiologist, patients may be enrolled. 20. Evidence of ongoing or recent (within 5 years) significant autoimmune disease requiring treatment with systemic immunosuppressive measures that may indicate risk of irAEs. The following are not excluded: vitiligo, remitted childhood asthma, hypothyroidism requiring hormone replacement only, type 1 diabetes, or psoriasis not requiring systemic treatment. 21. History of, or any evidence of, interstitial lung disease or active non-infectious pneumonia (within the past 5 years). 22. Moderate to severe pleural effusion as measured by baseline chest x-ray that may require thoracentesis within the next 4 weeks due to size or speed of expansion. An existing chest tube is acceptable if the patient meets all other inclusion / exclusion criteria. 23. Uncontrolled infection with human immunodeficiency virus, hepatitis B or C infection, or diagnosis of immunodeficiency. Note: - Patients with controlled HIV (either naturally occurring or on a stable antiviral regimen, with an undetectable viral load and a CD4 count greater than 350) will be allowed. - Patients with Hepatitis B who have controlled infection (HepBsAg+) (serum Hepatitis B virus DNA PCR below the limit of detection and receiving antiviral therapy for Hepatitis B) are allowed. - Patients who are hepatitis C virus antibody positive (HCV Ab+) and have controlled infection (undetectable HCV RNA by PCR, either spontaneously resolved or in response to a previous course of successful anti-HCV therapy) are admitted. 24. Active infection requiring systemic therapy. 25. Receipt of live vaccine within 30 days of planned study drug initiation. 26. Major surgical procedure, open biopsy, or significant traumatic injury within 2 weeks prior to enrollment. 27. Previous allogeneic stem cell transplant. 28. Intestinal obstruction within the past 3 months, or high risk of intestinal obstruction (in the opinion of the investigator) or current need for parenteral nutrition. 29. Any medical condition where, in the opinion of the investigator, study participation is not in the patient's best interest. 30. Documented allergic or acute hypersensitivity reactions resulting from antibody treatment. 31. Have known allergies or hypersensitivity to any component of the investigational drug. 32. Known psychiatric or substance abuse disorder that would prevent participation in the study requirements. 33. Clinical site study team members and / or their immediate families. 34. Pregnant or lactating women. 35.Continuous sexual activity in women of childbearing potential* who are not willing to use highly effective contraception prior to first dose / initiation of first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraceptive methods include: a. Stable use of a combined (estrogen- and progesterone-containing) hormonal contraceptive method associated with the inhibition of ovulation (oral, intravaginal, transdermal) or a progesterone-only hormonal contraceptive method (oral, injectable, implantable) initiated for at least two menstrual cycles prior to screening; b. Intrauterine devices (IUDs); Intrauterine hormone releasing systems (IUS); C. Tubal ligation; d. A vasectomized partner (provided that the male vasectomized partner is the WOCBP study participant's only sexual partner and that the vasectomized partner has undergone a medical evaluation of the surgical success of the procedure); and / or e. Sexual abstinence † ‡ *WOCBP are defined as women who are fertile after menarche until postmenopause, unless permanently infertile. Permanent methods of contraception include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. Postmenopausal status is defined as the absence of menses for 12 months without alternative medical causes. Elevated follicle-stimulating hormone (FSH) levels within the postmenopausal range may be used to confirm postmenopausal status in women who do not use hormonal contraception or hormone replacement therapy. However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient to determine the occurrence of postmenopausal status. The above definition follows guidance from the Clinical Trial Facilitation Group (CTFG). Pregnancy testing and contraception are not required for women with documented evidence of hysterectomy or tubal ligation. † Sexual abstinence is considered highly effective only when it is defined as abstinence from heterosexual intercourse for the entire period of risk associated with the study treatment. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the patient's preferred usual lifestyle. ‡ Periodic abstinence (calendar, symptom-temperature, or postovulatory methods), withdrawal (withdrawal), spermicide alone, and lactational amenorrhea method (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together. 36. Severe and / or uncontrolled hypertension at screening. Patients receiving antihypertensive medications must be on a stable antihypertensive regimen.

[0154] Treatment: Monotherapy (dose, route, and schedule)

[0155] Dose Escalation and Expansion Cohorts: REGN4018 is administered by IV infusion over a maximum of 4 hours ± 15 minutes (including flush) once weekly or once every three weeks.

[0156] Cohorts exploring SC administration of REGN4018 at week 1 (initial dose) and week 2 (transition dose): REGN4018 will be administered by SC injection once weekly in weeks 1 and 2. Subsequent doses (including the second transitional dose, and, if applicable, the full dose) will be administered by IV infusion once weekly over up to 4 hours (including flush).

[0157] A series of dose escalation cohorts will be used. The escalation scheme beyond DL4a will be selected based on tolerability of DL4 and DL4a (see below). [Table 5]

[0158] Treatment: Combination therapy (dose, route, and schedule)

[0159] Cemiplimab in all combination cohorts: Cemiplimab Q3W 350 mg will be administered by IV infusion over 30 minutes.

[0160] REGN4018 in Dose-Escalation and Dose-Expansion Combination Cohorts: REGN4018 will be administered by intravenous (IV) infusion once weekly over a maximum of 4 hours ± 15 minutes (including flush). If both agents are administered on the same day, cemiplimab will be administered first. A series of dose escalation cohorts will be used and will depend on the titration scheme selected after additional safety data has been collected. [Table 6]

[0161] Cohorts exploring SC administration of REGN4018 at week 1 (initial dose) and week 2 (transition dose): REGN4018 (2 mg) will be administered by SC injection in week 1, and REGN4018 (25 mg) will be administered subcutaneously in week 2. Subsequent doses (including the second transition dose, and all doses, if applicable) will be administered by IV infusion once weekly over a maximum of 4 hours ± 15 minutes (including flush). The three-arm randomized dose expansion includes: REGN4018 administered IV Q3W (250 mg monotherapy); REGN4018 administered IV Q3W (800 mg monotherapy); and REGN4018 administered IV Q3W (250 mg or highest tolerated dose in combination with cemiplimab) in combination with cemiplimab (350 mg IV Q3W). In each dose expansion cohort, patients will receive weekly step-up doses of REGN4018 for four weeks prior to Q3W dosing.

[0162] Study endpoints:

[0163] Dose Escalation Phase Primary endpoint These were: dose-limiting toxicities, treatment-emergent adverse events (TEAEs; including immune-related adverse events [irAEs]), serious AEs (SAEs), deaths, laboratory abnormalities (grade ≥ 3 by CTCAE), and PK as monotherapy and in combination with cemiplimab.

[0164] In the dose expansion phase, Primary endpoint is the ORR measured both as monotherapy and in combination with cemiplimab.

[0165] Dose escalation Secondary endpoints is ORR based on Response Evaluation Criteria in Solid Tumors (RESIST 1.1).

[0166] Dose expansion Secondary endpoints is as follows: 1) TEAEs (grade ≥3 by CTCAE), including immune-related, SAEs, deaths, and laboratory abnormalities; 2) concentrations of REGN4018 in serum over time; 3) change from baseline in QoL as measured by the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ)-C30 GHS / QoL score; 4) change from baseline in physical function as measured by EORTC QLQ-C30 physical function score; 5) change from baseline in abdominal symptoms as measured by the Ovarian Symptoms and Treatment Scale (MOST)-Abdominal Index score; 6) time to deterioration of GHS / QoL, physical function, and abdominal symptoms; and 7) Change from baseline in QoL as measured by EQ-5D.

[0167] (both in dose escalation and dose expansion phases) Secondary endpoints is as follows: 1) ORR based on iRECIST, best overall response (BOR), duration of response (DOR), disease control rate, CR rate, PFS based on RECIST 1.1 and iRECIST, and CA-125 response; and 2) Presence or absence of anti-drug antibodies against REGN4018 and cemiplimab.

[0168] Methodology and Evaluation:

[0169] Safety and tolerability of REGN4018 alone or in combination with cemiplimab will be monitored by clinical assessment of AEs and repeated measurements of clinical evaluations including vital signs (temperature, blood pressure, pulse, and respiration), physical examination (complete and limited), 12-lead electrocardiogram (ECG), echocardiogram, chest x-ray, and laboratory evaluations including standard hematology, chemistry, and urinalysis.

[0170] Since cytokine release after initial administration (initial dose and / or subsequent doses) has been observed with bispecific antibodies and similar molecules, certain measures were implemented in the study. These measures include an initial dose of 1 mg (day 1 of cycle 1) at DLC 4 or higher (monotherapy) and DLC 3 or higher (combination therapy), a transition dose of 20 mg (day 8 of cycle 1), the option of split doses on other dosing dates, required monitoring at selected dose administration, and the use of anti-IL-6 pathway therapy (e.g., tocilizumab) and corticosteroids for management of IRR / CRS. Additionally, a safety monitoring scheme will be implemented in the expansion cohort by including a stopping boundary based on the cumulative incidence of trigger events (cTE). Enrollment in a given cohort may be paused if the lower limit of the one-sided 80% confidence interval of the estimated cumulative incidence of TE (cTE) excludes 25%. For example, enrollment will be paused if 4 or more of 11 patients from both the dose escalation and expansion cohorts experience a TE. A discussion between the investigator and the Safety Monitoring Committee will determine whether enrollment can be resumed and, if so, whether at the same or a lower dose of REGN4018.

[0171] A baseline ocular examination is required for expression of MUC16 on the corneal and conjunctival epithelium.

[0172] Blood samples will be taken for measurement of drug concentrations and for ADA assessment.

[0173] Serum and plasma samples will be collected for analysis of additional biomarkers. Exploratory predictive and pharmacodynamic biomarkers related to REGN4018 treatment exposure, clinical activity, or underlying disease will be investigated from collected serum, plasma, whole blood, body fluids, archived tumor tissue, on-study tumor biopsy tissue, tumor DNA (including circulating tumor DNA), and tumor RNA samples.

[0174] Antitumor activity will be assessed by CT or MRI or PET-CT, as well as monitoring performance status and serum CA-125 levels.

[0175] Patient-reported outcomes (PROs) will be assessed during the dose expansion phase.

[0176] Statistical planning:

[0177] Dose Escalation Phase : There are no formal statistical hypotheses for the dose escalation phase of the study. Analyses for this phase are descriptive and exploratory in nature. For the dose escalation phase, DLTs observed during the DLT evaluation period will be summarized by dose cohort.

[0178] Dose expansion phase :

[0179] Statistical Hypothesis - For each dose expansion cohort (including each randomized arm of the second cohort), it is assumed that patients treated with REGN4018 monotherapy or REGN4018 / cemiplimab combination therapy will achieve an ORR of 25% (H1) or greater. Using a one-sided alpha of 0.05, 85% power, and optimal design, a total of 50 patients, including 20 patients in stage 1, are required for each cohort. Once 20 patients have been enrolled in stage 1 independently for each cohort, stage 2 will open enrollment only if 3 or more responders are observed over the first 6 months of treatment within the 20 patients enrolled in stage 1. The null hypothesis of 10% will be rejected if 9 or more responses are observed in 50 patients. Confirmed and unconfirmed responses will be combined to determine if a minimum number of responses has been achieved to proceed to stage 2.

[0180] Primary efficacy analysis - For a given expansion cohort, if the number of responders is equal to or greater than the minimum number of responders specified by the Simon two-stage design, the treatment will be considered effective and worthy of further investigation. ORR will be summarized by descriptive statistics with 95% confidence intervals. Patients in the Safety Analysis Set (SAF) who are not evaluable for ORR will be considered non-responders. Statistical analyses of efficacy for each expansion cohort will be performed and reported separately, i.e., efficacy results and clinical conclusions from each cohort will not affect the other cohort and vice versa.

[0181] Safety observations and measurements will be summarized, including drug exposure, AEs, clinical laboratory data, and vital signs.

[0182] Preliminary results: Initial safety, pharmacokinetic (PK), and efficacy data from the REGN4018 (ubamatamab) monotherapy portion of a first-in-human, Phase 1 dose-escalation study in patients with recurrent ovarian cancer are presented below.

[0183] Patients with recurrent, platinum-experienced, and / or intolerant ovarian cancer with elevated cancer antigen (CA)-125 levels received REGN4018 weekly intravenously (IV) at doses ranging from 0.1 to 800 mg. Dose escalation followed a modified 3+3 (4+3) design. Dose-limiting toxicity (DLT) periods were 28-35 days. Step-up dosing of the first two doses was utilized to reduce the risk of cytokine release syndrome (CRS) via escalating drug exposure. Primary endpoints included safety and PK. Secondary endpoints included preliminary efficacy as determined by objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.

[0184] Seventy-eight patients received REGN4018 monotherapy in the first phase of the study, with a median exposure of 12 weeks (range, 0.4-117). Based on histology, the 78 patients included 71 (91.0%) high-grade serous, 2 (2.6%) clear cell, 1 (1.3%) high-grade endometrioid, 1 (1.3%) low-grade serous, and 3 (3.8%) other. CA-125 baseline serum levels ranged from 107 to 10,000 with a median of 709 U / mL. Of the 78 patients, 26 (33%) had visceral metastases and 30 (58%) had >75% tumor cytology and 2+ baseline MUC16 IHC staining. The median number of prior therapies was 4.5 (range, 1-17). The most common treatment-emergent adverse events (TEAEs) were CRS (73.1%, all grade 1–2) and pain (87.2%, mostly grade 1–2), which occurred primarily during weeks 1–2 of the initial step-up dose. The most common grade ≥3 TEAEs were anemia (23.1%) and abdominal pain (19.2%). Objective responses were observed at doses of 20–800 mg (n=50). For those receiving a full dose of ≥1 (n=42), the ORR was 14.3% (95% CI 5.4–28.5) and the disease control rate (DCR) was 57.1% (41.0–72.3). In the subset of these without baseline visceral metastases (n=29), the ORR was 20.7% (8.0–39.7) and DCS was 72.4% (52.8–87.3). Median duration of response was 12.2 months. Exploratory analyses of patients (n=13) with >75% of tumor cells with baseline MUC16 immunohistochemical staining of 2+ and receiving ≥1 dose of ≥20 mg demonstrated an ORR of 30.8% (9.1-61.4) and DCR of 61.5% (31.6-86.1). 46.2% of patients with >75% of tumor cells with baseline MUC16 IHC staining of 2+ demonstrated a CA-125 response. Serum ubamatamab concentrations increased dose-proportionally and demonstrated linear pharmacokinetics. No definitive dose-response relationship was observed in safety or efficacy between 20 and 800 mg.

[0185] The REGN4018 safety profile was tolerable and demonstrated durable responses in this heavily pretreated population with ovarian cancer across a broad dose range. Data from this analysis support further investigation of REGN4018 in recurrent, platinum-experienced ovarian cancer.

[0186] In the second phase, up to 150 patients with progressive platinum-resistant OC and elevated serum CA-125 will be randomized into three IV arms (1:1:1) to receive ubamatamab (REGN4018) 250 mg IV Q3W or 800 mg IV Q3W as monotherapy, or ubamatamab 250 mg IV Q3W in combination with cemiplimab 350 mg Q3W. All arms include weekly step-up dosing of ubamatamab (1 mg in week 1, 20 mg in week 2, and full dose in weeks 3 and 4) to limit the risk of cytokine release syndrome before progressing to Q3W dosing. The expansion cohort will use a Simon two-stage trial design with an interim analysis after the first 20 patients. Any arm with ≥3 partial response or better will be expanded to 50 patients.

[0187] In this dose expansion phase, the primary endpoint is the objective response rate for each arm as defined by RECIST 1.1 criteria. Secondary endpoints include evaluation of duration of response and progression-free survival, as well as further evaluation of safety and pharmacokinetics. Exploratory endpoints include evaluation of baseline tumor MUC16 immunohistochemical expression and other biomarkers as predictors of response. The impact of ubamatamab on quality of life and physical function will also be evaluated.

[0188] In some cases, the 250 mg dose may be divided into two fractions, the first fraction containing 50 mg and the second fraction containing 200 mg.

[0189] Example 10: Translational Findings from a Study of REGN4018 in Patients with Recurrent Ovarian Cancer In a Phase 1 study detailed in Example 9, patients with ovarian cancer were administered ubamatamab intravenously as monotherapy (78 pts) or in combination with anti-PD-1 cemiplimab (27 pts). The first step-up dose in week 1 (W) was selected based on in vitro cytokine assay results. A mouse tumor regression model suggested an effective concentration to inhibit tumor growth. Monkey pharmacokinetic (PK) data was measured to predict drug exposure. A model of CD3 bispecific antibody + cemiplimab was used to set the administration sequence of cemiplimab + ubamatamab. Preclinical and clinical PK, cytokine, and efficacy data from dose escalation were integrated to determine the regimen in dose expansion. The regimens of interest were simulated by a population PK model.

[0190] In vitro data showed minimal and maximal cytokine release at concentrations of 0.01 mg / L and ≥0.1 mg / L. Data supported W1 step-up doses in the range of 0.1–1 mg, resulting in observed Cmax of 0.02–0.3 mg / L. Cmax values ​​at W1 in all cohorts of patients were well predicted from measurements (within ±30%). PK data from a mouse tumor regression model showed effective concentrations (0.5–50 mg / L) consistent with Cmin >4 mg / L at effective doses of ≥20 mg. PK simulations supported testing of ≥250 mg Q3W in expansion. As the model predicted, no significant cytokine release occurred when cemiplimab was combined with ubamatamab after induction of ubamatamab monotherapy.

[0191] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. ******** [Table 7]

Claims

1. A pharmaceutical composition comprising a bispecific antibody for use in a method of treating MUC16-expressing cancer in a subject requiring treatment for MUC16-expressing cancer, wherein the method comprises administering the bispecific antibody to the subject at a dose of at least 1 mg, and the bispecific antibody comprises a first antigen-binding domain that specifically binds to mucin 16 (MUC16) and a second antigen-binding domain that specifically binds to human CD3.

2. (i) The cancer is ovarian cancer, fallopian tube cancer, or primary peritoneal cancer; and / or (ii) The pharmaceutical composition according to claim 1, wherein the cancer is resistant to platinum-based chemotherapy, or the subject has been previously treated with platinum-based chemotherapy.

3. The first antigen-binding domain is as follows: (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 1, and (b) A light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 2, comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), Optional, (i) The first antigen-binding domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 8, HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and HCDR3 containing the amino acid sequence of SEQ ID NO: 10; and / or (ii) The first 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; and / or (iii) The first antigen-binding domain comprises an HCVR containing the amino acid sequence of SEQ ID NO: 1 and an LCVR containing the amino acid sequence of SEQ ID NO: 2 The pharmaceutical composition according to claim 2.

4. The second antigen-binding domain is as follows: (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (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 the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 2 It includes, and is optional, (i) 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; and / or (ii) 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; and / or (iii) The second antigen-binding domain comprises an HCVR containing the amino acid sequence of SEQ ID NO: 3 and an LCVR containing the amino acid sequence of SEQ ID NO: 2 The pharmaceutical composition according to claim 3.

5. The aforementioned bispecific antibody contains the human IgG heavy chain constant region, and optionally, (i) The human IgG heavy chain constant region is isotype IgG1, and optionally the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype; or (ii) The constant region of the human IgG heavy chain is isotype IgG4, and optionally the bispecific antibody includes a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype. A pharmaceutical composition according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, wherein the first or second heavy chain comprises a CH3 domain having H435R (EU numbering) modification and Y436F (EU numbering) modification, but not both.

7. (i) The bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 29; or (ii) The bispecific antibody comprises a second heavy chain having the amino acid sequence of SEQ ID NO: 31; or (iii) The bispecific antibody 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. A pharmaceutical composition according to any one of claims 1 to 4.

8. (i) The subject has a serum CA-125 level of 60 U / ml or higher; and / or (ii) The method further comprises administering a second therapeutic agent or therapeutic regimen, optionally comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, The anti-PD-1 antibody or antigen-binding fragment is (a) Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 33, and (b) comprising three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34, and further optionally, (I) The anti-PD-1 antibody or antigen-binding fragment is HCDR1 containing the amino acid sequence of SEQ ID NO: 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and SEQ ID NO: 37 HCDR3 containing the amino acid sequence of; and / or (II) The anti-PD-1 antibody and 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; and / or (III) The anti-PD-1 antibody or antigen-binding fragment comprises 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, and optionally, the anti-PD-1 antibody and antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a light chain containing the amino acid sequence of SEQ ID NO:

42. A pharmaceutical composition according to any one of claims 1 to 4.

9. (i) The bispecific antibody is administered in a dosing regimen that includes a divided initial dose; and / or (ii) The bispecific antibody is administered to the subject weekly at a dose of 10 mg to 1000 mg, and optionally, the bispecific antibody is administered to the subject weekly at a dose of approximately 250 mg, and optionally, the dose is divided into a first fraction of approximately 50 mg and a second fraction of approximately 200 mg, or the bispecific antibody is administered to the subject weekly at a dose of approximately 800 mg, and optionally, the dose is divided into a first fraction of approximately 50 mg and a second fraction of approximately 750 mg; or (iii) The bispecific antibody is administered to the subject at a dose of 10 mg to 1000 mg once every three weeks, and optionally, the bispecific antibody is administered to the subject at a dose of approximately 250 mg once every three weeks, and optionally, the dose is divided into a first fraction of approximately 50 mg and a second fraction of approximately 200 mg, or the bispecific antibody is administered to the subject at a dose of approximately 800 mg once every three weeks, and optionally, the dose is divided into a first fraction of approximately 50 mg and a second fraction of approximately 750 mg. A pharmaceutical composition according to any one of claims 1 to 4.

10. The bispecific antibody may be administered in the following ways: (i) 1 mg of the bispecific antibody in the first week, optionally divided into a first fraction of approximately 0.5 mg and a second fraction of approximately 0.5 mg; (ii) 20 mg of the bispecific antibody in the second week, optionally divided into a first fraction of approximately 10 mg and a second fraction of approximately 10 mg; and (iii) 250 mg of the bispecific antibody in the third week. The pharmaceutical composition according to any one of claims 1 to 4, wherein the dose is optionally divided into a first fraction of about 50 mg and a second fraction of about 200 mg, and the bispecific antibody is administered in an administration regimen that optionally includes administering the bispecific antibody once a week from the fourth week onward in a dose of about 250 mg, or once every three weeks from the fourth week onward in a dose of about 250 mg, or once every three weeks from the fourth week onward in a dose of about 800 mg.

11. (i) The bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 29; or (ii) The bispecific antibody comprises a second heavy chain having the amino acid sequence of SEQ ID NO: 31; or (iii) The bispecific antibody 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. The pharmaceutical composition according to claim 10.

12. The pharmaceutical composition according to claim 8, wherein the anti-PD-1 antibody is administered to the subject once every three weeks at a dose of 300 mg to 400 mg, and optionally, the anti-PD-1 antibody is administered to the subject once every three weeks at a dose of 350 mg.

13. (i) The subject has stable disease, partial response, or complete response after administration of the bispecific antibody at a dose of 1 to 800 mg for at least one week; or (ii) The subject has stable disease status, partial response, or complete response after administration of the bispecific antibody at a dose of 20 to 800 mg for at least one week. A pharmaceutical composition according to any one of claims 1 to 4.

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

15. (i) MUC16 is highly expressed in ≥75% of tumor cells in the subject, as determined by immunohistochemical staining, or The aforementioned subject is, - Baseline MUC16 immunohistochemical staining score of 2 in MUC16-expressing tumors; or • Baseline MUC16 immunohistochemical staining score of 2+ in MUC16-expressing tumors; or - Baseline MUC16 immunohistochemical staining score of 3 in MUC16-expressing tumors; or • Baseline MUC16 immunohistochemical staining score of 3+ in MUC16-expressing tumors; or - Baseline MUC16 immunohistochemical staining score of 4 in MUC16-expressing tumors; or • Baseline MUC16 immunohistochemical staining score of 4+ in MUC16-expressing tumors; or - Baseline MUC16 immunohistochemical staining score of 5 in MUC16-expressing tumors; or - Tumors in which ≥50% of tumor cells express MUC16; or - Tumors in which ≥55% of tumor cells express MUC16; or - Tumors in which ≥60% of tumor cells express MUC16; or - Tumors in which ≥65% of tumor cells express MUC16; or - Tumors in which ≥70% of tumor cells express MUC16; or Tumors in which ≥75% of tumor cells express MUC16, Having; and / or (ii) The bispecific antibody is administered intravenously or subcutaneously. A pharmaceutical composition according to any one of claims 1 to 4.

16. The pharmaceutical composition according to claim 12, wherein the anti-PD-1 antibody or antigen-binding fragment is administered intravenously.

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