Methods for treating recurrent epithelioid sarcoma with a bispecific anti-MUC16x 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 epithelioid sarcoma). The methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody that specifically binds to 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 to the programmed death 1 (PD-1) receptor.

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 11138WO01_Sequence, created on Mar. 16, 2023, and containing 54,705 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 hyperglycosylated integral membrane glycoprotein highly expressed in epithelioid sarcoma (Hoshino, M. et al., 2010, J Cancer Res Clin Oncol., 136(3):457-64). 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 the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in cancers including epithelioid sarcoma, 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; Hoshino, M. et al., 2010, Cancer Res Clin Oncol., 136 (3): 457-64). 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 cancer using antibodies against MUC16 are being investigated. Oregovomab and abgovomab are anti-MUC16 antibodies that have had 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. Additionally, bispecific antibodies capable of binding to 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-1 (PD-1) receptor 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 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 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] Epithelioid sarcoma is a rare, high-grade soft tissue tumor with a known tendency for local recurrence, regional lymph node involvement, and distant metastasis. (Sobanko et al., J Clin Aesthet Dermatol.2(5):49-54,2009). Most cases begin in the soft tissues under the skin of the fingers, hands, forearms, lower legs, or feet, but the tumor can begin to grow in other areas of the body. Because epithelioid sarcoma exists harmlessly, malignancy inherently portends a poor prognosis. Misdiagnosis of this tumor can lead to delayed or inappropriate treatment, adversely affecting patient survival. Current treatments for epithelioid sarcoma include radical tumor resection, adjuvant chemotherapy, sentinel lymph node biopsy, and radiation therapy. (Casanova et al., Cancer. 106(3):708-17, 2006; Sobanko et al., J Clin Aesthet Dermatol. 2(5):49-54, 2009). While the majority of patients respond to initial treatment, most experience disease recurrence, resulting in repeated cycles of surgery and additional rounds of chemotherapy. Although recurrent epithelioid sarcomas can respond to further treatments, nearly all of them eventually become resistant to currently available therapies. Despite recent advances in therapy, recurrent metastatic epithelioid sarcoma remains a disease of high unmet need.

[0007] Evidence suggests that epithelioid sarcoma may be amenable to some forms of immunotherapy. For example, tumor specimens from patients with epithelioid sarcoma show high levels of PD-L1 expression and CD3 + / CD8 + It has been previously detected in T-lymphocyte infiltration (Gong et al., Front Oncol., 11:728437, 2021). Blockade of the PD-1 / PD-L1 checkpoint pathway may be beneficial in epithelioid sarcoma. However, blocking agents of this pathway alone are not sufficient.

[0008] Thus, given this high unmet need, additional therapies are needed to target epithelioid sarcoma. Summary of the Invention

[0009] In one aspect, the disclosure includes a method of treating a MUC16-expressing cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody 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 is administered to the subject at a dose of at least 1 mg (e.g., weekly).

[0010] In some embodiments, the MUC16-expressing cancer is epithelioid sarcoma. In some cases, the subject has metastatic epithelioid sarcoma.

[0011] In some embodiments, MUC16 expressing cancer is MUC16 expressing sarcoma.In some cases, sarcoma is epithelioid sarcoma, rhabdoid tumor of kidney and soft tissue, rhabdomyosarcoma, Ewing's sarcoma, or soft tissue sarcoma.In some cases, MUC16 expressing sarcoma is epithelioid sarcoma, or rhabdoid tumor of kidney and soft tissue.

[0012] In some embodiments, the MUC16-expressing cancer (eg, a sarcoma) lacks expression of functional integrase interactor 1 protein.

[0013] In some embodiments, the subject has been previously treated with anti-cancer therapy. In some cases, the subject is resistant to, inadequately responsive to, or has relapsed after the previous therapy. In some cases, the subject has been previously treated with chemotherapy, radiation therapy, surgery, or immunotherapy. In some cases, the chemotherapy is tazemetostat. In some cases, the immunotherapy is a PD1 inhibitor or a CTLA4 inhibitor. In some cases, the immunotherapy is pembrolizumab, nivolumab, or ipilimumab. In some cases, the MUC16-expressing cancer is resistant to, or inadequately responsive to, the previous therapy, or has relapsed after the previous therapy.

[0014] In some embodiments, the bispecific antibody 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.

[0015] In some embodiments, including those in which the first antigen-binding domain is as described above, the bispecific antibody 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 cases, the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:3, and an LCVR comprising the amino acid sequence of SEQ ID NO:2.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 serum CA-125 level that is greater than 92 U / ml.

[0021] 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.

[0022] 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 cases, the anti-PD-1 antibody or antigen-binding fragment comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 33, and an LCVR comprising the amino acid sequence of SEQ ID NO: 34. In some instances, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody that comprises 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.

[0023] In some embodiments, the bispecific antibody is administered in a dosing regimen that includes a split initial dose (e.g., an initial dose of 1 mg is divided into two equal fractions of 0.5 mg administered on two consecutive days). In some embodiments, the bispecific antibody is administered to the subject at a dose of 1 mg to 1000 mg weekly. In some cases, the bispecific antibody is administered to the subject at a dose of 250 mg weekly. In some cases, the bispecific antibody is administered to the subject as frequently as every three weeks. In some cases, the bispecific antibody is administered to the subject via intravenous administration. In some cases, the bispecific antibody is administered to the subject via subcutaneous administration. In some cases, the anti-PD-1 antibody is administered to the subject before, simultaneously with, or after the bispecific antibody.

[0024] 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 1-250 mg. In some cases, the epithelioid sarcoma in the subject's breast tissue decreases in size after at least one week of administration of the bispecific antibody at a dose of 1-250 mg.

[0025] In some embodiments of the method, the bispecific antibody is administered in a dosing regimen comprising administering an initial dose of 1 mg of the bispecific antibody during week 1 of the dosing regimen, administering a transition dose of 20 mg of the bispecific antibody during week 2 of the dosing regimen, and administering a total dose of 250 mg of the bispecific antibody during week 3 of the dosing regimen. In some cases, the initial dose is divided into two equal fractions administered on consecutive days. In some cases, the transition dose is divided into two equal fractions administered on consecutive days. In some cases, the total dose is divided into two fractions administered on consecutive days. In some embodiments, the two fractions of the total dose comprise a 50 mg fraction and a 250 mg fraction. In some embodiments, the dosing regimen further comprises administering a maintenance dose of 250 mg of the bispecific antibody administered during week 4 of the dosing regimen. In some cases, the maintenance dose is administered weekly during subsequent weeks of the dosing regimen. In some cases, maintenance doses are administered every other week (Q2W) during subsequent weeks of the dosing regimen.

[0026] Other embodiments of the invention will become apparent from consideration of the detailed description that follows. [Brief description of the drawings]

[0027] [Figure 1] Figure 1 shows the binding of various concentrations of anti-MUC16 clones 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because they may vary. It should also be understood that the terms used herein are used only to describe 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 described above or discussed herein may be combined with another related value described above or discussed 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.

[0029] 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 can 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 (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0030] 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.

[0031] Methods for Treating or Inhibiting Cancer Growth - Patent application The present invention includes methods for treating, ameliorating, or reducing the severity of at least one symptom or sign of, or inhibiting the growth of, a cancer (e.g., recurrent epithelioid sarcoma) in a subject. Methods according to embodiments of the invention include administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody 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.

[0032] 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 epithelioid sarcoma, 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 distension, chest pain / tightness, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, and itching. This phrase includes subjects with primary or established epithelioid sarcoma. In specific embodiments, this phrase includes human subjects with epithelioid sarcoma or another tumor expressing MUC16 and in need of treatment thereof. In other specific embodiments, this phrase includes subjects with MUC16+ tumors (e.g., tumors with MUC16 expression as determined by flow cytometry). In certain embodiments, the term "subject in need thereof" includes patients with epithelioid sarcoma that is resistant or refractory to previous therapy (e.g., treatment with conventional anticancer drugs) or not adequately controlled by previous therapy. For example, the term includes subjects treated with chemotherapy, such as chemotherapeutic agents (e.g., tazemetostat) or taxol compounds (e.g., docetaxel). The term also includes subjects with epithelioid sarcoma for which conventional anticancer 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 epithelioid sarcoma that have been treated but have subsequently recurred or metastasized. For example, patients with epithelioid sarcoma who have undergone treatment with one or more anticancer drugs and may have experienced tumor regression, but who subsequently recur with cancer resistant to one or more anticancer drugs (e.g., chemotherapy-resistant cancer), are treated with the methods of the present invention.

[0033] The term "subject in need thereof" also includes subjects at risk of developing epithelioid sarcoma, such as those with a family history of epithelioid sarcoma, those with a past medical history of an infection associated with epithelioid sarcoma, those with a mutation in the SMARCB1 gene, or those with a compromised immune system due to HIV infection or immunosuppressant drugs.

[0034] In certain embodiments, the methods of the invention can be used to treat patients who exhibit elevated levels of one or more cancer-associated biomarkers (e.g., programmed death-ligand 1 (PD-L1), 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 in combination with a bispecific anti-MUC16 / anti-CD3 antibody to a patient with elevated levels of PD-L1 and / or CA125.

[0035] In certain embodiments, the methods of the present invention are used in subjects with epithelioid sarcoma. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "epithelioid sarcoma" refers to a tumor in the soft tissue under the skin, for example, of the fingers, hands, forearms, lower legs, or feet, although tumors may also begin to grow in other areas of the body.

[0036] According to certain embodiments, the invention includes methods for treating tumors or slowing or inhibiting tumor growth. 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 a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody, to a subject in need thereof, where each antibody is administered to the subject in multiple doses, e.g., as part of a particular therapeutic dosing regimen. For example, the therapeutic dosing regimen may include administering one or more doses of an anti-MUC16xCD3 antibody 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 are administered in combination with one or more therapeutically effective doses of a bispecific anti-MUC16 / anti-CD3 antibody, and the one or more doses of an anti-PD-1 antibody are administered to a subject approximately 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.

[0037] In certain embodiments, each dose of anti-MUC16 / anti-CD3 antibody is administered in more than one fraction, e.g., 2-5 fractions ("split doses"), within a given administration period. Anti-MUC16 / anti-CD3 bispecific antibodies 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-associated 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 in combination with one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody, where the dose of the bispecific antibody 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 is split into two or more fractions, with each fraction containing an equal amount of antibody as the other fractions. For example, a dose of anti-MUC16 / anti-CD3 antibody comprising 1000 micrograms may be administered once a week, the dose being administered in two fractions within the week, each fraction comprising 500 micrograms. In certain embodiments, a dose of a bispecific antibody is administered divided into two or more fractions, the fractions comprising unequal amounts of antibody, e.g., more or less than the first fraction. For example, a dose of anti-MUC16 / anti-CD3 antibody comprising 1000 micrograms may be administered once a week, the dose being administered in two fractions within the week, the first fraction comprising 700 micrograms and the second fraction comprising 300 micrograms. As another example, a dose of anti-MUC16 / anti-CD3 antibody containing 1000 micrograms may be administered once every two weeks, with 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.

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

[0039] In a specific embodiment, the invention provides a method for increasing anti-tumor efficacy or increasing tumor inhibition. According to this aspect of the invention, the method comprises administering to a subject having epithelioid sarcoma ovarian cancer a therapeutically effective amount of an anti-PD-1 antibody followed by a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, where the anti-PD-1 antibody 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. In certain embodiments, the method provides an increase in tumor inhibition of, for example, 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 prior to the anti-PD-1 antibody.

[0040] In certain embodiments, the methods of the invention include administering to a subject with epithelioid sarcoma a therapeutically effective amount of a bispecific anti-CD3xMUC16 antibody, alone or in combination with an anti-PD-1 antibody. In further embodiments, the epithelioid sarcoma is indolent or aggressive. In certain embodiments, the subject is unresponsive to previous therapy or has relapsed after previous therapy. In some embodiments, the subject has a CA-125 level that is greater than or equal to two times the upper limit of normal (ULN) (e.g., greater than about 92 U / ml). In various embodiments, the subject's serum CA-125 level (pre-treatment) is greater than or equal to 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 U / ml. In certain embodiments, the methods of the invention further include 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 to a subject with a MUC16+ cancer. In a specific embodiment, the cancer is epithelioid sarcoma. In further embodiments, the epithelioid sarcoma is indolent or aggressive. In some embodiments, the epithelioid sarcoma is present in the chest wall, lung, and / or breast tissue of the patient. In some embodiments, the patient with epithelioid sarcoma has elevated serum CA-125 levels (e.g., at least 2xULN). In some embodiments, the cancer is resistant to a chemotherapeutic agent. In certain embodiments, the subject is not responsive to a previous therapy or has relapsed after a previous therapy (e.g., chemotherapy).

[0042] In certain embodiments, the methods of the invention comprise administering an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody to a subject in need thereof as a "first line" treatment (e.g., initial treatment). In other embodiments, the anti-PD-1 antibody in combination with the bispecific anti-MUC16 / anti-CD3 antibody is administered as a "second line" treatment (e.g., after previous therapy). For example, the anti-PD-1 antibody in combination with the bispecific anti-MUC16 / anti-CD3 antibody is administered as a "second line" treatment to a subject who has relapsed after previous therapy, e.g., with 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, alone or in combination with an anti-PD-1 antibody, 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, alone or in combination with an anti-PD-1 antibody, and optionally, a third therapeutic agent. The third therapeutic agent can be, for example, radiation, chemotherapy, surgery, a cancer vaccine, a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody), a LAG3 inhibitor (e.g., an anti-LAG3 antibody), a CTLA-4 inhibitor (e.g., an 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., tazemetostat, 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, alone or in combination with an anti-PD-1 antibody. When a combination is administered, administration of the antibodies 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 as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody leads to increased tumor regression, tumor shrinkage and / or elimination. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody 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 as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody prevents tumor recurrence and / or increases the survival of the subject, e.g., increases 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 administered either antibody as monotherapy. In certain embodiments, administration of the antibody combination increases progression-free survival or overall survival. In certain embodiments, administration of an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody 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% over untreated subjects or subjects receiving either antibody as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a subject with epithelioid sarcoma leads to the complete disappearance of all evidence of tumor cells (a "complete response").In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a subject with epithelioid sarcoma 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 and a bispecific anti-MUC16 / anti-CD3 antibody to a subject with epithelioid sarcoma leads to complete or partial disappearance of tumor cells / lesions, including new measurable lesions. Tumor reduction can be measured by any of the methods known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody 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 combination is safe and well tolerated by patients, with no increase in adverse side effects (e.g., increased cytokine release ("cytokine storm") or increased T cell activation) compared to patients receiving the bispecific antibody 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, as well as 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), interspersed with more conserved regions, 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 parallel 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 the antibody variable domains and, optionally, the 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 the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody, or constrained FR3-CDR3-FR4 peptides. 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 term "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 Domain-associated V H For antigen-binding fragments containing domains, V 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 V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-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 LIn 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 be comprised 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 L It 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) between the domains.

[0053] The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or 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 present invention using routine techniques available in the art. For example, the present 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, 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, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab.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 with 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 in vitro random or site-specific mutagenesis or by in vivo somatic mutation), for example, in CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences. 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 mouse, are grafted onto human framework sequences.

[0055] The antibody used in the method of the present invention may be a recombinant human antibody. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (discussed in more detail below), antibodies isolated from a recombinant, combinatorial human antibody library (discussed in more detail below), antibodies isolated from an animal (e.g., a mouse) that is 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 to 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 using human Ig transgenic animals), 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 method 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 the 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 An isolated antibody that specifically binds human PD-1 may, however, have cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0057] According to certain exemplary embodiments of the 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 set forth in U.S. Patent Publication No. 20150203579. 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 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 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 is the fully human anti-PD-1 antibody known as REGN2810 (also known as cemiplimab). According to certain exemplary embodiments, the methods of the invention include the use of REGN2810, or a biological equivalent thereof. As used herein, the term "biological equivalent" 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 a rate and / or extent of absorption that does not differ significantly from that of REGN2810 when administered in the same molar dose under similar experimental conditions, either in a single dose or multiple doses.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 REGN2810.

[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] 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 DFor example, an antibody or antigen-binding fragment thereof may be used that has 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.

[0061] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH compared to neutral pH. In addition, modification 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 CDR) with histidine residues, an antibody can be obtained with 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 antibody According to certain exemplary embodiments of the invention, the methods include administering a therapeutically effective amount of a bispecific antibody that specifically binds CD3 and MUC16. Such antibodies may be referred to herein, for example, as "anti-MUC16 / anti-CD3," or "anti-MUC16xCD3," or "MUC16xCD3" bispecific antibodies, 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 of which comprises 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 (C H2 -C H3 The Fc portion of an immunoglobulin (including a domain), for example, 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 antibody of the invention typically comprises two multimerization domains, e.g., two Fc domains, each of which is part of an individually distinct 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 antigen-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 antibodies of the invention, the Fc domain may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the 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 decreased) 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. 20150266966, 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 IgG1 domain, and the first Ig C domain is a IgG2 domain. The first Ig C domain is a IgG1 ... H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or eliminates Protein A binding, for example the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further contain a Y96F modification (Y436F according to IMGT and according to the EU). See, e.g., U.S. Patent No. 8,586,713. HAdditional 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, the 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 specific 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 present invention are described in U.S. Patent Publication No. 20140243504, which is incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding, thereby affecting 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. 20180112001. 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) of a HCVR (B-HCVR) 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 a HCVR comprising SEQ ID NO:1 (A-HCVR) and a LCVR comprising SEQ ID NO:2 (A-LCVR), and (b) a second antigen-binding arm comprising a HCVR comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 (B-HCVR) 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 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 Publication No. 20180112001.

[0073] Combination therapy According to certain embodiments, the methods of the invention comprise administering to a subject an anti-MUC16 / anti-CD3 bispecific antibody in combination with an anti-PD-1 antibody. In certain embodiments, the methods of the invention comprise administering the antibodies for additive or synergistic activity to treat cancer, preferably epithelioid sarcoma. As used herein, the term "in combination with" means that the anti-MUC16 / anti-CD3 bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. The term "in combination with" also includes sequential or simultaneous administration of the anti-PD-1 antibody and the bispecific anti-MUC16 / anti-CD3 antibody. For example, when administered "prior to" the bispecific anti-MUC16 / anti-CD3 antibody, the anti-PD-1 antibody 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. When administered "after" the bispecific anti-MUC16 / anti-CD3 antibody, the anti-PD-1 antibody 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 72 hours after administration of the bispecific anti-MUC16 / anti-CD3 antibody. "Concurrent administration" with the bispecific anti-MUC16 / anti-CD3 antibody means that the anti-PD-1 antibody is administered to the subject in a separate dosage form within less than 5 minutes of (before, after, or simultaneously with) administration of the bispecific anti-MUC16 / anti-CD3 antibody, or is administered to the subject as a single, combined dosage formulation including both the anti-PD-1 antibody and the bispecific anti-MUC16 / anti-CD3 antibody.

[0074] In certain embodiments, the methods of the invention include administration of a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer agent. In certain embodiments, the methods of the invention include administering an anti-PD-1 antibody and an anti-MUC16 / anti-CD3 bispecific antibody in combination with radiation therapy to produce 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 and the bispecific anti-MUC16 / anti-CD3 antibody to the cancer patient. For example, radiation therapy may be administered in one or more doses to the tumor lesion after administration of one or more doses of the antibody. In some embodiments, radiation therapy may be administered locally to the tumor lesion after systemic administration of the anti-PD-1 antibody and / or the bispecific anti-MUC16 / anti-CD3 antibody to enhance the 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 a bispecific anti-MUC16 / anti-CD3 antibody alone or in combination with an anti-PD-1 antibody to a subject, the antibodies being contained in separate pharmaceutical compositions or in a 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, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption 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.

[0077] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J.Biol.Chem.262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and can 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 empty of 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, Burgdorf, 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 ... STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices that have application in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are by no means limited to, the SOLOSTAR™ Pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to name just a few.

[0080] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used. In another embodiment, polymeric materials can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, controlled release systems 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, infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, for example, 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)]. The oily medium may be, for example, sesame oil, soybean oil, and the like, 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 active ingredients.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 and / or an anti-PD-1 antibody 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, alone or in combination with an anti-PD-1 antibody, 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, alone or in combination with one or more doses of an anti-PD-1 antibody. As used herein, "sequentially administering" means that each dose of the antibody is administered to the subject at different times, e.g., on different days separated by a predefined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient one initial dose of the antibody, followed by one or more secondary doses of the antibody, and optionally further one or more tertiary doses of the antibody.

[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 antibody (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 may be administered to a patient with epithelioid sarcoma 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.

[0086] In an exemplary embodiment of the invention, each secondary and / or tertiary dose is administered ½ to 14 weeks after the immediately preceding dose (e.g., ½ week, 1 week, 1 1 / 2 weeks, 2 weeks, 2 1 / 2 weeks, 3 weeks, 3 1 / 2 weeks, 4 weeks, 4 1 / 2 weeks, 5 weeks, 5 1 / 2 weeks, 6 weeks, 6 1 / 2 weeks, 7 weeks, 7 1 / 2 weeks, 8 weeks, 8 1 / 2 weeks, 9 weeks, 9 1 / 2 weeks, 10 weeks, 10 1 / 2 weeks, 11 weeks, 11 1 / 2 weeks, 12 weeks, 12 1 / 2 weeks, 13 weeks, 13 1 / 2 weeks, 14 weeks, 14 1 / 2 weeks, or more). As used herein, the phrase “immediately preceding dose” refers to a dose of bispecific anti-MUC16 / anti-CD3 (and / or anti-PD-1 antibody) in a series of multiple doses that is administered to a patient prior to administration of the immediately succeeding dose in the series, without any intervening doses.

[0087] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of the bispecific anti-MUC16 / anti-CD3 antibody (and / or anti-PD-1 antibody) 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 the bispecific anti-MUC16 / anti-CD3 antibody (e.g., and anti-PD-1 antibody) are administered on a more frequent basis (twice weekly, once weekly, or once every two weeks) at the start of the treatment regimen as an "induction dose", followed by subsequent doses ("consolidation doses" or "maintenance doses") administered on a less frequent basis (e.g., once every 4-12 weeks).

[0090] The present invention includes methods comprising sequential administration of a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody, to a patient for treating epithelioid sarcoma. In some embodiments, the methods comprise administering one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody, optionally followed by one or more doses of an anti-PD-1 antibody. In certain embodiments, the methods comprise administering a single dose of an anti-PD-1 antibody, followed by one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody. In some embodiments, one or more doses of an anti-PD-1 antibody at about 0.1 mg / kg to about 20 mg / kg can be administered, followed by one or more doses of a bispecific antibody at about 0.1 mg / kg to about 20 mg / kg to inhibit tumor growth and / or prevent tumor recurrence in a subject with epithelioid sarcoma. In some embodiments, administration of one or more doses of an anti-PD-1 antibody followed by one or more doses of a bispecific antibody 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 antibody as monotherapy). Alternative embodiments of the invention relate to the co-administration of an anti-PD-1 antibody and a bispecific antibody administered in separate doses at a similar or different frequency compared to the anti-PD-1 antibody. In some embodiments, the bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. In certain embodiments, the bispecific antibody is administered in a single dosage formulation with the anti-PD-1 antibody.

[0091] Dosage The amount of bispecific anti-MUC16 / anti-CD3 antibody, and optionally anti-PD-1 antibody, 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 antibody (anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody) that results in one or more of the following, compared to untreated subjects or subjects administered either antibody as monotherapy: (a) a reduction in the severity or duration of symptoms of cancer (e.g., epithelioid sarcoma); (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 or a delay or cessation of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) an increase in survival of subjects with cancer (e.g., epithelioid sarcoma); 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] In the case of a bispecific anti-MUC16 / anti-CD3 antibody, a therapeutically effective amount can 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 40 mg, about 60 mg, about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of a bispecific anti-MUC16 / anti-CD3 antibody.

[0093] In the case of an anti-PD-1 antibody, 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, and the like. The amount of anti-PD-1 antibody 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 anti-PD-1 antibody is administered.

[0094] The amount of bispecific anti-MUC16 / anti-CD3 antibody, and optionally anti-PD-1 antibody, contained in an individual dose may be expressed in milligrams per kilogram (i.e., mg / kg) of the subject's body weight. In certain embodiments, the bispecific anti-MUC16 / anti-CD3 antibody, and optionally anti-PD-1 antibody 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 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 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 skilled 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 allowed 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.

[0097] 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 CD3 and MUC16, such bispecific antigen-binding molecules are also referred to herein as "anti-MUC16 / anti-CD3 or anti-MUC16xCD3 bispecific molecules." 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 anti-MUC16 specific binding domains and anti-CD3 specific binding domains are constructed using standard methodologies, 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 illustrated 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 known to be promiscuous or known to pair effectively with various 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. US20140243504A1, 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 hours 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 1x10 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 diluted in PBS at 4000 units / mL and diluted in PBS at 4000 units / mL in 96-well microtiter plates. °C overnight. Plates were then washed with PBST and blocked with 0.5% BSA in PBS for 1 h. Biotinylated BSMUC16 / CD3-001, MUC16 parent 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, USA). 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 anti-tumor activity of anti-MUC16xCD3 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 cells previously passaged in vivo (day 0) were injected intraperitoneally (IP) into immunodeficient NSG mice 13 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 alone or in combination with 100ug of REGN2810 on days 5 and 8. 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 a significant anti-tumor effect as determined by BLI measurements on day 25, and combination with REGN2810 (anti-PD-1) further enhanced the anti-tumor effect. All groups had similar tumor burden assessed by BLI prior to dosing. There were no significant differences in tumor burden between 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 CD3 binding control (5mg / kg IV) along with isotype control or in combination with anti-PD-1. Treatment with BSMUC16 / CD3-001 extended median survival compared to the group that received the CD3 binding control, while 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 a 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 more than 20% 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 a CD3 binding control (1 mg / kg IV) along with an isotype control or an 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 10x10 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 10x10 6ID8-VEGF-huMUC16Δ tumor cells were implanted subcutaneously. Tumor-bearing mice were treated with a mean tumor size of 150 mm 3 If so, 20 days after transplantation 89 Zr-radiolabeled antibody was administered.

[0116] PET and 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 value by the respective weight 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 subjected to a 12-week 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 showed 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 during the treatment or recovery period, and no changes in urinalysis parameters, peripheral blood immunophenotyping, food consumption, or body weight. 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, nor were any macroscopic changes observed, either at terminal or recovery necropsies. Although a dose-related, reversible increase in circulating inflammatory markers (C-reactive protein (CRP) and IL-6) was observed within 1 day of the first dose of 1.0 or 0.1 mg / kg, these increases 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) were administered control substance (diluted placebo) or BSMUC16 / CD3-001 (0.01, 0.1, or 1 mg / kg) via 30-minute IV infusion once a week. 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 Roche Modular 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 administration 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 bearing only OVCAR3 / 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 4 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 in the trachea, as well as at low levels 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. Immunohistochemical staining 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) Vs 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 found in these tissues after BSMUC16 / CD3-001 administration (data not shown). Histopathological examination revealed that there was no inflammation or infiltration of MUC16-expressing tissues in mice at the time points examined after BSMUC16 / CD3-001 administration.

[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 1x10 6 BSMUC16 / CD3-001 cells / mL in RPMI medium supplemented with 100% IgG4 / mL ...

[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 Epithelioid Sarcoma with Anti-MUC16xAnti-CD3 Bispecific Antibody Alone or in Combination with Anti-PD-1 Antibody A single patient with recurrent epithelioid sarcoma who had exhausted all existing treatment options was treated with REGN4018, an anti-MUC16 x anti-CD3 bispecific antibody.

[0133] In this study, treatment assessment was based on tumor response. Tumor response assessment was 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.

[0134] Objective: Explore exploratory objectives.

[0135] This test exploratory purpose is as follows: Collection of blood for cytokine profiling, pharmacokinetics, and tumor tissue by biopsy is optional. When available, these samples will be used for the following exploratory purposes: (1) To evaluate 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, including cytokines -Changes in gene expression in tumors - Tumor expression levels of proteins such as MUC16.

[0136] Patient demographics: -Age: 20 -Gender: Female

[0137] Diagnosis: The patient has multiple recurrent metastatic epithelioid sarcoma with disease in the chest wall and lungs. Tumor samples have been shown to express MUC16 by immunohistochemistry while CA-125 (the shed extracellular portion of MUC16) was elevated in serum samples.

[0138] History of Disease Progression to Date: The patient was initially diagnosed with epithelioid sarcoma of the right radius. She underwent radiation therapy (50.4 Gy) to the right upper extremity and local surgery with negative margins. The first recurrence occurred with a nodal recurrence in the mid-arm that was treated with surgical resection and further radiation therapy (54 Gy). A further recurrence was detected with PET-avid nodal disease in the axilla and treated with surgery. There was a further disease recurrence in the right elbow within the previous radiation field. The patient was treated with tazemetostat in an earlier study, which was discontinued due to tolerability and compliance issues. With evidence of further disease progression in the arm and evidence of pulmonary nodules, the patient was treated with pembrolizumab. She had a long period of disease stability before being withdrawn from the study in the context of disease progression. The patient was then treated with nivolumab / ipilimumab and underwent a palliative shoulder girdle dissection of the right arm given worsening tumor-related ulceration. Disease evaluation showed stable disease in the chest, but in light of immune-related pneumonitis, immune checkpoint inhibitor therapy was discontinued. The patient was retreated with tazemetostat, which had been discontinued in association with disease progression. She was subsequently treated with CLR-131 (targeted radiation therapy), but there was further disease progression. Most recently, the patient underwent radiation therapy (20 Gy in 5 fractions) to the right breast for palliation of ulcerative skin lesions.

[0139] Currently, the patient has evidence of bilateral chest wall / breast tissue disease and multiple measurable lesions in both lungs. She is clinically stable with a Karnofsky score of 70 (primarily the result of the amputation). Currently, there are no clinical trial options or alternative "standard" therapies available for her disease in this setting.

[0140] Current Study Design and Treatment Procedure: REGN4018 is an investigational agent currently being evaluated in an ongoing adult phase 1 / 2 study in patients with platinum-experienced and / or intolerant ovarian, fallopian tube, or primary peritoneal cancer.

[0141] REGN4018 is administered by IV infusion over 4 hours (including flush) weekly. Treatment is continuous (once weekly) with cycles lasting 6 weeks (42 days). During cycle 1, patients received escalating doses of REGN4018 to reach a total dose according to the table below. The initial dose of drug was administered in divided doses on consecutive days (see below). Any dose may be divided at any time to improve tolerability. [Table 5]

[0142] Rationale for dose selection -REGN4018 is an investigational agent currently being evaluated in an ongoing adult Phase 1 / 2 study. Patients with this SPS will be treated at one dose level below the dose currently being evaluated in the monotherapy cohort of this Phase 1 / 2 study (450 mg), i.e., 250 mg, which is the highest dose level already demonstrated to be tolerable. The dose may then be adjusted based on new information at the physician's discretion to the patient's clinical benefit.

[0143] REGN4018 Administration Procedure -REGN4018 product is supplied as a lyophilized single-use product in 5 mg / vial and / or 50 mg / vial for administration by IV infusion. REGN4018 is administered by IV weekly. REGN4018 is administered by IV infusion over 4 hours (including flush). Patients must be observed in a monitored setting for at least 24 hours for the following doses / conditions: a) Cycle 1 - First dose on Day 1 of Cycle 1 - Cycle 1, split transition dose on days 8 / 9 - Cycle 1, first total dose split on days 15 / 16 - First 250 mg dose on Day 22 of Cycle 1 b) Escalation to the new highest tolerated dose level. c) Any subsequent dose of REGN4018 occurring following a Grade ≧2 cytokine release syndrome (CRS) AE is observed. d) Upon resumption of investigational drug after a prolonged drug-free period (e.g., >21 days off investigational drug).

[0144] During the inpatient observation period, hospitalization alone is insufficient to classify an AE as serious. Prolonged hospital stay due to an AE (beyond the protocol-mandated 24 hours) will be considered serious.

[0145] If the dose is split, monitoring times on the second day should follow the same guidelines as for day 1 of infusion. Cycle 1 Day 9, Cycle 1 Day 16, and Cycle 1 Day 23 (if appropriate) should be 24-hour monitoring.

[0146] After the patient tolerates the first full dose, the patient may then receive study therapy in an outpatient infusion unit, with the investigator having the option to shorten the duration of the REGN4018 infusion (including flush) to 3 hours, then 2 hours, 1 hour, and 30 minutes, with each well-tolerated weekly infusion of REGN4018. REGN4018 will be administered intravenously in either an inpatient or outpatient setting with immediate access to an acute care unit.

[0147] Premedication is not required and will be at the investigator's discretion.

[0148] Dose escalation - Dose escalation / reascalation may be performed at the investigator's discretion based on updated information from ongoing Phase 1 / 2 studies. Specifically, higher doses may be introduced into this SPS if they are determined to be tolerable in Phase 1 / 2 studies.

[0149] Dose modification - Dose modifications / reductions of REGN4018 may be performed at the investigator's discretion and in the patient's best interest. Adverse events (AEs) will be treated symptomatically.

[0150] Treatment Duration: With REGN4018 therapy, each cycle is 6 weeks (42 days) long. Treatment will continue until unacceptable toxicity, lack of clinical benefit, withdrawal of patient consent, physician's judgment (e.g., based on unacceptable AEs, quality of life, new treatment options), or lack of drug availability. In the setting of disease progression, treatment may be continued at the physician's discretion if the patient is tolerating treatment without disease progression and, in the investigator's opinion, the patient is deriving clinical benefit from continued study treatment. Clinical benefit will be objectively assessed at regular intervals outlined in this protocol or, if clinically indicated, with a final scheduled imaging reassessment (and compared to baseline).

[0151] After at least 24 weeks of treatment, if there is a clinical or radiological response, patients may elect to switch to a Q2W (every two weeks) dosing schedule of REGN4018 and continue all relevant study evaluations. Upon subsequent disease progression, patients may resume treatment at a dose level (including a new higher dose level) selected by the investigator for the patient's clinical benefit. Post-dose follow-up will be within 30 days of the last dose and also 90 days (± 10 days) after the last dose to complete end-of-study safety evaluations.

[0152] Safety Data Available for REGN4018 Therapy: An adult Phase 1 / 2 trial of REGN4018 in patients with ovarian cancer is ongoing and is currently enrolling at 450 mg (total dose, level 8a). The previous dose level of 250 mg (dose level 7a) is the proposed dose for use in this SPS and has been demonstrated to be tolerable. Currently available safety data, including treatment-emergent adverse events (TEAEs) and treatment-related TEAEs, are as follows:

[0153] Treatment-emergent adverse eventsThe most frequently reported TEAEs (all grades in ≥ 10% of patients) were: cytokine release syndrome (n = 20; 69%); abdominal pain (n = 18; 62.1%), nausea (n = 13; 44.8%), back pain (n = 10; 34.5%), anemia and fatigue (n = 9 each; 31 each), vomiting (n = 8; 27.6%), diarrhea and cough (n = 7 each; 24.1 each), pyrexia and hypomagnesemia (n = 5; 17.2 each), constipation, dyspnea, dry eyes, ocular discharge, decreased appetite, headache, hypotension, infusion-related reactions and dyspepsia, pleuritic pain (n = 4 each; 13.8 each), asthenia, flank pain, noncardiac chest pain, musculoskeletal pain, ocular pruritus, ocular congestion, conjunctivitis, sinus tachycardia, and chills (n = 3 each; 10.3% each). The incidence of TEAEs was highest during the first week of treatment and decreased in subsequent weeks.

[0154] Treatment-related TEAEs - Twenty-eight of 29 patients (96.6%) experienced at least one treatment-related TEAE as assessed by the investigator during the treatment period. The most frequently reported treatment-related TEAEs (all grades in ≥10% of patients) as assessed by the investigator were cytokine release syndrome (n=20, 69%), abdominal pain (n=16, 55.2%), nausea (n=7, 24.1%), back pain (n=6, 20.7%), fatigue and anemia (n=5 each, 17.2% each), vomiting, pleuritic pain, hypotension, infusion-related reaction and pyrexia (n=4 each, 13.8% each), diarrhea, dyspepsia, cough, dry eye, conjunctivitis and flank pain (n=3 each, 10.3% each). Eleven patients (37.9%) experienced a total of 17 grade 3 treatment-related TEAEs (Table 9). The most frequently reported grade 3 treatment-related adverse event was abdominal pain (n=6; 20.7%, 2 patients in DL3 and 4 patients in DL4). There were no grade 4 or 5 treatment-related TEAEs.

[0155] Potential efficacy of REGN4018 in the patient studied in this SPS: Immunohistochemistry confirms that the patient's cancer expresses MUC16, a relevant target of REGN4018. Additionally, there is an elevation of CA-125 (the shed extracellular portion of MUC16) in serum samples. The potential benefit is that the investigational drug may halt or shrink the patient's cancer growth for a period of time. Symptoms such as pain caused by cancer may be reduced.

[0156] Identified Potential Risks: Below are important identified and potential risks of treatment based on the drug's safety profile and patient medical history.

[0157] Key identified risks: infusion-related reactions and cytokine release syndrome -Infusion-related reactions (IRR) and cytokine release syndrome (CRS) are associated with typical signs and symptoms including, but not limited to, flushing, tachycardia, hypotension, dyspnea, bronchospasm, back pain, fever, urticaria, edema, nausea, and rash.

[0158] Infusion-related reactions are common adverse drug reactions (ADRs) observed with monoclonal antibodies. Symptoms are temporally related to drug administration and can range from symptomatic discomfort to fatal events. Numerous reported terms are used to identify IRRs, with significant overlapping symptoms, while each has a very different etiology. Such terms include anaphylaxis, anaphylactoid reactions, CRS, and complement activation-associated pseudoallergy (CARPA). The primary reason for attempting to identify the etiology of symptoms and the actual diagnosis is to ensure the identification of reactions that are likely to recur or worsen with subsequent exposure to the drug (e.g., anaphylaxis) as opposed to reactions that are likely to improve with repeated exposure (anaphylactoid reactions and CRS).

[0159] An IRR is defined as an adverse reaction occurring during the infusion and for 2 hours thereafter. In the context of this study, signs and symptoms of an IRR occurring more than 2 hours after completion of the infusion will be identified as CRS.

[0160] Clinically, CRS is a syndrome that includes symptoms of fever, hypotension, tachycardia, and hypoxia. In addition, there may be neurological findings that reflect immune effector cell-associated neurotoxicity syndrome (ICANS), including delirium, encephalopathy, aphasia, lethargy, agitation, tremors, and seizures.

[0161] Risk Mitigation Measures: IRR and CRS 1) the use of initial and transition doses to gradually approach total dose levels; 2) inpatient monitoring for at least 24 hours during initial dose escalation and as otherwise specified; 3) Infusion-related reactions and cytokine release syndrome should be managed with symptomatic and supportive care according to institutional practice standards.

[0162] Evaluation of Drug Response: The safety and tolerability of REGN4018 in patients will be monitored by clinical evaluation of AEs and by repeated measurements of clinical assessments including vital signs (temperature, blood pressure, pulse, oxygen saturation, and respiration), physical examination (complete and limited), 12-lead electrocardiogram (ECG), echocardiogram, chest x-ray, and laboratory evaluations including standard hematology, blood chemistry, and urinalysis.

[0163] Because cytokine release after initial administration (initial dose and / or subsequent doses) has been observed with bispecific antibodies and similar molecules, specific measures were implemented in this study, including an initial dose of 1 mg (day 1 of cycle 1), a transition dose of 20 mg (day 8 of cycle 1), the option of split doses, 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.

[0164] Tumor evaluations will include all known or suspected disease sites. Tumor evaluations will be performed prior to the start of Cycle 1 (baseline), then prior to Cycle 2 (± 7 days), then prior to each even-numbered cycle (± 7 days). Additional tumor evaluations may be performed given clinical indications. Tumor evaluations should be repeated at the end of treatment, if necessary, if more than 2 cycles have elapsed since the last evaluation. Evaluations will be performed according to institutional practice standards and will be compared to baseline measurements obtained within 28 days of starting study therapy.

[0165] A baseline ocular examination is required for expression of MUC16 on the corneal and conjunctival epithelium.

[0166] Blood samples will be taken for measurement of drug concentrations and for ADA assessment.

[0167] A simple neurological examination is required within 24 hours after administration of the study drug.

[0168] 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.

[0169] Antitumor activity will be assessed by CT or MRI or PET-CT, as well as monitoring of performance status and serum CA-125 levels.

[0170] Permitted and prohibited concomitant medications and rationale:

[0171] Prohibited and discouraged drugs -During participation in this study, patients may not receive any standard or investigational agent for the treatment of their tumor other than REGN4018 as monotherapy, with the exception of permitted local palliative external beam radiation therapy.

[0172] It is recommended that patients not receive systemic corticosteroids such as hydrocortisone, prednisone, prednisolone (Solu-Medrol®) or dexamethasone (Decadron®) at any time throughout the study, except for life-threatening emergencies and / or to treat IRR / CRS not responsive or predicted to be not responsive to anti-IL-6 pathway therapy (e.g., tocilizumab), or irAEs severe enough to require corticosteroids. Inhaled, topical, ophthalmic, or intranasal steroids are permitted.

[0173] Permitted Drugs - Focal palliative therapy (e.g., radiation therapy) may be permitted. Pre-infusion medications are at the investigator's discretion, e.g., antihistamines, acetaminophen, nonsteroidal anti-inflammatory drugs (e.g., ketorolac or ibuprofen), and / or oral opioids (e.g., hydromorphone). Specific premedications are not mandated by the protocol.

[0174] Allow systemic corticosteroids in physiologic replacement doses, even if >10 mg / day prednisone equivalent. Allow short-term corticosteroids for prevention (e.g., contrast allergy) or treatment (e.g., delayed-type hypersensitivity reactions triggered by contact allergens) of non-autoimmune conditions.

[0175] Any other medications deemed necessary for the patient's welfare and expected not to interfere with the evaluation of the investigational drug may be administered at the discretion of the investigator. Treatment of bone metastases (bisphosphonates, denosumab) and hormonal therapy are permitted.

[0176] Study endpoints:

[0177] The primary endpoints of the study were dose-limiting toxicity, treatment-emergent adverse events (TEAEs; including immune-related adverse events [irAEs]), serious AEs (SAEs), deaths, laboratory abnormalities (grade ≥3 by CTCAE), and PK of therapy.

[0178] The secondary endpoint of the study was ORR based on Response Evaluation Criteria in Solid Tumors (RECIST).

[0179] Preliminary Results: Safety and efficacy data from a REGN4018 monotherapy study in a single patient with recurrent epithelioid sarcoma are presented below.

[0180] Patients with recurrent epithelioid sarcoma with elevated cancer antigen (CA)-125 levels received REGN4018 administered intravenously (IV) weekly at doses ranging from 1 to 250 mg. Step-up dosing of the first two doses was utilized to reduce the risk of cytokine release syndrome (CRS) via a gradual increase in drug exposure. Primary endpoints included safety and PK. Secondary endpoints included efficacy as determined by objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.

[0181] The patient experienced grade 2 cytokine release syndrome on C1D2 and C1D9. Other treatment-emergent adverse events included grade 1 cough, and grade 2 intermittent hypoxia, myalgia, constipation, pericardial effusion, and pleural effusion.

[0182] Upon treatment, the patient's CA-125 levels decreased and target lesions on CT scans showed a partial response by RECIST v1.1, sustained for 60 weeks, from a baseline sum of 89mm in diameter to 60mm by cycle 4 imaging. Additionally, the patient's skin cancer ulcers on her right breast decreased in size. These lesions improved significantly with treatment with REGN4018, resulting in near complete wound healing and significant improvement in pain caused by such lesions.

[0183] REGN4018 was tolerable to patients in this single-patient study with evidence of clinical activity as judged by ORR based on Response Evaluation Criteria in Solid Tumors (RECIST).Data from this analysis support the treatment of recurrent epithelioid sarcoma with REGN4018 (anti-MUC16 x anti-CD3) bispecific antibody.

[0184] Treating epithelioid sarcoma with REGN4018 in combination with cemiplimab: It is contemplated that this patient with recurrent epithelioid sarcoma may be administered an anti-PD-1 antibody (e.g., cemiplimab) in combination with a REGN4018 bispecific antibody (i.e., an anti-MUC16x anti-CD3 antibody) to treat the tumor.

[0185] Example 10: Semi-quantitative MUC16 immunohistochemical expression across different subtypes of pediatric sarcomas We investigated semi-quantitative MUC16 immunohistochemistry (IHC) expression across different subtypes of pediatric sarcomas. Between 2015 and 2021, 72 specimens were collected from patients <25 years old at Children's Hospital and included epithelioid sarcoma (n=6), rhabdoid tumor of kidney and soft tissue (n=4), rhabdomyosarcoma (n=23), Ewing sarcoma (n=19), and other soft tissue sarcomas (n=19). IHC was performed on 4 μm thick formalin-fixed paraffin-embedded tissue sections from a representative block of each case or specimen. The assay was performed on a Dako Omnis staining platform using an anti-CA125 antibody (clone M11, monoclonal mouse, ready to use, DAKO, catalog no. GA701) and the Envision Flex detection kit (DAKO, catalog no. GV800). Appropriate positive and negative controls were used. Immunoreactivity was assessed using the H-score method.

[0186] Of the six epithelioid sarcoma specimens selected for MUC16 IHC, three showed diffuse and intense staining reaction (H score 300), one showed heterogeneous staining (H score 110), and the remaining two were negative (H score 0). Of the remaining tumor entities, MUC16 expression was present in two of four kidney and soft tissue rhabdoid tumor samples (H score 300 and 170, respectively) and was absent in the remaining 62 cases (H score 0).

[0187] MUC16 expression is a frequent feature in epithelioid sarcoma and can be identified in other INI-1 (SMARCB1)-deficient malignancies.

[0188] 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 6] *******

Claims

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

2. The pharmaceutical composition according to claim 1, wherein the MUC16-expressing sarcoma is an epithelioid sarcoma, a rhabdoid tumor of the kidney and soft tissue, a rhabdomyosarcoma, a Ewing sarcoma, or a soft tissue sarcoma.

3. The pharmaceutical composition according to claim 1, wherein the method is for treating MUC16-expressing epithelioid sarcoma in a subject, and optionally the subject has metastatic epithelioid sarcoma.

4. The pharmaceutical composition according to claim 1, wherein the MUC16-expressing sarcoma lacks the expression of functional integrase interactor 1.

5. The aforementioned subject is, (a) previously treated with anticancer therapy, and / or (b) The pharmaceutical composition according to claim 1, which is resistant to or has an insufficient response to prior therapy, or has relapsed after prior therapy.

6. The first antigen-binding domain 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: 1, Optionally, three heavy chain complementarity determining regions, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 8, HCDR2 contains the amino acid sequence of SEQ ID NO: 9, and HCDR3 contains the amino acid sequence of SEQ ID NO: 10, 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, Optionally, three light chain complementarity determination regions: LCDR1 contains the amino acid sequence of SEQ ID NO: 11, LCDR2 contains the amino acid sequence of SEQ ID NO: 12, and LCDR3 contains the amino acid sequence of SEQ ID NO:

13. A pharmaceutical composition according to claim 1, comprising:

7. The pharmaceutical composition according to claim 6, wherein 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.

8. The second antigen-binding domain 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: 3, Optionally, three heavy chain complementarity determining regions, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 14, HCDR2 contains the amino acid sequence of SEQ ID NO: 15, and HCDR3 contains the amino acid sequence of SEQ ID NO: 16, 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, Optionally, three light chain complementarity determination regions: LCDR1 contains the amino acid sequence of SEQ ID NO: 11, LCDR2 contains the amino acid sequence of SEQ ID NO: 12, and LCDR3 contains the amino acid sequence of SEQ ID NO:

13. The pharmaceutical composition according to claim 6, comprising:

9. The pharmaceutical composition according to claim 7, wherein 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.

10. The aforementioned bispecific antibody contains the human IgG heavy chain constant region, and optionally, (a) The human IgG heavy chain constant region is isotype IgG1 or isotype IgG4, and optionally the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype, and / or (b) The pharmaceutical composition according to claim 9, wherein the first or second heavy chain comprises a CH3 domain comprising H435R (EU numbering) modification and Y436F (EU numbering) modification, but not both.

11. The aforementioned bispecific antibody (a) The first heavy chain containing the amino acid sequence of SEQ ID NO: 29, (b) A second heavy chain containing the amino acid sequence of SEQ ID NO: 31, and / or (c) 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 9, comprising:

12. (a) The subject has a serum CA-125 level greater than 92 U / ml, (b) The bispecific antibody is administered in a dosing regimen that includes a divided initial dose, and / or (c) The bispecific antibody is administered to the subject at a dose of 1 mg to 1000 mg per week, or at a dose of 250 mg per week. A pharmaceutical composition according to any one of claims 1 to 5.

13. The method further comprises administering a second therapeutic agent or therapeutic regimen. Optionally, the second therapeutic agent or therapeutic regimen comprises an anti-PD-1 antibody or its antigen-binding fragment. Furthermore, optional, (a) The anti-PD-1 antibody or antigen-binding fragment is (i) 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, Optionally, three heavy chain complementarity determining regions, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 35, HCDR2 contains the amino acid sequence of SEQ ID NO: 36, and HCDR3 contains the amino acid sequence of SEQ ID NO: 37, and (ii) 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, Optionally, three light chain complementarity determining regions, wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 38, LCDR2 contains the amino acid sequence of SEQ ID NO: 39, and LCDR3 contains the amino acid sequence of SEQ ID NO:

40. (iii) HCVR containing the amino acid sequence of SEQ ID NO: 33, and LCVR containing the amino acid sequence of SEQ ID NO: 34, Optionally, the anti-PD-1 antibody or 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. including and / or (b) The anti-PD-1 antibody is administered to the subject before, simultaneously with, or after the bispecific antibody. A pharmaceutical composition according to any one of claims 1 to 5.

14. (a) The subject has been previously treated with chemotherapy drugs, radiotherapy, surgery, or immunotherapy drugs, and at their discretion, (i) The chemotherapy drug is tazemetostat, or (ii) The immunotherapy drug is a PD1 inhibitor or a CTLA4 inhibitor, (iii) The immunotherapy drug is pembrolizumab, nivolumab, or ipilimumab. (b) The subject has stable disease status, partial response, or complete response after administration of the bispecific antibody at a dose of 1 to 250 mg for at least one week. (c) The epithelioid sarcomas in the breast tissue of the subject decrease in size and / or decrease in size after administration of the bispecific antibody at a dose of 1 to 250 mg for at least one week. (d) The antibody is administered intravenously or subcutaneously to the subject. A pharmaceutical composition according to any one of claims 1 to 5.

15. The bispecific antibody is administered in a regimen comprising: administering an initial dose of 1 mg of the bispecific antibody during the first week of the regimen; administering a transition dose of 20 mg of the bispecific antibody during the second week of the regimen; and administering a total dose of 250 mg of the bispecific antibody during the third week of the regimen. Optional, (a) The initial dose is divided into two equal fractions to be administered on consecutive days. (b) The transfer dose is divided into two equal fractions to be administered on consecutive days. (c) The total dose is divided into two fractions, optionally 50 mg and 250 mg, to be administered on consecutive days, and / or (d) The administration regimen further comprises administering a maintenance dose of 250 mg of the bispecific antibody during the fourth week of the administration regimen, and optionally, the maintenance dose is administered weekly during the weeks following the administration regimen. A pharmaceutical composition according to any one of claims 1 to 5.

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