WO2023177772A1 - Methods of treating recurrent epithelioid sarcoma with bispecific anti-muc16 x anti-cd3 antibodies alone or in combination with anti-pd-1 antibodies - Google Patents

Abstract

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

Description

METHODS OF TREATING RECURRENT EPITHELIOID SARCOMA WITH BISPECIFIC ANTI- MUC16 x ANTI-CD3 ANTIBODIES ALONE OR IN COMBINATION WITH ANTI-PD-1 ANTIBODIES

REFERENCE TO A SEQUENCE LISTING

[0001] This application incorporates by reference a computer readable Sequence Listing in ST.26 XML format, titled 11138WO01_Sequence, created on March 16, 2023 and containing 54,705 bytes.

FIELD OF THE INVENTION

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

BACKGROUND

[0003] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a single transmembrane domain highly glycosylated integral membrane glycoprotein that is highly expressed in epithelioid sarcoma (Hoshino, M. et al., 2010, J Cancer Res Clin Oncol., 136(3):457-64). MUC16 consists 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 comprises a segment of the transmembrane region and a short cytoplasmic tail. Proteolytic cleavage results in shedding of 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 uterine cervix, and adenocarcinoma of the gastric tract, and in diseases and conditions including inflammatory bowel disease, liver cirrhosis, cardiac 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 is shown to protect tumor cells from the immune system. (Felder, M. et al., 2014, Molecular Cancer, 13:129) Methods for treating cancer using antibodies to MUC16 have been investigated. Oregovomab and abgovomab are anti-MUC16 antibodies which have had limited success. (Felder, supra, Das, S. and Batra, S.K. 2015, Cancer Res. 75:4660-4674.)

[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. The CD3 dimeric arrangements include gamma/epsilon, delta/epsilon and zeta/zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes involving the activation of T cells. In addition, bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving 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 allowing tumor cells to escape immune surveillance 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 has demonstrated the clinical activity of PD- 1 blockade in patients with aggressive NHL and Hodgkin's lymphoma (Lesokhin, et al. 2014, Abstract 291 , 56th ASH Annual Meeting and Exposition, San Francisco, Calif.; Ansell et al. 2015, N. Engl. J. Med. 372(4) :311-9).

[0006] Epithelioid sarcoma is a rare, highgrade, soft tissue tumor that has a known propensity for local recurrence, regional lymph node involvement, and distant metastases. (Sobanko et al., J Clin Aesthet Dermatol. 2(5):49-54, 2009). Most cases begin in the soft tissue under the skin of a finger, hand, forearm, lower leg or foot, though the tumor can begin to grow in other areas of the body. Because epithelioid sarcoma presents innocuously, the malignancy inherently portends a poor prognosis. Misdiagnosis of this tumor can lead to delayed and improper treatment, adversely affecting patient survival. The current treatment modalities for epithelioid sarcoma include radical tumor excision, 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). Whilst the majority of patients respond to initial treatment, most experience a recurrence of the disease, resulting in a cycle of repeated surgeries and additional rounds of chemotherapy. Although recurrent epithelioid sarcoma may respond to further treatment, virtually all of them will ultimately 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 in epithelioid sarcoma patients have previously been detected with high levels of PD-L1 expression and CD3⁺/CD8⁺ 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, blockade of this pathway alone may not be sufficient.

[0008] Thus, in view of this high unmet need, additional therapies for targeting epithelioid sarcoma are needed.

BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, the present 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 mucin 16 (MUC16) on a target tumor cell, and a second antigen-binding domain that specifically binds 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, the MUC16-expressing cancer is a MUC16-expressing sarcoma. In some cases, the sarcoma is epithelioid sarcoma, rhabdoid tumor of kidney and soft tissue, rhabdomysarcoma, Ewing’s sarcoma, or a soft tissue sarcoma. In some cases, the MUC16- expressing sarcoma is epithelioid sarcoma or rhabdoid tumor of kidney and soft tissue.

[0012] In some embodiments, the MUC16-expressing cancer (e.g., a sarcoma) is deficient in expression of functional integrase interactor 1 protein.

[0013] In some embodiments, the subject has previously been treated with an anti-cancer therapy. In some cases, the subject is resistant to, inadequately responsive to, or relapsed after, prior therapy. In some cases, the subject has previously been treated with a chemotherapy drug, radiation therapy, surgery, or an immunotherapy drug. In some cases, the chemotherapy drug is tazemetostat. In some cases, the immunotherapy drug is a PD1 inhibitor or a CTLA4 inhibitor. In some cases, the immunotherapy drug is pembrolizumab, nivolumab or ipilimumab. In some cases, the MUC16-expressing cancer is resistant or inadequately responsive to, or relapsed after, prior 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) contained 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) contained 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some cases, the first antigen-binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some cases, the first antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1 , and a 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 discussed above, the bispecific antibody comprises a second antigen-binding domain comprising: (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: 3; 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: 2. In some cases, the second antigen-binding domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16. In some cases, the second antigen-binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 , a LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some cases, the second antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 3, and a LCVR comprising the amino acid sequence of SEQ ID NO: 2.

[0016] In some embodiments, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype lgG1. In some cases, the human IgG heavy chain constant region is isotype lgG4.

[0017] In some embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fey receptor binding relative to a wild-type hinge of the same isotype.

[0018] In some embodiments, the first heavy chain or the second heavy chain of the bispecific antibody, but not both, comprises a CH3 domain comprising a 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 at least two times the upper limit of normal. In some embodiments, the subject has a serum CA-125 level of greater than 92 U/ml.

[0021] In some embodiments, the method further comprises administering a second therapeutic agent or therapeutic regimen. In some cases, the second therapeutic agent or therapeutic 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some cases, the anti-PD-1 antibody or antigenbinding fragment comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some cases, the anti-PD-1 antibody or antigenbinding fragment comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 33, and a LCVR comprising the amino acid sequence of SEQ ID NO: 34. In some cases, the anti- PD-1 antibody or antigen-binding fragment is an anti-PD-1 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.

[0023] In some embodiments, the bispecific antibody is administered in a dosing regimen comprising a split initial dose (e.g., an initial dose of 1 mg is split into two equal fractions of 0.5 mg administered over two consecutive days). In some embodiments, the bispecific antibody is administered to the subject at a dose of from 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 at a frequency of every 3 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 prior to, concurrent 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 following administration of the bispecific antibody for at least one week at a dose of 1-250 mg. In some cases, the epithelioid sarcoma of the breast tissue of the subject decreases in size following administration of the bispecific antibody for at least one week at a dose of 1-250 mg.

[0025] In some embodiments of the method, the bispecific antibody is administered in a dosing regimen comprising: administration of an initial dose of 1 mg of the bispecific antibody during week 1 of the dosing regimen; administration of a transitional dose of 20 mg of the bispecific antibody during week 2 of the dosing regimen; and administration of a full dose of 250 mg of the bispecific antibody during week 3 of the dosing regimen. In some cases, the initial dose is split into two equal fractions administered on consecutive days. In some cases, the transitional dose is split into two equal fractions administered on consecutive days. In some cases, the full dose is split into two fractions administered on consecutive days. In some embodiments, the two fractions of the full dose comprise a 50 mg fraction and a 250 mg fraction. In some embodiments, the dosing regimen further comprises administration of 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, the maintenance dose is administered every other week (Q2W) during subsequent weeks of the dosing regimen.

[0026] Other embodiments of the present invention will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 illustrates the binding of various concentrations of anti-MUC16 clone 3A5 and BSMUC16/CD3-001 to CA125, as determined by ELISA (described in Example 2 herein). BSMUC16/CD3-001 and its MUC16 parental antibody displayed a markedly reduced binding signal at all concentrations tested in comparison to an anti-MUC16 clone 3A5 that binds to the repeat region of MUC16.

[0028] Figure 2 illustrates the mean tumor growth curves for groups of mice (5 per group) treated with CD3-binding control + isotype control (A), BSMUC16/CD3-005 + isotype control (□), CD3-binding control + anti-PD-1 (A), and BSMUC16/CD3-005 + anti-PD-1 (■) (as described in Example 3 herein). The combination of an anti-PD-1 antibody and an anti-CD3xMUC16 bispecific antibody synergistically inhibited tumor growth.

[0029] Figure 3 illustrates the impact of T cell incubation with BSMUC16/CD3-001 on the percentage of PD-1 positive T cells.

DETAILED DESCRIPTION

[0030] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiments or features of embodiments can be combined with one another, and such combinations are expressly encompassed within the scope of the present invention. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1 , 99.2, 99.3, 99.4, etc.).

[0032] 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 in this specification are incorporated herein by reference in their entireties.

Methods for Treating or Inhibiting the Growth of Cancers

[0033] The present invention includes methods for treating, ameliorating or reducing the severity of at least one symptom or indication, or inhibiting the growth of a cancer (e.g., recurrent epithelioid sarcoma) in a subject. The methods according to this aspect of the invention comprise administering 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 PD-1 to a subject in need thereof. As used herein, the terms "treat", "treating", or the like, mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, to delay or inhibit tumor growth, to reduce tumor cell load or tumor burden, to promote tumor regression, to cause tumor shrinkage, necrosis and/or disappearance, to prevent tumor recurrence, and/or to increase duration of survival of the subject.

[0034] As used herein, the expression "a subject in need thereof" means a human or nonhuman mammal that exhibits one or more symptoms or indications of cancer, and/or who has been diagnosed with cancer, including an epithelioid sarcoma and who needs treatment for the same. In many embodiments, the term "subject" may be interchangeably used with the term "patient". For example, a human subject may be diagnosed with a primary or a metastatic tumor and/or with one or more symptoms or indications including, but not limited to, enlarged lymph node(s), swollen abdomen, chest pain/pressure, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlargement of spleen, itching. The expression includes subjects with primary or established epithelioid sarcoma. In specific embodiments, the expression includes human subjects that have and need treatment for epithelioid sarcoma or another tumor expressing MUC16. In other specific embodiments, the expression includes subjects with MUC16+ tumors (e.g., a tumor with MUC16 expression as determined by flow cytometry). In certain embodiments, the expression "a subject in need thereof" includes patients with an epithelioid sarcoma that is resistant to or refractory to or is inadequately controlled by prior therapy (e.g., treatment with a conventional anti-cancer agent). For example, the expression includes subjects who have been treated with chemotherapy, such as a chemotherapeutic agent (e.g., tazemetostat) or a taxol compound (e.g., docetaxel). The expression also includes subjects with an epithelioid sarcoma for which conventional anticancer therapy is inadvisable, for example, due to toxic side effects. For example, the expression includes patients who have received one or more cycles of chemotherapy with toxic side effects. In certain embodiments, the expression "a subject in need thereof" includes patients with an epithelioid sarcoma which has been treated but which has subsequently relapsed or metastasized. For example, patients with an epithelioid sarcoma that may have received treatment with one or more anti-cancer agents leading to tumor regression; however, subsequently have relapsed with cancer resistant to the one or more anti-cancer agents (e.g., chemotherapy-resistant cancer) are treated with the methods of the present invention. [0035] The expression "a subject in need thereof" also includes subjects who are at risk of developing epithelioid sarcoma, e.g., persons with a family history of epithelioid sarcoma, persons with a past history of infections associated with epithelioid sarcoma, persons with mutations in the SMARCB1 gene, or persons with an immune system compromised due to HIV infection or due to immunosuppressive medications.

[0036] In certain embodiments, the methods of the present invention may be used to treat patients that show 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 present invention comprise 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 an elevated level of PD-L1 and/or CA125.

[0037] In certain embodiments, the methods of the present invention are used in a subject with an epithelioid sarcoma. The terms "tumor", "cancer" and "malignancy" are interchangeably used herein. The term "epithelioid sarcoma", as used herein, refers to tumors of soft tissue, e.g., under the skin of a finger, hand, forearm, lower leg or foot; although the tumor may begin to grow in other areas of the body as well.

[0038] According to certain embodiments, the present invention includes methods for treating, or delaying or inhibiting the growth of a tumor. In certain embodiments, the present invention includes methods to promote tumor regression. In certain embodiments, the present invention includes methods to reduce tumor cell load or to reduce tumor burden. In certain embodiments, the present invention includes methods to prevent tumor recurrence. The methods, according to this aspect of the invention, comprise 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, wherein each antibody is administered to the subject in multiple doses, e.g., as part of a specific therapeutic dosing regimen. For example, the therapeutic dosing regimen may comprise administering one or more doses of an anti-MUC16 x CD3 antibody to the subject at a frequency of about 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, the one or more doses of anti-PD-1 antibody are administered in combination with one or more doses of a therapeutically effective amount of a bispecific anti-MUC16/anti- CD3 antibody, wherein the one or more doses of the anti-PD-1 antibody are administered to the subject at a frequency of about 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.

[0039] In certain embodiments, each dose of the anti-MUC16/anti-CD3 antibody is administered in more than 1 fractions, e.g., in 2-5 fractions ("split dosing") within the given dosing period. The anti-MUC16/anti-CD3 bispecific antibody may be administered in split doses to reduce or eliminate the cytokine "spikes" induced in response to administration of the antibody. Cytokine spikes refer to the clinical symptoms of the cytokine release syndrome ("cytokine storm") and infusion related reactions. In certain embodiments, the methods of the present invention comprise administering one or more doses of anti-PD-1 antibody in combination with one or more doses of a bispecific anti-MUC16/anti-CD3 antibody to a subject in need thereof, wherein a dose of the bispecific antibody is administered as split doses, or in more than 1 fractions, e.g., as 2 fractions, as 3 fractions, as 4 fractions or as 5 fractions within the given dosing period. In certain embodiments, a dose of the bispecific antibody is split into 2 or more fractions, wherein each fraction comprises an amount of the antibody equal to the other fractions. For example, a dose of anti-MUC16/anti-CD3 antibody comprising 1000 micrograms may be administered once a week, wherein the dose is administered in 2 fractions within the week, each fraction comprising 500 micrograms. In certain embodiments, a dose of the bispecific antibody is administered split into 2 or more fractions, wherein the fractions comprise unequal amounts of the antibody, e.g., more than 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, wherein the dose is administered in 2 fractions within the week, wherein the first fraction comprises 700 micrograms and the second fraction comprises 300 micrograms. As another example, a dose of anti-MUC16/anti-CD3 antibody comprising 1000 micrograms may be administered once in 2 weeks, wherein the dose is administered in 3 fractions within the 2-week period, wherein the first fraction comprises 400 micrograms, the second fraction comprises 300 micrograms and the third fraction comprises 300 micrograms.

[0040] In certain embodiments, the present invention includes methods to inhibit, retard or stop tumor metastasis or tumor infiltration into peripheral organs. The methods, according to this aspect, comprise 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.

[0041] In specific embodiments, the present invention provides methods for increased anti- tumor efficacy or increased tumor inhibition. The methods, according to this aspect of the invention, comprise administering to a subject with an epithelioid sarcoma a therapeutically effective amount of an anti-PD-1 antibody prior to administering a therapeutically effective amount of a bispecific anti-MUC16/anti-CD3 antibody, wherein the anti-PD-1 antibody may 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 methods provide for increased tumor inhibition, e.g., by about 20%, more than 20%, more than 30%, more than 40% more than 50%, more than 60%, more than 70% or more than 80% as compared to a subject administered with the bispecific antibody prior to the anti-PD-1 antibody.

[0042] In certain embodiments, the methods of the present invention comprise administering a therapeutically effective amount of a bispecific anti-CD3xMUC16 antibody alone, or in combination with an anti-PD-1 antibody to a subject with an epithelioid sarcoma. In further embodiments, the epithelioid sarcoma is indolent or aggressive. In certain embodiments, the subject is not responsive to prior therapy or has relapsed after prior therapy. In some embodiments, the subject has a CA-125 level that is equal to or greater than 2 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 (prior to treatment) is at or greater than 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 present invention further comprise administering an additional therapeutic agent to the subject.

[0043] In certain embodiments, the methods of the present invention comprise administering a therapeutically effective amount of a bispecific anti-MUC16/anti-CD3 antibody to a subject with a MUC16+ cancer. In specific embodiments, the cancer is an epithelioid sarcoma. In further embodiments, the epithelioid sarcoma is indolent or aggressive. In some embodiments, the epithelioid sarcoma is present in chest wall, lungs, and/or breast tissue of the patient. In some embodiments, the patient with epithelioid sarcoma has elevated levels of serum CA-125 (e.g., at least 2x ULN). In some embodiments, the cancer is resistant to chemotherapy drugs. In certain embodiments, the subject is not responsive to prior therapy or has relapsed after prior therapy (e.g., chemotherapy).

[0044] In certain embodiments, the methods of the present 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, an anti- PD-1 antibody in combination with a bispecific anti-MUC16/anti-CD3 antibody is administered as a "second line" treatment (e.g., after prior therapy). For example, an anti-PD-1 antibody in combination with a bispecific anti-MUC16/anti-CD3 antibody is administered as a "second line" treatment to a subject that has relapsed after prior therapy with, e.g., chemotherapy.

[0045] In certain embodiments, the methods of the present invention are used to treat a patient with a MRD-positive disease. Minimum residual disease (MRD) refers to small numbers of cancer cells that remain in the patient during or after treatment, wherein the patient may or may not show symptoms or signs of the disease. Such residual cancer cells, if not eliminated, frequently lead to relapse of the disease. The present invention includes methods to inhibit and/or eliminate residual cancer cells in a patient upon MRD testing. MRD may be assayed according to methods known in the art (e.g., MRD flow cytometry). The methods, according to this aspect of the invention, comprise administering a bispecific anti-MUC16/anti-CD3 antibody alone, or in combination with an anti-PD-1 antibody to a subject in need thereof.

[0046] The methods of the present 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 may be an agent selected from the group consisting of, e.g., 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 to 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, a cytotoxin, a chemotherapeutic agent, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21 , and IL-15, an anti-inflammatory drug such as corticosteroids, and non-steroidal anti-inflammatory drugs, and a dietary supplement such as anti-oxidants. In certain embodiments, the antibodies may be administered in combination with 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 antibodies are administered to the subject at the same time as, just before, or just after administration of the third therapeutic agent. In certain embodiments, the third therapeutic agent is administered as a co-formulation with the antibodies.

[0047] In certain embodiments, the methods of the present 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. Where the combination is administered, the administration of the antibodies leads to 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% as compared to an untreated subject or a subject administered with either antibody as monotherapy. In certain embodiments, the administration of an anti-PD-1 antibody and a bispecific anti-MUC16/anti-CD3 antibody leads to increased tumor regression, tumor shrinkage and/or disappearance. In certain embodiments, the administration of an anti-PD-1 antibody and a bispecific anti-MUC16/anti-CD3 antibody leads to delay in tumor growth and development, 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 as 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 duration of survival of the subject, e.g., increases duration of survival by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months than an untreated subject or a subject which is administered either antibody as monotherapy. In certain embodiments, administration of the antibodies in 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 response and duration of response in a subject, e.g., by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% over an untreated subject or a subject which has received 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 an epithelioid sarcoma leads to complete disappearance of all evidence of tumor cells ("complete response"). In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti- MUC16/anti-CD3 antibody to a subject with an epithelioid sarcoma leads to at least 30% or more decrease 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 an 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, e.g., X-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analyses. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti- MUC16/anti-CD3 antibody produces a synergistic anti-tumor effect that exceeds the combined effects of the two agents when administered alone.

[0048] In certain embodiments, the combination of administered antibodies is safe and well- tolerated by a patient wherein there is no increase in an adverse side effect (e.g., increased cytokine release ("cytokine storm") or increased T-cell activation) as compared to a patient administered with the bispecific antibody as monotherapy.

[0049] In certain cases, the response of a subject to therapy is categorized as a complete response (CR), a partial response (PR), progressive disease (PD), or as stable disease (SD). A CR is defined as disappearance of all target lesions, and a reduction in short axis of any pathological lymph nodes (whether target or non-target) to <10 mm (<1 cm). A PR is defined as an at least 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters. PD is defined as an at least 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm). (Note: the appearance of one or more new lesions is also considered a progression). SD is defined as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.

Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof

[0050] According to certain exemplary embodiments of the present invention, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody or antigenbinding fragment thereof. The term "antibody," as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V 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 sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0051] The term "antibody," as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0052] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as 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 shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0053] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V_H domain associated with a V domain, the V_H and V_L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V_H-V_H, V_H-V or V -V dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or L domain.

[0054] In certain embodiments, an antigen-binding fragment of an antibody may contain 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 present invention include: (i) V_H-C_H1 ; (ii) V_H-C_H2; (iii) V_H-C_H3; (iv) V_H-CH1-C_H2; (V) _H-C_H1-CH2-C_H3; (vi) V_H-C_H2-C_H3; (vii) V_H-C_L; (viii) V_L-C_H1; (ix) V_L-C_H2; (x) V_L- C_H3; (xi) V_L-C_H1-CH2; (xii) _L-CH1-CH2-C_H3; (xiii) V_L-C_H2-C_H3; and (xiv) V -C_L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V_H or V_L domain (e.g., by disulfide bond(s)).

[0055] The term "antibody," as used herein, also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format may be adapted for use in the context of an antibody or antigenbinding fragment of an antibody of the present invention using routine techniques available in the art. For example, the present invention includes methods comprising the use of bispecific antibodies wherein one arm of an immunoglobulin is specific for PD-1 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG- scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab.sup.2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which 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]).

[0056] The antibodies used in the methods of the present invention may be human antibodies. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0057] The antibodies used in the methods of the present invention may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_H and V sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0058] According to certain embodiments, the antibodies used in the methods of the present invention specifically bind PD-1. The term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic 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, an antibody that "specifically binds" PD- 1 , as used in the context of the present invention, includes antibodies that bind PD-1 or portion thereof with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay. 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.

[0059] According to certain exemplary embodiments of the present invention, the anti-PD-1 antibody, or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising any of the amino acid sequences of the anti-PD-1 antibodies as set forth in US Patent Publication No. 20150203579. In certain exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof that can be used in the context of the methods of the present invention comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and the light chain complementarity determining regions (LCDRs) of a 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), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 35; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 36; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 37; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 38; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 39; and the 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 an HCVR comprising SEQ ID NO: 33 and an LCVR comprising SEQ ID NO: 34. In certain embodiments, the methods of the present invention comprise the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, 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 present invention comprise the use of REGN2810, or a bioequivalent thereof. The term "bioequivalent", as used herein, refers to anti- PD-1 antibodies or PD-1 -binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives whose rate and/or extent of absorption do not show a significant difference with that of REGN2810 when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose. In the context of the invention, the term refers to antigen-binding proteins that bind to PD-1 which do not have clinically meaningful differences with REGN2810 in their safety, purity and/or potency.

[0060] Other anti-PD-1 antibodies that can be used in the context of the methods of the present invention include, e.g., the antibodies referred to and known in the art as 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 as set forth in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0061] The anti-PD-1 antibodies used in the context of the methods of the present invention may have pH-dependent binding characteristics. For example, an anti-PD-1 antibody for use in the methods of the present invention may exhibit reduced binding to PD-1 at acidic pH as compared to neutral pH. Alternatively, an anti-PD-1 antibody of the invention may exhibit enhanced binding to its antigen at acidic pH as compared to neutral pH. The expression "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 expression "neutral pH" means 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.

[0062] In certain instances, "reduced binding to PD-1 at acidic pH as compared to neutral pH" is expressed in terms of a ratio of the K_D value of the antibody binding to PD-1 at acidic pH to the K_D value of the antibody binding to PD-1 at neutral pH (or vice versa). For example, an antibody or antigen-binding fragment thereof may be regarded as exhibiting "reduced binding to PD-1 at acidic pH as compared to neutral pH" for purposes of the present invention if the antibody or antigen-binding fragment thereof exhibits an acidic/neutral K_D ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic/neutral K_D ratio for an antibody or antigen-binding fragment of the present invention 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 greater.

[0063] Antibodies with pH-dependent binding characteristics may be obtained, e.g., by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigen-binding domain at the amino acid level may yield antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced antigen-binding at acidic pH relative to neutral pH may be obtained. As used herein, the expression "acidic pH" means a pH of 6.0 or less.

Bispecific Anti-MUC16/Anti-CD3 Antibodies

[0064] According to certain exemplary embodiments of the present invention, the methods comprise administering a therapeutically effective amount of a bispecific antibody that specifically binds CD3 and MUC16. Such antibodies may be referred to herein as, e.g., "anti- MUC16/anti-CD3," or "anti-MUC16xCD3" or "MUC16xCD3" bispecific antibodies, or other similar terminology.

[0065] 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 a first antigen (e.g., MUC16), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., CD3). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each comprising three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix "A" and the CDRs of the second antigen-binding domain may be designated with the prefix "B". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1 , A-HCDR2, and A-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1 , B-HCDR2, and B-HCDR3.

[0066] 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 constitution. In the context of the present invention, the multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes lgG1 , lgG2, I gG3, and lgG4, as well as any allotype within each isotype group. [0067] Bispecific antibodies of the present invention typically comprise two multimerizing domains, e g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1/lgG1 , lgG2/lgG2, lgG4/lgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1/lgG2, lgG1/lgG4, lgG2/lgG4, etc. [0068] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats).

[0069] In the context of bispecific antibodies of the present invention, Fc domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a CH2 or a CHS region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in US Patent Publication No. 20150266966, incorporated herein in its entirety.

[0070] The present invention also includes bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). See, for example, US Patent No. 8,586,713. Further modifications that may be found within the second C_H3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of lgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of lgG4 antibodies.

[0071] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a C_H2 sequence derived from a human I gG 1 , human lgG2 or human lgG4 CH2 region, and part or all of a CH3 sequence derived from a human lgG1 , human lgG2 or human lgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence, derived from a human lgG1 , a human lgG2 or a human lgG4 hinge region, combined with a "lower hinge" sequence, derived from a human I gG 1 , a human lgG2 or a human lgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG4 Cn1]-[lgG4 upper hinge]-[lgG2 lower hinge]-[lgG4 CH2]-[lgG4 CH3], Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG1 Cn1]-[lgG1 upper hinge]-[lgG2 lower hinge]-[lgG4 CH2]-[lgG1 CH3], These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in US Patent Publication No. 20140243504, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function.

[0072] According to certain exemplary embodiments of the present invention, the bispecific anti- MUC16/anti-CD3 antibody, or antigen-binding fragment thereof comprises heavy chain variable regions (A-HCVR and B-HCVR), light chain variable regions (A-LCVR and B-LCVR), and/or complementarity determining regions (CDRs) comprising any of the amino acid sequences of the bispecific anti-MUC16/anti-CD3 antibodies as set forth in US Patent Publication No. 20180112001. In certain exemplary embodiments, the bispecific anti-MUC16/anti-CD3 antibody or antigen-binding fragment thereof that can be used in the context of the methods of the present invention comprises: (a) a first antigen-binding arm comprising the 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 the 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 the heavy chain CDRs (B-HCDR1, B-HCDR2 and B- HCDR3) of a HCVR (B-HCVR) comprising an 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 the 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, the A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 8; the A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 9; the A- HCDR3 comprises the amino acid sequence of SEQ ID NO: 10; the A-LCDR1 comprises the amino acid sequence of SEQ ID NO: 11 ; the A-LCDR2 comprises the amino acid sequence of SEQ ID NO: 12; the A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; the 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; the 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 the 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 the B-LCDR1 comprises the amino acid sequence of SEQ I D NO: 11 ; the B-LCDR2 comprises the amino acid sequence of SEQ I D NO: 12; the 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 (A-HCVR) comprising SEQ ID NO: 1 and a LCVR (A-LCVR) comprising SEQ ID NO: 2; and (b) a second antigen-binding arm comprising a HCVR (B-HCVR) comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and a LCVR (B-LCVR) comprising SEQ ID NO: 2. 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. [0073] In certain embodiments, the anti-tumor activity of the bispecific anti-CD3xMUC16 antibodies of the present invention is not substantially impeded by the presence of high levels (e.g., up to 10,000 U/ml) of circulating CA125. Serum levels of CA125 are increased in the serum of the majority of ovarian cancer patients (median published levels are about 656 U/ml). As demonstrated in Example 2, below, high levels of CA125 in serum or ascites will not significantly interfere with the anti-tumor profile of the bispecific antibodies of the present invention.

[0074] Other bispecific anti-MUC16/anti-CD3 antibodies that can be used in the context of the methods of the present invention include, e.g., any of the antibodies as set forth in US Patent Publication No. 20180112001.

Combination Therapies

[0075] The methods of the present invention, according to certain embodiments, comprise administering to the subject an anti-MUC16/anti-CD3 bispecific antibody in combination with an anti-PD-1 antibody. In certain embodiments, the methods of the present invention comprise administering the antibodies for additive or synergistic activity to treat cancer, preferably an epithelioid sarcoma. As used herein, the expression "in combination with" means that the anti- MUC16/anti-CD3 bispecific antibody is administered before, after, or concurrent with the anti- PD-1 antibody. The term "in combination with" also includes sequential or concomitant administration of anti-PD-1 antibody and a bispecific anti-MUC16/anti-CD3 antibody. For example, when administered "before" 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 prior to the 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 the administration of the bispecific anti-MUC16/anti-CD3 antibody. Administration "concurrent" 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 (before, after, or at the same time) of administration of the bispecific anti-MUC16/anti-CD3 antibody, or administered to the subject as a single combined dosage formulation comprising both the anti-PD-1 antibody and the bispecific anti- MUC16/anti-CD3 antibody.

[0076] In certain embodiments, the methods of the present invention comprise administration of a third therapeutic agent wherein the third therapeutic agent is an anti-cancer drug. In certain embodiments, the methods of the invention comprise administering an anti-PD-1 antibody and an anti-MUC16/anti-CD3 bispecific antibody in combination with radiation therapy to generate long-term durable anti-tumor responses and/or enhance survival of patients with cancer.

[0077] In some embodiments, the methods of the invention comprise administering radiation therapy prior to, concomitantly or after administering an anti-PD-1 antibody and a bispecific anti- MUC16/anti-CD3 antibody to a cancer patient. For example, radiation therapy may be administered in one or more doses to tumor lesions after administration of one or more doses of the antibodies. In some embodiments, radiation therapy may be administered locally to a tumor lesion to enhance the local immunogenicity of a patient's tumor (adjuvinating radiation) and/or to kill tumor cells (ablative radiation) after systemic administration of an anti-PD-1 antibody and/or a bispecific anti-MUC16/anti-CD3 antibody.

Pharmaceutical Compositions and Administration

[0078] The present invention includes methods which comprise administering a bispecific anti- MUC16/anti-CD3 antibody alone, or in combination with an anti-PD-1 antibody to a subject wherein the antibody or antibodies are contained within separate or a combined (single) pharmaceutical composition. The pharmaceutical compositions of the invention may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous 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 Sei Technol 52:238-311.

[0079] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., 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 may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.

[0080] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a 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 that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains 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 comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0081] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

[0082] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may 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, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., 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.

[0083] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0084] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

Administration Regimens

[0085] The present invention includes methods comprising administering to a subject a bispecific anti-MUC16 x CD3 antibody and/or an anti-PD-1 antibody at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.

[0086] According to certain embodiments of the present invention, multiple doses of a 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 methods according to this aspect of the invention comprise sequentially administering to a subject one or more doses of a 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 a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an antibody, followed by one or more secondary doses of the antibody, and optionally followed by one or more tertiary doses of the antibody.

[0087] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the antibody (anti-PD-1 antibody or bispecific antibody). In certain embodiments, however, the amount contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses"). For example, an anti-PD-1 antibody may be administered to a patient with an epithelioid sarcoma at a loading dose of about 1-3 mg/kg followed by one or more maintenance doses of about 0.1 to about 20 mg/kg of the patient's body weight.

[0088] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1/2 to 14 (e.g., 1/2, 1 , 11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11 , 111/2, 12, 121/2, 13, 131/2, 14, 141/2, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of a bispecific anti-MUC16/anti-CD3 (and/or anti-PD-1 antibody) which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0089] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of bispecific anti-MUC16/anti-CD3 antibody (and/or an anti-PD-1 antibody). 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. Likewise, 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. [0090] In embodiments involving 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 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving 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 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

[0091] In certain embodiments, one or more doses of a bispecific anti-MUC16/anti-CD3 antibody (e.g., and an anti-PD-1 antibody) are administered at the beginning of a treatment regimen as "induction doses" on a more frequent basis (twice a week, once a week or once in 2 weeks) followed by subsequent doses ("consolidation doses" or "maintenance doses") that are administered on a less frequent basis (e.g., once in 4-12 weeks).

[0092] 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 to treat an epithelioid sarcoma. In some embodiments, the present 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 present 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 about 0.1 mg/kg to about 20 mg/kg of an anti-PD-1 antibody may be administered followed by one or more doses of about 0.1 mg/kg to about 20 mg/kg of the bispecific antibody to inhibit tumor growth and/or to prevent tumor recurrence in a subject with an epithelioid sarcoma. In some embodiments, the anti-PD-1 antibody is administered at one or more doses followed by one or more doses of the bispecific antibody resulting in increased anti-tumor efficacy (e.g., greater inhibition of tumor growth, increased prevention of tumor recurrence as compared to an untreated subject or a subject administered with either antibody as monotherapy). Alternative embodiments of the invention pertain to concomitant administration of anti-PD-1 antibody and the bispecific antibody which is administered at a separate dosage at a similar or different frequency relative to the anti-PD-1 antibody. In some embodiments, the bispecific antibody is administered before, after or concurrently with the anti-PD-1 antibody. In certain embodiments, the bispecific antibody is administered as a single dosage formulation with the anti-PD-1 antibody.

Dosage

[0093] 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 present invention is, generally, a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of antibody (anti-PD-1 antibody or bispecific anti- MUC16/anti-CD3 antibody) that results in one or more of: (a) a reduction in the severity or duration of a symptom of a cancer (e.g., epithelioid sarcoma); (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and development; (d) inhibit or retard or stop tumor metastasis; (e) prevention of recurrence of tumor growth; (f) increase in survival of a subject with cancer (e.g., epithelioid sarcoma); and/or (g) a reduction in the use or need for conventional anti-cancer therapy (e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents) as compared to an untreated subject or a subject administered with either antibody as monotherapy.

[0094] 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 the bispecific anti-MUC16/anti-CD3 antibody.

[0095] In the case of an anti-PD-1 antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, 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, of the anti-PD-1 antibody. In certain embodiments, 350 mg of an anti-PD-1 antibody is administered.

[0096] The amount of bispecific anti-MUC16/anti-CD3 antibody and optionally anti-PD-1 antibody contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of subject body weight (/.e., mg/kg). In certain embodiments, the bispecific anti-MUC16/anti-CD3 antibody, and optionally the anti-PD-1 antibody, used in the methods of the present invention may be administered to a subject at a dose of about 0.0001 to about 100 mg/kg of subject 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 a patient's body weight, and the optional anti-PD-1 antibody may be administered at dose of about 0.1 mg/kg to about 20 mg/kg of a patient's body weight.

[0097] A summary of the sequences and the corresponding SEQ ID NOs referenced herein is shown in Table 1 , below.

Table 1 : Summary of Sequences

EXAMPLES

[0098] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 : Generation of Bispecific Antibodies that Bind Ovarian Cell-Specific (MUC16) and CD3

[0099] The present invention 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 that express 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 a tumor cell and CD3 on a T-cell facilitates directed killing (cell lysis) of the targeted tumor cell by the activated T-cell.

[0100] Bispecific antibodies comprising an anti-MUC16-specific binding domain and an anti- CD3-specific binding domain were constructed using standard methodologies, wherein the anti- MUC16 antigen binding domain and the anti-CD3 antigen binding domain each comprise different, distinct HCVRs paired with a common LCVR. In exemplified bispecific antibodies, the molecules were constructed utilizing a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody and a common light chain from the anti-MUC16 antibody. In other instances, the 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 pair effectively with a variety of heavy chain arms.

[0101] Exemplified bispecific antibodies were manufactured having an I gG 1 Fc domain (BSMUC16/CD3-001 , -002, -003, and -004) or a modified (chimeric) lgG4 Fc domain (BSMUC16/CD3-005) as set forth in US Patent Application Publication No. US20140243504A1 , published on August 28, 2014.

[0102] A summary of the component parts of the antigen-binding domains of the various anti- MUC16xCD3 bispecific antibodies constructed is set forth in Table 2.

Table 2: Summary of Component Parts of Anti-MUC16xCD3 Bispecific Antibodies

Example 2: CA-125 Does Not Interfere with Anti-MUC16xCD3 Antibody Activity In Vitro [0103] The impact of soluble CA-125 (the 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 the majority of ovarian cancer patients and circulating levels could impact any MUC16-targeted therapy by acting as an antigen sink. The levels of CA-125 used in the assay (10,000 ll/rnl) greatly exceed the median published levels 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 (MUC16A) was carried out at an Effector/Target ratio of 4:1 with a fixed concentration of BSMUC16/CD3-001 or CD3-binding control antibody (100pM), and a serial dilution of either MUC16-1 H or MUC16A for 72 hours at 37°C. In order to monitor the specific killing of MUC16-bearing target cells, OVCAR-3 cells were labeled with 1uM of Violet Cell Tracker. After labeling, cells were plated overnight at 37°C. Separately, human PBMCs were plated in supplemented RPMI media at 1x10⁶ cells/mL and incubated overnight at 37°C in order to enrich for lymphocytes by depleting adherent cells. The next day, target cells were coincubated with adherent cell-depleted naive PBMC (Effector/Target cell ratio 4:1) and a serial dilution of either BSMUC16/CD3-001 or the CD3-binding control for 72 hours at 37°C. Cells were removed from cell culture plates using trypsin, and analyzed by FACS. For FACS analysis, cells were stained with a dead/live far red cell tracker (Invitrogen). For the assessment of specificity of killing, cells were gated on Violet cell tracker labeled populations. Percent of live target cells was reported for the calculation of adjusted survival as follows: Adjusted survival=(R1/R2)*100, where R1= % live target cells in the presence of antibody, and R2= % live target cells in the absence of test antibody. T cell activation was assessed by incubating cells with directly conjugated antibodies to CD2, CD69, and CD25, and by reporting the percent of activated (CD69+) T cells or (CD25+) T cells out of total T cells (CD2+).

[0104] The binding of BSMUC16/CD3-001 and an antibody known to bind CA-125 (clone 3A5) to CA125 obtained from human ascites fluid was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, soluble CA-125 (creative Biomart, NY, USA) at a concentration of 4000 units/mL in PBS was passively adsorbed to a 96-well microtiter plates overnight at 4°C. The plates were then washed with PBST and blocked with 0.5% BSA in PBS for 1 hour. Biotinylated BSMUC16/CD3-001 , the MUC16 parental antibody, a-MUC16 3A5 and non-binding controls (BSMUC16/CD3-001 isotype control and a-MUC16 3A5 isotype control), were added to plate at concentrations of 10, 1 , 0.3, or 0.1 nM in 0.5% BSA in PBS for 1 hour, followed by a wash with PBST. Streptavidin conjugated with horseradish peroxidase (SA-HRP) (ThermoFisher Scientific, Waltham, MA, USA) at 1 :10000 dilution of 1.0 mg/mL stock solution was added to the wells and incubated for 1 hour to detect plate-bound biotinylated antibodies. The plate was washed and developed with 3-3’, 5-5’-tetramethylbenzidine (BD Biosciences, Franklin Lakes, NJ, USA) substrate according the manufacturer's instructions. Absorbance at 450 nm was recorded for each well on a Victor Multilabel Plate Reader (Perkin Elmer; Melville, NY). Data were analyzed with GraphPad Prism software.

[0105] Excess CA-125 had minimal impact on BSMUC16/CD3-0001 binding to OVCAR-3 cells suggesting minimal binding to CA-125 (Figure 1). In contrast, CA-125 greatly inhibited the ability of a comparator antibody that likely binds to the repeat region of MUC16 (in-house version of antibody clone 3A) (Figure 1). Further, a soluble MUC16 construct containing the membrane-proximal region up to the 5^th SEA domain of MUC16 (MUC16A) dramatically inhibited binding of BSMUC16/CD3-001 , showing that BSMUC16/CD3-001 binds a membrane proximal region, as discussed in greater detail in WO 2018/067331 , which is herein incorporated by reference. In alignment 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 a high concentration of MUC16A (data not shown). Thus, BSMUC16/CD3-001 can bind to MUC16 and induce T cell redirected killing even in the presence of high concentrations of CA-125.

Example 3: PD-1 Blockade Enhances Anti-Tumor Activity of Anti-MUC16xCD3 Bispecific Antibodies in Xenogenic and Syngeneic Tumor Models [0106] The in vivo efficacy of an anti-MUC16/anti-CD3 bispecific antibody in combination with PD-1 blockade was evaluated in xenogenic and syngeneic tumor models.

A. Xenogenic Model - OVCAR-3/Luc

[0107] For the xenogenic model, immunodeficient NSG mice were injected intraperitoneally (IP) with OVCAR-3/Luc cells previously passaged in vivo (Day 0) thirteen days after engraftment with human PBMCs. Mice were treated IP with 12.5ug/mouse BSMUC16/CD3-001 , or administered 12.5ug CD3-binding control alone or in combination with 100ug REGN2810 on Days 5 and 8. Tumor burden was assessed by BLI on Days 4, 8, 12, 15, 20 and 25 post tumor implantation. As determined by BLI measurements on Day 25, treatment with 12.5ug of BSMUC16/CD3-001 resulted in significant anti-tumor efficacy as determined by BLI measurements and combination with REGN2810 (anti-PD-1) further enhanced the anti-tumor efficacy. All groups had similar tumor burden as assessed by BLI before dosing started. There was no significant difference in tumor burden between groups.

[0108] BSMUC16/CD3-001 significantly reduces tumor burden at 12.5ug and addition of anti- PD-1 enhances the anti-tumor efficacy over that of BSMUC16/CD3-001 alone. NSG mice engrafted with human T cells were implanted with human OVCAR-3/Luc cells. Mice were treated on Days 5 and 8 with 12.5ug BSMUC16/CD3-001 administered IV or treated with a CD3-binding control or non-binding control (12.5ug IV). Data shown in Table 3, below, is tumor burden as assessed by BLI on Day 25 post tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. Treatment with BSMUC16/CD3-001 +/- REGN2810 was compared to the 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 combination with REGN2810 (# p < 0.05).

Table 3: Bioluminescence on Day 25 post tumor implantation

B. Syngeneic Model - ID8-VEGF/huMUC16

[0109] To examine efficacy in an immune-competent model, the murine CD3 gene was replaced with human CD3 and a portion of the mouse MLIC16 gene was replaced with the human sequence. The replacements resulted in a mouse whose T cells express human CD3 and that expresses a chimeric MUC16 molecule containing a portion of human MUC16 where the BSMUC16/CD3-001 and BSMUC16/CD3-005 bispecific antibodies bind.

[0110] For this first syngeneic tumor model, the ID8-VEGF cell line engineered to express the portion of human MUC16 was used. Mice were implanted with the ID8-VEGF/huMUC16 cells IP and treated with 5mg/kg of BSMUC16/CD3-001 or CD3-binding control with isotype control or in combination with anti-PD-1 (5mg/kg IV) three days after implantation. Treatment with BSMUC16/CD3-001 extended the median survival compared to the group that received the CD3-binding control but the addition of anti-PD-1 blockade also resulted in survival of 50% of the mice.

[0111] BSMUC16/CD3-001 significantly increases median survival time in an ID8-VEGF ascites model and addition of PD-1 (REGN2810) blockade allows survival of several mice. Mice expressing human CD3 in place of mouse CD3 and a chimeric MUC16 molecule were implanted with the murine ovarian tumor line expressing a portion of human MUC16. Mice were administered BSMUC16/CD3-001 (5mg/kg IV) or administered CD3-binding control (5mg/kg IV) with isotype control or with anti-PD-1 on day 3 post implantation. Mice were treated on Days 3, 7, 10, 14, 17 post tumor implantation. Data shown is median survival. Mice were sacrificed when they had a with weight-gain of more than 20% due to ascites-induced abdominal distension. Statistical significance was determined using the Mantel-Cox method. Both BSMUC16/CD3-001 and BSMUC16/CD3-001 + anti-PD-1 treatment resulted in an increase in median survival time and the combination of BSMUC16/CD3-001 + anti-PD-1 resulted in 50% survival, demonstrating a synergistic effect between the MUC16xCD3 bispecific antibody and the anti-PD-1 antibody. Results are shown in Table 4, below.

Table 4: Median Survival in the ID8-VEGF/huMUC16 model

[0112] Similar results were observed when BSMUC16/CD3-001 was administered at 1 mg/kg in combination with the anti-PD-1 antibody.

C. Syngeneic Model - MC38/huMUC16 [0113] As discussed above, the mice used in this experiment were engineered so that the murine CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with the human sequence. The replacements resulted in a mouse whose T cells express human CD3 and that expresses a chimeric MUC16 molecule containing a portion of human MUC16 where the BSMUC16/CD3-001 and BSMUC16/CD3-005 bispecific antibody binds.

[0114] For this second syngeneic tumor model, the MC38 line engineered to express the portion of human MUC16 was used. Mice were implanted with MC38/huMUC16 cells SC and treated with BSMUC16/CD3-005 or CD3-binding control with isotype control (1 mg/kg IV) or in combination with anti-PD-1 (5mg/kg IV) on Day 7 post tumor implantation. The anti-PD-1 antibody used in this experiment was a commercially available murine antibody (clone RMP1- 14, BioXCell). The combination of BSMUC16/CD3-005 and anti-PD-1 showed a synergistic anti-tumor effect.

[0115] The combination of BSMUC16/CD3-005 and anti-PD-1 blockade resulted in better anti-tumor efficacy than BSMUC16/CD3-005 alone in a MC38 SC model. Mice expressing human CD3 in place of mouse CD3 and a chimeric MUC16 molecule were implanted with the murine tumor line MC38 expressing a portion of human MUC16. Mice were administered BSMUC16/CD3-005 or administered CD3-binding control (1mg/kg IV) with isotype control or with anti-PD-1 antibody (5mg/kg IV) on day 7 post implantation. Mice were treated on Days 7, 11 and 14 post tumor implantation. The results are illustrated in Figure 2. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparison test. BSMUC16/CD3- 005 plus anti-PD-1 significantly, and synergistically, inhibited tumor growth over the CD3- binding control.

Example 4: Immuno-PET Imaging in Engineered Mice Showed Localization of the Anti- MUC16xCD3 Bispecific Antibody to T Cell-Rich Organs

[0116] The in vivo localization of BSMUC16/CD3-001 and BSMUC16/CD3-005 and the expression of MUC16 protein were assessed in wild type and genetically humanized mice using PET imaging. The biodistribution of the ⁸⁹Zr-labelled anti-MUC16 antibody (bivalent anti-MUC16 antibody generated using the same anti-MUC16 heavy and light chain as the bispecifics, herein referred to as “parental”) was similar in both wild type and humanized mice, suggesting low expression/availability of the humanized MUC16 protein to the antibody. In contrast, when mice were administered therapeutically relevant doses of a ⁸⁹Zr-labelled BSMUC16/CD3-001 bispecific antibody, 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 analyses in individual tissues confirmed localization to lymph nodes and spleen (data not shown). Uptake of ⁸⁹Zr-labelled BSMUC16/CD3-005 bispecific antibody in lymphoid tissues was greatly reduced relative to BSMUC16/CD3-001 due to its lower affinity for CD3. To assess whether BSMUC16/CD3-001 and BSMUC16/CD3-005 can accumulate in MUC16-expressing tumors, ⁸⁹Zr-labelled BSMUC16/CD3-001 and ⁸⁹Zr-labelled BSMUC16/CD3-005 were administered to mice bearing ID8-VEGF-huMUC16A tumors. Tumor uptake between the bispecific antibodies was not significantly different despite the higher lymphoid uptake of BSMUC16/CD3-001 (data not shown).

[0117] Preparation of immunoconjuqate and small animal PET: BSMUC16/CD3-001 and control antibody were conjugated with DFO to glutamine residues at position 295 via transamidation by microbial transglutaminase following deglycosylation of the antibodies with PNGase F. DFO conjugated antibodies were then chelated with Zirconium-89 (⁸⁹Zr). Mice received antibody at a final dose of 0.5mg/kg via tail vein injection. PET imaging was then performed to assess in vivo localization of the radioimmunoconjugate at day 6 post dosing, prior to ex vivo biodistribution studies. For experiments in tumor-bearing mice, mice were implanted subcutaneously with 10x10® ID8-VEGF-huMUC16A tumor cells. Tumor bearing mice were dosed with ⁸⁹Zr radiolabeled antibodies 20 day post implantation when tumors averaged 150mm³.

[0118] A pre-calibrated Sofie Biosciences G8 PET/CT instrument (Sofie Biosciences (Culver city, CA) and Perkin Elmer) was used to acquire PET and CT images. The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm at the center of the field of view. On day 6 post dosing, mice underwent induction anesthesia using isoflurane and were kept under continuous flow of isoflurane during a 10-minute static PET acquisition. CT images were acquired following PET acquisition. The PET image was subsequently reconstructed using pre-configured settings. Decay-corrected PET data and CT data were processed using VivoQuant software (inviCRO Imaging Services) into false-colored co-registered PET-CT maximum intensity projections on a color scale calibrated to indicate a signal range of 0 to 30% of injected dose per volume, expressed as %ID/g. For ex vivo biodistribution analysis, mice were euthanized following imaging on day 6 post dosing. Blood was collected via cardiac puncture into counting tubes. Normal tissues (inguinal and axillary lymph nodes, thymus, spleen, heart, lungs, stomach, small intestine, liver, kidneys, bone and ovary) were then excised and placed into counting tubes. Tumors were similarly collected into counting tubes. All tubes had been pre-weighed and were subsequently re-weighed to determine the weight of the blood and tissues. The y-emission radioactivity for all samples were then counted on an automatic gamma counter (Wizard 2470, Perkin Elmer) and results reported in in counts per minute (cpm). The %ID for each sample was the determined using samples counts relative to dose-standards counts prepared from the original injected material. Subsequently, the individual %l D/g values were derived by dividing the %ID value by the respective weight of the appropriate blood, tissues or tumor sample.

[0119] ⁸⁹Zr-labeled BSMUC16/CD3-001 and ⁸⁹Zr-labeled BSMUC16/CD3-005 demonstrated specific localization to MUC16+ tumors and CD3+ lymphoid tissues, with lymphoid distribution correlating to relative CD3 affinity. Both MUC16xCD3 bispecifics demonstrated equivalent tumor localization in the presence of CD3+ tissues.

Example 5: Toxicology Studies in Cynomolgus Monkeys Showed No Overt Toxicity for the Anti-MUC16xCD3 Bispecific Antibody

[0120] BSMUC16/CD3-001 cross-reacts with monkey MUC16 and CD3. To determine the safety and tolerability, and characterize the pharmacokinetics of the bispecific antibody, a multidose toxicity study was conducted in cynomolgus monkeys. Six monkeys/sex/group received weekly administration of BSMUC16/CD3-001 for a total of five doses at 0.01, 0.1 or 1 mg/kg. At the completion of the dosing period, 3 animals/sex/group were euthanized and tissues examined for microscopic finding, while the remaining three animals/sex/group underwent 12 weeks of treatment-free recovery to assess the reversibility or persistence of any BSMUC16/CD3-001-related effects. BSMUC16/CD3-001 was well tolerated, and all animals survived to the time of scheduled necropsy. Toxicokinetic analysis demonstrated doseproportional exposures and linear kinetics across the dose groups, with no gender differences observed (data not shown). Continuous exposure to BSMUC16/CD3-001 was observed throughout the dosing phase, and BSMUC16/CD3-001 exposure was maintained until the end of the recovery phase in all (n=6) and 50% of animals in the 0.1 and 1 .0 mg/kg groups, respectively. BSMUC16/CD3-001 was not detected in the serum in any animal in the 0.01 mg/kg group after recovery week 8. The elimination half-life of BSMUC16/CD3-001 was approximately 10 days.

[0121] There were no BSMUC16/CD3-001-related clinical observations, nor any changes in urinalysis parameters, peripheral blood immunophenotyping, food consumption, or body weight during the dosing or recovery periods. Importantly, BSMUC16/CD3-001 administration did not result in any changes in respiratory, neurologic, or cardiovascular safety pharmacology evaluations, including no changes in ECG parameters. No BSMUC16/CD3-001-related changes in organ weight were found, nor were any macroscopic changes noted at either terminal or recovery necropsy. Dose-related, reversible elevations of circulating inflammatory markers (C- reactive protein (CRP) and IL-6) were observed within 1 day after the initial dose of either 1.0 or 0.1 mg/kg, but these elevations were not apparent after subsequent doses (data not shown). In accordance with the minimal increase of serum cytokines, T cell redistribution was not detected after BSMUC16/CD3-001 administration (data not shown), in contrast to what has been described for several CD3 bispecific molecules against hematological tumors.

[0122] The cynomolgus monkey study was conducted in accordance to guidelines of the IACUC. Cynomolgus monkeys (6 animals/sex/group) were administered control article (diluted placebo) or BSMUC16/CD3-001 (0.01 , 0.1 , or 1 mg/kg) once weekly via a 30-minute IV infusion. The control article was 10mM histidine with 10% sucrose and 0.05% polysorbate 20, pH 6, diluted with 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 concentration was 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 quantitated using flow cytometry. Briefly, blood was collected in potassium EDTA tubes, lysed, stained for CD3, CD4 and CD8 (BD Biosciences) and relative values for each phenotype are determined using a FACS Canto II. These values are then multiplied by the absolute lymphocyte values (via hematology analysis) to enumerate absolute cell counts for each phenotype.

[0123] Immunohistochemical staining for MUC16 was present in expected tissues: pancreas (mesothelium, ductal epithelium), heart and ovary (data not shown) as well as salivary gland (goblet cells), liver (mesothelium, bile duct), lung (mesothelium, bronchiolar/bronchial epithelium), small intestine (mesothelium), testis (mesothelium, rete testis/efferent duct) and tonsil (epithelium, mucous glands) (not shown). BSMUC16/CD3-001 -related microscopic changes, evaluated by hematoxylin and eosin (H&E) histologic staining, included inflammation (infiltration of white blood cells) and increased mesothelial cell size and cellularity leading to non-adverse thickening of the serosal lining and/or submesothelial connective tissue of multiple thoracic and peritoneal organs. These changes were generally focal or multi-focal in nature and were minimal to slight in severity and were considered to be on-target for BSMUC16/CD3-001 , resulting from engagement of MUC16 expressed on serosal epithelial (mesothelial) cells and activation of T cells. Importantly, the serosal changes were reversed or trended towards reversal at the end of the recovery period (data not shown). [0124] Toxicology 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.

Example 6: Assessment of Serum Cytokine Induction in Tumor-Bearing Mice

[0125] Because cytokine release syndrome (CRS) is a frequent serious side effect of CD3 bispecific and CAR T cell therapies, a study to monitor serum cytokines in relevant models following treatment with BSMUC16/CD3-001 was conducted. In genetically humanized MUC16/CD3 mice without tumors, no serum cytokine response was evident upon BSMUC16/CD3-001 administration.

[0126] To assess in vivo T cell activation by BSMUC16/CD3-001 , serum cytokine levels from tumor-bearing mice were measured. Serum samples were collected 4 hours after the first antibody dose in the 0.5 mg/kg BSMUC16/CD3-001 , CD3-binding control, and non-binding control groups. Treatment with BSMUC16/CD3-001 activated T cells as determined by induction of I FNy, TNFa, IL-2, IL-6, IL-8, and IL-10, compared to the non-binding control and the CD3- binding control (data not shown). BSMUC16/CD3-001-induced cytokine response required the presence of both T cells as well as OVCAR-3/Luc cells, as mice bearing only OVCAR3/Luc cells did not have detectable human I FNy in the serum, and mice without tumor cells to provide MUC16 for cross-linking did not show an increase in serum IFNy in response to BSMUC16/CD3-001 (data not shown).

[0127] Measurement of serum cytokine levels: T cell activation in response to treatment with BSMUC16/CD3-001 was assessed by measuring the serum concentrations of interferon y (IFNy), tumor necrosis factor a (TNFa), interleukin-2 (IL-2), IL-4, IL-6, IL-8, IL-10, IL-12p70, IL- 13, and IL-1 B four hours after the first 0.5 mg/kg dose. Cytokine levels were analyzed using V- plex Human Prolnflammatory-10 Plex kit following the manufacturer’s instructions (Meso Scale Diagnostics, Rockville, MA). Cytokines were measured in two separate studies with 4-6 mice per group.

Example 7: MUC16 Expression in Humanized Mice and Effect of Anti-MUC16xCD3 Bispecific Antibodies on MUC16-Positive Tissues

[0128] To investigate the antitumor efficacy of BSMUC16/CD3-001 in a mouse with a fully intact immune system, mice were genetically engineered to express human CD3 on T cells and a region of MUC16 covering the antibody binding region, both in the endogenous murine loci (knock-in mice). To validate these mice, MUC16 expression was examined by both RT-PCR and IHC. RNA expression was detected in the trachea as well as low levels in the lung, heart, ovary, pancreas and bladder (data not shown), similar to published data on murine MLIC16 expression. To assess MUC16 protein expression, IHC was performed on selected tissues using an anti-human MUC16 antibody that recognizes a membrane-proximal region of MUC16. MUC16 protein expression was confirmed in the surface epithelium of the ovary and stomach in these mice. MUC16 was also observed in the tracheal lining/epithelium as well as the submucosal glands, as has been described in humans (data not shown).

[0129] Histology on mouse tissues: Tissues from humanized or WT mice were harvested and stained with an anti-MUC16 antibody binding the membrane proximal domain of MUC16 by IHC using the Ventana Discovery XT (Ventana; Tucson, AZ). 5pm Paraffin sections were cut onto Superfrost PLUS slides and baked for an hour at 60°C. The immunohistochemical staining was performed on the Discovery XT Automated IHC staining system using the Ventana DAB Map detection kit. Deparaffinization was performed using EZ Prep solution at 75°C for 8 minutes. Mild antigen retrieval was performed (95°C, 8 minutes followed by 100°C, 24 minutes) using Tris-EDTA buffer pH 9 (CC1) from Ventana. This was followed by multiple blocking steps. Tissue sections were incubated with the anti-MUC16 antibody (2pg/ml) for 8 hours at RT. An isotype control antibody recognizing an irrelevant non-binding antibody was used as the negative control. Primary antibody and negative control were applied manually. Biotinylated Goat Anti-Human IgG (Jackson ImmunoResearch) was used as the secondary antibody (1 pg/ml) and samples were incubated for an hour at RT. The chromogenic signal was developed using the Ventana DAB MAP Kit. Slides were manually counterstained with Hematoxylin (2 minutes), dehydrated and coverslipped. Images were acquired on the Aperio AT 2 slide scanner (Leica Biosystems; Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs; Corrales, NM). H&E staining were performed by Histoserv, Inc (Germantown, MD, USA).

[0130] The T cells in these mice are polyclonal, as assessed by T cell receptor (TCR) VB usage, express human CD3, and are present in similar numbers to wildtype 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 T cell numbers in blood, serum cytokines, and histopathology were then examined. Although T cells can be activated by an anti-human CD3 antibody (OKT3) as measured by T cell margination from the blood and increased levels of serum cytokines (data not shown), BSMUC16/CD3-001 did not induce any such effects, suggesting limited accessibility of the MUC16 target (data not shown). To determine whether BSMUC16/CD3-001 induced any microscopic changes in MUC16-expressing tissues, MUC16 and CD3 humanized mice received two doses of BSMUC16/CD3-001 at 10 mg/kg on Day 0 and Day 3. On day 5, several MUC16-expressing tissues (trachea, stomach and ovary) were examined, and no cellular infiltration or necrosis was seen in these tissues following BSMUC16/CD3-001 administration (data not shown).

Histopathology examination revealed no inflammation or infiltration into MUC16-expressing tissues in mice after BSMUC16/CD3-001 administration at the time examined.

[0131] The results of this study, as well as the cynomolgus monkey study discussed in Example 5, demonstrate the safety profile of BSMUC16/CD3-001. BSMUC16/CD3-001 induced only minimal serum cytokines and, while there was focal induction of inflammation and thickening of the serosal lining in MUC16-expressing suggesting on-target activity, these effects were resolving by the end of the recovery period and consistent with inflammation and increased cellularity indicative of repair. The observed serosal changes were not correlated with any clinical observations, clinical pathology (except inflammatory response), or microscopic changes to the underlying parenchyma. Thus, studies in both genetically humanized mice and cynomolgus monkey show BSMUC16/CD3-001 was well-tolerated.

Example 8: Monitoring PD-1 Expression in a FACS-Based Cytotoxicity Assay Using Naive Human Effector Cells

[0132] In order to monitor the specific killing of Muc16-bearing target cells by flow cytometry, the ovarian cell line OVCAR-3 was labeled with 1uM of Violet Cell Tracker. After labeling, cells were plated overnight at 37°C. Separately, human PBMCs were plated in supplemented RPMI media at 1x10⁶ cells/mL and incubated overnight at 37°C in order to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, target cells were co-incubated with adherent cell-depleted naive PBMC (Effector/Target cell 4:1) and a serial dilution of either BSMUC16/CD3-001 or the CD3-binding control for 72 hours at 37°C. Cells were removed from cell culture plates using trypsin, and analyzed by FACS. For FACS analysis, cells were stained with a dead/live far red cell tracker (Invitrogen). For the assessment of specificity of killing, cells were gated on Violet cell tracker labeled populations.

[0133] PD-1 expression was assessed by incubating cells with directly conjugated antibodies to CD2, CD4, CD8, and PD-1 by reporting the percent of PD-1/CD4 positive T cells or PD-1/CD8 positive T cells out of 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. Results are shown in Figure 3. Example 9: Methods of Treating Epithelioid Sarcoma with Anti-MUC16 x Anti-CD3 Bispecific Antibodies Alone or in Combination with Anti-PD-1 Antibody

[0134] A single patient with relapsed epithelioid sarcoma who has exhausted all existing therapeutic options, was treated with REGN4018 (anti-MUC16 x anti-CD3 bispecific antibody). [0135] In this study, treatment assessment was based on tumor response. The tumor response assessment was based on the levels of CA-125, a tumor marker in patients with and without measurable disease. Additionally, biomarker analysis includes peripheral T-cell phenotyping as REGN4018 treatment is expected to transiently reduce the population of peripheral CD3 T cells. Further analysis will include biomarkers such as tumor expression of proteins such as MUC16 and PD-L1 and may include ctDNA, tumor (RNA and somatic DNA sequencing) genetic analyses for variations that impact the clinical course of underlying disease or modulate treatment side effects.

[0136] Objectives: Exploratory objectives will be explored.

[0137] The exploratory objectives of the study are:

Collection of blood for cytokine profiling, pharmacokinetics and tumor tissue via biopsy is optional. If available, these samples will be used for the following exploratory objectives: (1) To evaluate biomarkers that may correlate with mechanism of action, increased understanding of disease/target, observed toxicity, and potential anti-tumor activity including, but not limited, to:

- Circulating proteins, including cytokines

- Gene expression changes in tumor

- Tumor expression levels of proteins such as MUC16.

[0138] Patient demographic information:

- Age: 20 years

- Sex: female

[0139] Diagnosis: Patient has multiple recurrent metastatic epithelioid sarcoma with disease in chest wall and lungs. Tumor specimen has been shown to express MUC16 by immunohistochemistry while there was elevated CA-125 (the shed extracellular portion of MUC16) in serum samples.

[0140] History of disease progression to date: The patient was initially diagnosed with epithelioid sarcoma of the right radius. She underwent radiation therapy (50.4Gy) to the right upper limb and local surgery with negative margins. First relapse occurred with a nodal recurrence in the mid-arm treated with surgical excision and further radiotherapy (54Gy). A further recurrence was detected with PET-avid nodal disease in the axilla and was treated with surgery. There was further disease recurrence at the right elbow within the previous radiation field. The patient was treated with tazemetostat on an early phase trial but discontinued due to problems with tolerability and compliance. With evidence of further disease progression in the arm and evidence of pulmonary nodules, the patient was treated with pembrolizumab. She had a prolonged period of disease stability, before coming off stud in the context of progressive disease. The patient was then treated with nivolumab/ipilimumab and underwent a palliative forequarter amputation of her right arm in view of worsening tumor-related ulceration. Disease evaluation showed stable disease in the chest, but in view of immune-related pneumonitis, immune checkpoint inhibitor therapy was discontinued. The patient was retreated with tazemetostat, discontinuing in the context of progressive disease. She was subsequently treated with CLR-131 (molecularly targeted radiotherapy); but had further disease progression. Most recently, the patient received radiotherapy (20Gy in 5 fractions) to the right breast for palliation of ulcerative skin lesions.

[0141] Currently, the patient has evidence of disease in chest wall/breast tissue bilaterally and multiple measurable lesions in both lungs. She is clinically stable, with a Karnofsky score 70 (mainly as a result of her amputation). There are no currently available clinical trial options or alternative ‘standard’ therapies for her disease in this context.

[0142] 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.

[0143] REGN4018 will be administered by IV infusion over 4 hours (including flush) weekly. Treatment will be given continuously (weekly) in cycles lasting 6 weeks (42 days). During cycle 1 , the patient received escalating doses of REGN4018 to reach the full dose as per the table below. Initial doses of the drug were given in split consecutive days (see below). Any dose may be split at any time point to improve tolerability.

Table 5: Dose Scheme for REGN4018 Therapy

[0144] Rationale for dose selection - REGN4018 is an investigational agent currently being evaluated in an ongoing adult phase 1/2 study. The patient for this SPS was treated at the dose level one dose level below the dose (450 mg) currently being evaluated in the monotherapy cohorts in this phase 1/2 study; i.e. 250 mg, which is the highest dose level that has already been demonstrated to be tolerable. The dose may subsequently be adjusted based on new information at the discretion of the physician to the clinical benefit of the patient.

[0145] REGN4018 Administration Procedure - REGN4018 drug product is supplied as lyophilized single-use product at 5 mg/vial and/or 50 mg/vial for administration by IV infusion. REGN4018 will be administered by IV weekly. REGN4018 will be administered by IV infusion over 4 hours (including flush). The patient must be observed in a monitored setting for at least 24 hours for the following doses/circumstances: a) Cycle 1

- Initial dose on cycle 1, day 1

- Transitional dose split on cycle 1 , days 8/9

- First full dose split on cycle 1, days 15/16

- First 250 mg dose on cycle 1, day 22 b) Escalation to a new highest tolerated dose level. c) Any subsequent dose of REGN4018 that occurs after a grade >2 cytokine release syndrome (CRS) AE is observed. d) At restart of study drug after a prolonged drug-free period (e.g. more than 21 days off study drug).

[0146] During the in-hospital observation periods, hospitalization alone will not be sufficient to categorize an AE as serious. Prolongation of hospitalization (beyond the protocol-mandated 24 hours) due to an AE will be considered serious.

[0147] If a dose is split, time of monitoring on the second day should follow the same guidelines for the first day of infusion. For Cycle 1 Day 9, Cycle 1 Day 16, Cycle 1 Day 23 (if appropriate) this should be 24h of monitoring.

[0148] After the patient tolerates the first full dose, the patient may subsequently receive study therapy at an outpatient infusion unit and investigator will have the option of shortening the duration of 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 the in-patient or day hospital setting with immediate access to the acute care hospital unit.

[0149] Pre-medications are not required and are at the discretion of the investigator.

[0150] Dose Escalation - Dose escalation/re-escalation may be implemented at the discretion of the investigator, based on latest information from the ongoing phase 1/2 study. Specifically, if a higher dose is determined to be tolerable in the phase 1/2 study, that higher dose may be introduced into this SPS.

[0151] Dose Modification - Dose modification/reduction of REGN4018 may be implemented at the discretion of the investigator and taking into account the best interests of the patient. Adverse events (AE) will be treated symptomatically.

[0152] Duration of Therapy: In REGN4018 therapy, each cycle will be 6 weeks (42 days) long. The treatment will be continued until unacceptable toxicity, lack of clinical benefit, patient withdrawal of consent, physician discretion (e.g., based on intolerable AEs, quality of life, new treatment options) or lack of availability of the drug supply. In the context of disease progression, if the patient is tolerating treatment without disease progression and, in the opinion of the investigator, the patient is deriving clinical benefit from continuing study treatment; the treatment may be continued at the discretion of the physician. Clinical benefit will be assessed objectively with planned imaging re-evaluations (and compared to baseline) at the end at a regular interval outlined in this protocol, or if clinically indicated.

[0153] After a minimum of 24 weeks of treatment if there is a clinical or radiological response, the patient may elect to switch to a Q2W (every two weeks) dosing schedule of REGN4018 and continue with all relevant study assessments. Upon subsequent disease progression, the patient may resume treatment at a dose level selected by the investigator for the clinical benefit of the patient (including a new, higher dose level). Post-treatment follow up will be within 30 days of the last dose, and again 90 days (±10 days) following the last dose in order to complete end-of-study safety assessments.

[0154] Available Safety Data for REGN4018 Therapy: Adult phase 1/2 study of REGN4018 in patients with ovarian cancer is ongoing and is currently enrolling at 450mg (full dose; level 8a). The previous dose level of 250mg (dose level 7a) is the dose proposed 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:

[0155] Treatment-Emergent Adverse Events - The most commonly 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 hypomagnesaemia (n=5; 17.2% each); constipation, dyspnea, dry eye, eye discharge, decreased appetite, headache, hypotension, infusion-related reaction and dyspepsia, pleuritic pain (n=4 each; 13.8% each); asthenia, flank pain, non-cardiac chest pain, musculoskeletal pain, eye pruritis, ocular hyperaemia, conjunctivitis, sinus tachycardia and chills (n=3 each; 10.3% each). The incidence of TEAEs was highest during the first week of therapy and declined in subsequent weeks.

[0156] T reatment-related TEAEs - Twenty-eight of 29 patients (96.6%) experienced at least one treatment-related TEAE as assessed by investigator during the treatment period. The most commonly reported treatment-related TEAE (all grades in >10% of patients) as assessed by 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 at DL3 and 4 patients at DL4). There were no Grade 4 or 5 treatment-related TEAEs.

[0157] Potential for REGN4018 efficacy in the patient considered in this SPS:

Immunohistochemistry confirms that this patient’s cancer expresses MUC16, the relevant target for REGN4018. Additionally, there is elevated CA-125 (the shed extracellular portion of MUC16) in serum samples. The potential benefit is that the study drug may cause patient’s cancer to stop growing or to shrink for a period of time. It may lessen the symptoms, such as pain, that are caused by the cancer.

[0158] Identified and potential risks: Following are the important identified risks and the potential risks of the therapy based on drug safety profile and patient history:

[0159] Important 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 rashes.

[0160] Infusion-related reactions are common adverse drug reactions (ADRs) observed with monoclonal antibodies. Symptoms are temporally related to drug administration and may range from symptomatic discomfort to fatal events. A number of reported terms are used to identify IRRs and while there is considerable overlapping symptomatology, the aetiology of each is very different. Such terms include, anaphylaxis, anaphylactoid reactions, CRS, and complement activation-related pseudoallergy (CARPA). The key reason for trying to identify the etiology of the symptoms and the actual diagnosis is to ensure identification of reactions that are likely to recur or even worsen with subsequent exposure to the medication (eg, anaphylaxis), as opposed to those that are likely to improve with repeated exposure (anaphylactoid reactions and CRS).

[0161] An IRR is defined as an adverse reaction that occurs during the infusion and the subsequent 2 hours. In the context of this study, signs and symptoms of IRR that occur more than 2 hours after the infusion has completed are identified as CRS.

[0162] Clinically, CRS is a syndrome that includes symptoms of fever, hypotension, tachycardia and hypoxia. Additionally, there may be neurological findings, including delirium, encephalopathy, aphasia, lethargy, agitation, tremor and seizures, that reflect immune effector cell-associated neurotoxicity syndrome (ICANS).

[0163] Risk mitigation measures: IRR and CRS

1) Use of initial and transitional doses to gradually approach a full dose level;

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 treatment and supportive care as per institutional standards

[0164] Assessment of Drug Response: The safety and tolerability of REGN4018 in the patient will be monitored by clinical assessment of AEs and by repeated measurements of clinical evaluation including vital signs (temperature, blood pressure, pulse, oxygen saturation, and respiration), physical examinations (complete and limited), 12-lead electrocardiograms (ECGs), echocardiogram, chest x-ray, and laboratory assessment including standard hematology, blood chemistry, and urinalysis.

[0165] Because cytokine release following initial dosing (first dose and/or subsequent doses) has been observed with bispecific antibodies and similar molecules, specific measures have been implemented for this study. These measures include: 1 mg initial dose (cycle 1 day 1), a transitional dose (cycle 1 day 8) of 20 mg, the option for a split dose, required monitoring on select dose administrations, and use of anti-IL-6 pathway therapy (e.g., tocilizumab) and corticosteroids for management of IRR/CRS.

[0166] Tumor assessments will include all known or suspected disease sites. Tumor assessment will be performed prior to starting cycle 1 (baseline), and then prior to Cycle 2 (± 7 days) and then prior to each even-numbered cycles (± 7 days). Additional tumor assessments may be done upon clinical indication. Tumor assessment should be repeated at the end of treatment, if appropriate, if more than 2 cycles have passed since the last evaluation.

Assessment will be performed as per institutional standards and will be compared to baseline measurements taken within 28 days of beginning study therapy. [0167] A baseline eye examination is required due to expression of MLIC16 on the corneal and conjunctival epithelium.

[0168] Blood samples for measurement of drug concentration and for ADA assessment will be collected.

[0169] A brief neurologic exam is required within 24 hours following study drug administration. [0170] 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 fluid, archived tumor tissue, on-study tumor biopsy tissue, tumor DNA (including circulating tumor DNA), and tumor RNA samples.

[0171] Anti-tumor activity will be assessed by CT or MRI or PET-CT, and monitoring of performance status and serum CA-125 levels.

[0172] Allowed and prohibited concomitant therapies and rationale:

[0173] Prohibited and discouraged medications - While participating in the current study, the patient may not receive any standard or investigational agent for treatment of a tumor other than REGN4018 as monotherapy, with the exception of localised palliative external-beam radiotherapy, which is permitted.

[0174] It is recommended that the patient does not receive systemic corticosteroids such as hydrocortisone, prednisone, prednisolone (Solu-Medrol®) or dexamethasone (Decadron®) at any time throughout the study except in the case of a life-threatening emergency and/or to treat an IRR/CRS that has not or is not expected to respond to anti-IL-6 pathway therapy (e.g., tocilizumab) or an irAE severe enough to require corticosteroids. Inhaled, topical, ophthalmologic, or intranasal steroids are allowed.

[0175] Allowed medications - Focal palliative treatment (e.g. radiotherapy) may be permitted. Medication before infusions are at the discretion of the investigator e.g., anti-histamines, acetaminophen, nonsteroidal anti-inflammatory drugs (e.g., ketorolac or ibuprofen), and/or oral opioids (e.g., hydromorphone). No specific pre-medications are mandated per protocol.

[0176] Physiologic replacement doses of systemic corticosteroids are permitted, even if >10 mg/day prednisone equivalents. A brief course of corticosteroids for prophylaxis (e.g., contrast dye allergy) or for treatment of non-autoimmune conditions (e.g., delayed-type hypersensitivity reaction caused by contact allergen) is permitted.

[0177] Any other medication which is considered necessary for the patient’s welfare, and which is not expected to interfere with the evaluation of the study drug, may be given at the discretion of the investigator. Treatments for bone metastases (bisphosphonates, denosumab) and hormonal therapies are permitted.

[0178] Study Endpoint(s):

[0179] The primary endpoints of the study are dose-limiting toxicities, treatment-emergent adverse events (TEAEs; including immune-related adverse events [irAEs]), serious AEs (SAEs), deaths, laboratory abnormalities (grade 3 or higher per CTCAE), and PK for therapy.

[0180] The secondary endpoint of the study is ORR based on Response Evaluation Criteria in Solid Tumors (RECIST).

[0181] Preliminary Results: The safety and efficacy data from the REGN4018 monotherapy study in the single patient with recurrent epithelioid sarcoma is presented below.

[0182] The patient with recurrent epithelioid sarcoma with elevated cancer antigen (CA)-125 levels was administered REGN4018 intravenously (IV) weekly, at a dose range of 1-250 mg. Step-up dosing for the initial two doses was utilized to mitigate risk of cytokine release syndrome (CRS) via gradual increase of drug exposure. Primary endpoints included safety and PK. Secondary endpoints included efficacy as determined by objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.

[0183] The patient experienced grade 2 cytokine release syndrome on C1 D2 and C1 D9. Other treatment emergent adverse events were Grade 1 cough and Grade 2 intermittent hypoxia, myalgia, constipation, pericardial and pleural effusion.

[0184] Upon treatment patient’s CA-125 levels dropped and target lesions on CT scan showed a partial response by RECIST v1.1 , ongoing for 60 weeks, from a baseline sum of diameters of 89 mm to 60 mm by cycle 4 imaging. Additionally, the patient had skin cancer ulcers on the right breast, which decreased in size. These lesions improved significantly with treatment with REGN4018, leading to almost complete wound healing and significant pain improvement caused by such lesions.

[0185] REGN4018 was tolerable by the patient in this single patient study with signs of clinical activity as determined by ORR based on Response Evaluation Criteria in Solid Tumors (RECIST). Data from this analysis supports treatment of recurrent epithelioid sarcoma with REGN4018 (anti-MUC16 x anti-CD3) bispecific antibody.

[0186] Treatment of epithelioid sarcoma with REGN4018 in combination with cemiplimab:

It is contemplated that this patient with recurrent epithelioid sarcoma could be administered an anti-PD-1 antibody (e.g., cemiplimab) in combination with the REGN4018 bispecific antibody (i.e., anti-MUC16 x anti-CD3 antibody) to treat the tumor. Example 10: Semiquantitative MUC16 Immunohistochemistry Expression Across Different Subtypes of Pediatric Sarcoma

[0187] Semiquantitative MUC16 immunohistochemistry (IHC) expression across different subtypes of pediatric sarcoma was investigated. Seventy-two samples were collected between 2015 and 2021 from patients <25 years old at the Hospital for Sick Children, and included epithelioid sarcoma (n=6), rhabdoid tumor of kidney and soft tissue (n=4), rhabdomyosarcoma (n=23), Ewing’s sarcoma (n=19), and other soft tissue sarcoma (n=19). IHC was performed on 4-pm-thick formalin-fixed, paraffin-embedded tissue sections from a representative block for each case or sample. The assay was conducted on the DAKO Omnis staining platform using anti-CA125 antibody (clone M11 , monoclonal mouse, Ready-to-Use, DAKO, Cat. No. GA701) and the Envision Flex detection kit (DAKO, Cat. No. GV800). Appropriate positive and negative controls were used. The immunoreactivity was evaluated using H-score methodology.

[0188] Among the six epithelioid sarcoma samples selected for MUC16 IHC, three showed diffuse and strong staining reaction (H-score 300), one showed heterogenous staining (H-score 110), and the remaining two were negative (H-score 0). Among the remaining tumor entities, MUC16 expression was present in two of four rhabdoid tumor samples of kidney and soft tissue (H-score 300 and 170, respectively) and absent in the remaining 62 cases (H-score 0).

[0189] MUC16 expression is a frequent feature in epithelioid sarcoma and may be identified in other INI-1 (SMARCBI)-deficient malignancies.

[0190] 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 fall within the scope of the appended claims.

Table 6: Sequences Excluded from ST.26-Formatted Sequence Listing

Claims

What is claimed is:

1. A method of treating a MUC16-expressing epithelioid sarcoma in a subject in need thereof, comprising administering to the subject a bispecific antibody comprising a first antigen-binding domain that specifically binds mucin 16 (MUC16) on a target tumor cell, and a second antigen-binding domain that specifically binds human CD3 on a T cell.

2. The method of claim 1 , wherein the subject has metastatic epithelioid sarcoma.

3. A method of treating a MUC16-expressing sarcoma in a subject in need thereof, comprising administering to the subject a bispecific antibody comprising a first antigenbinding domain that specifically binds mucin 16 (MUC16) on a target tumor cell, and a second antigen-binding domain that specifically binds human CD3 on a T cell.

4. The method of claim 3, wherein the MUC16-expressing sarcoma is epithelioid sarcoma, rhabdoid tumor of kidney and soft tissue, rhabdomysarcoma, Ewing’s sarcoma, or a soft tissue sarcoma.

5. The method of claim 4, wherein the MUC16-expressing sarcoma is epithelioid sarcoma or rhabdoid tumor of kidney and soft tissue.

6. The method of any one of claims 1-5, wherein the MUC16 expressing sarcoma is deficient in expression of functional integrase interactor 1.

7. The method of any one of claims 1-6, wherein the subject has previously been treated with an anti-cancer therapy.

8. The method of any one of claims 1-7, wherein the subject is resistant or inadequately responsive to, or relapsed after, prior therapy.

9. The method of any one of claims 1-8, wherein the first antigen-binding domain 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: 1 ; 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: 2.

10. The method of claim 9, wherein the first antigen-binding domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 10.

11 . The method of claim 9 or 10, wherein the first antigen-binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 , a LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 13.

12. The method of any one of claims 9-11 , wherein the first antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1 , and a LCVR comprising the amino acid sequence of SEQ ID NO: 2.

13. The method of any one of claims 1-12, wherein the second antigen-binding domain 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: 3; 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: 2.

14. The method of claim 13, wherein the second antigen-binding domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

15. The method of claim 13 or 14, wherein the second antigen-binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 , a LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 13.

16. The method of any one of claims 13-15, wherein the second antigenbinding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 3, and a LCVR comprising the amino acid sequence of SEQ ID NO: 2.

17. The method of any one of claims 1-16, wherein the bispecific antibody comprises a human IgG heavy chain constant region.

18. The method of claim 17, wherein the human IgG heavy chain constant region is isotype lgG1.

19. The method of claim 17, wherein the human IgG heavy chain constant region is isotype lgG4.

20. The method of claim 18 or 19, wherein the bispecific antibody comprises a chimeric hinge that reduces Fey receptor binding relative to a wild-type hinge of the same isotype.

21 . The method of any one of claims 17-20, wherein the first heavy chain or the second heavy chain, but not both, comprises a CH3 domain comprising a H435R (EU numbering) modification and a Y436F (EU numbering) modification.

22. The method of any one of claims 1-16, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29.

23. The method of any one of claims 1-16, wherein the bispecific antibody comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31.

24. The method of any one of claims 1-16, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31 , and a common light chain comprising the amino acid sequence of SEQ ID NO: 30.

25. The method of any one of claims 1-24, wherein the subject has a serum CA-125 level of greater than 92 U/ml.

26. The method of any one of claims 1-25, further comprising administering a second therapeutic agent or therapeutic regimen.

27. The method of claim 26, wherein the second therapeutic agent or therapeutic regimen comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

28. The method of claim 27, wherein 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.

29. The method of claim 28, wherein the anti-PD-1 antibody or antigen-binding fragment comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 37.

30. The method of claim 28 or 29, wherein the anti-PD-1 antibody or antigenbinding fragment comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 40.

31 . The method of any one of claims 28-30, wherein the anti-PD-1 antibody or antigen-binding fragment comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 33, and a LCVR comprising the amino acid sequence of SEQ ID NO: 34.

32. The method of claim 31 , wherein the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 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.

33. The method of any one of claims 1-32, wherein the bispecific antibody is administered in a dosing regimen comprising a split initial dose.

34. The method of any one of claims 1-33, wherein the bispecific antibody is administered to the subject at a dose of from 1 mg to 1000 mg weekly.

35. The method of claim 34, wherein the bispecific antibody is administered to the subject at a dose of 250 mg weekly.

36. The method of any one of claims 27-35, wherein the anti-PD-1 antibody is administered to the subject prior to, concurrent with or after the bispecific antibody.

37. The method of any one of claims 1-36, wherein the subject has previously been treated with a chemotherapy drug, radiation therapy, surgery, oran immunotherapy drug.

38. The method of claim 37, wherein the chemotherapy drug is tazemetostat.

39. The method of claim 37, wherein the immunotherapy drug is a PD1 inhibitor or a CTLA4 inhibitor.

40. The method of claim 37, wherein the immunotherapy drug is pembrolizumab, nivolumab or ipilimumab.

41 . The method of any one of claims 1-36, wherein the subject has stable disease, a partial response, or a complete response following administration of the bispecific antibody for at least one week at a dose of 1-250 mg.

42. The method of any one of claims 1-36, wherein the epithelioid sarcoma of the breast tissue of the subject decreases in size following administration of the bispecific antibody for at least one week at a dose of 1-250 mg.

43. The method of any one of claims 1-36, wherein the antibodies are administered to the subject intravenously or subcutaneously.

44. The method of any one of claims 1-43, wherein the bispecific antibody is administered in a dosing regimen comprising: administration of an initial dose of 1 mg of the bispecific antibody during week 1 of the dosing regimen; administration of a transitional dose of 20 mg of the bispecific antibody during week 2 of the dosing regimen; and administration of a full dose of 250 mg of the bispecific antibody during week 3 of the dosing regimen.

45. The method of claim 44, wherein the initial dose is split into two equal fractions administered on consecutive days.

46. The method of claim 44 or 45, wherein the transitional dose is split into two equal fractions administered on consecutive days.

47. The method of any one of claims 44-46, wherein the full dose is split into two fractions administered on consecutive days.

48. The method of claim 47, wherein the two fractions of the full dose comprise a 50 mg fraction and a 250 mg fraction.

49. The method of any one of claims 44-48, wherein the dosing regimen further comprises administration of a maintenance dose of 250 mg of the bispecific antibody administered during week 4 of the dosing regimen.

50. The method of claim 49, wherein the maintenance dose is administered weekly during subsequent weeks of the dosing regimen.