Methods of treating recurrent epithelioid sarcoma using bispecific anti-MUC16*anti-CD3 antibodies alone or in combination with anti-PD-1 antibodies

Abstract

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

Description

[0001] Reference to sequence listing

[0002] This application incorporates by reference a computer-readable sequence listing in ST.26 XML format, named 11138WO01_Sequence, created on March 16, 2023, and containing 54,705 bytes. Technical Field

[0003] The present invention relates to methods for treating cancer using bispecific antibodies that bind to mucin 16 (MUC16) and CD3, alone or in combination with an anti-PD-1 antibody. Background Art

[0004] Mucin 16 (MUC16), also known as cancer antigen 125, cancer antigen 125, carbohydrate antigen 125 or CA-125, is a highly glycosylated integral membrane glycoprotein with a single transmembrane domain 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, an enterokinase and agrin (SEA) domain, and a carboxyl-terminal domain containing a segment of a transmembrane region and a short cytoplasmic tail. Proteolytic cleavage results in the 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, mass-forming intrahepatic bile duct carcinoma, cervical adenocarcinoma, and gastric adenocarcinoma, as well as in diseases and conditions including inflammatory bowel disease, cirrhosis, heart failure, peritoneal infection, and abdominal surgery. (Haridas, D. et al., 2014, FASEB J., 28:4183-4199; Hoshino, M. et al., 2010, Cancer Res Clin Oncol., 136(3):457-64). Expression on cancer cells has been shown to protect tumor cells from the immune system. (Felder, M. et al., 2014, Molecular Cancer, 13:129) Methods of using antibodies to MUC16 to treat cancer have been studied. Oregovomab and abgovomab are anti-MUC16 antibodies, but with limited success. (Felder, supra, Das, S. and Batra, SK 2015, Cancer Res. 75:4660-4674.)

[0005] CD3 is a homodimer or heterodimer antigen expressed on T cells associated with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the association of dimers of two of four different chains: ε, ζ, δ and γ. CD3 dimer arrangements include γ / ε, δ / ε and ζ / ζ. It has been shown that antibodies against CD3 aggregate CD3 on T cells, thereby causing T cell activation in a manner similar to that of MHC molecules loaded with peptides engaging TCR. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. In addition, bispecific antibodies capable of binding CD3 and target antigens have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing target antigens.

[0006] 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. Blocking of the PD-1 signaling pathway has shown clinical activity in patients with a variety of tumor types, and antibody therapies that block PD-1 (e.g., nivolumab and pembrolizumab) have been approved for the treatment of metastatic melanoma and metastatic squamous non-small cell lung cancer. Recent data have demonstrated the clinical activity of PD-1 blockade in patients with aggressive NHL and Hodgkin's lymphoma (Hodgkin's lymphoma) (Lesokhin, et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exhibition, San Francisco, Calif.; Ansell et al. 2015, N. Engl. J. Med. 372 (4): 311-9).

[0007] Epithelioid sarcoma is a rare, high-grade soft tissue tumor with a known tendency to local recurrence, regional lymph node involvement, and distant metastasis. (Sobanko et al., J Clin Aesthet Dermatol. 2(5):49-54, 2009). Most cases begin in the soft tissue under the skin of the fingers, hands, forearms, lower legs, or feet, but tumors can also begin to grow in other parts of the body. Although epithelioid sarcoma appears harmless, its malignancy itself indicates a poor prognosis. Misdiagnosis of this tumor may lead to delayed and inappropriate treatment, which has an adverse effect on patient survival. Current treatments for epithelioid sarcoma include radical tumor resection, adjuvant chemotherapy, sentinel lymph node biopsy, and radiation therapy. (Casanova et al., Cancer. 106(3):708-17, 2006; Sobanko et al., J Clin Aesthet Dermatol. 2(5):49-54, 2009). Although most patients respond to initial treatment, the majority experience recurrence of the disease, leading to cycles of repeated surgeries and additional rounds of chemotherapy. Although recurrent epithelioid sarcomas may respond to further treatment, nearly all epithelioid sarcomas eventually become resistant to currently available therapies. Despite recent advances in therapy, recurrent metastatic epithelioid sarcoma remains a disease with a high unmet need.

[0008] There is some evidence that epithelioid sarcoma may be amenable to certain forms of immunotherapy. For example, tumor specimens from patients with epithelioid sarcoma have previously been detected to have high levels of PD-L1 expression and CD3 + / CD8 + T lymphocyte infiltration (Gong et al., Front Oncol., 11:728437, 2021). Blocking the PD-1 / PD-L1 checkpoint pathway can be beneficial for epithelioid sarcoma. However, blocking this pathway alone may not be enough.

[0009] Therefore, given the high unmet need, additional therapies targeting epithelioid sarcoma are needed. Summary of the invention

[0010] In one aspect, the present disclosure includes a method of treating a MUC16-expressing cancer in a subject in need thereof, the method comprising administering to the subject a bispecific antibody comprising a first antigen binding domain that specifically binds to mucin 16 (MUC16) on a target tumor cell, and a second antigen binding domain that specifically binds to human CD3 on a T cell. In some embodiments, the bispecific antibody is administered to the subject at a dose of at least 1 mg (e.g., weekly).

[0011] In some embodiments, the cancer expressing MUC16 is epithelioid sarcoma. In some cases, the subject has metastatic epithelioid sarcoma.

[0012] In some embodiments, the cancer expressing MUC16 is a sarcoma expressing MUC16. In some cases, the sarcoma is an epithelioid sarcoma, a rhabdoid tumor of the kidney and soft tissue, a rhabdomyosarcoma, an Ewing sarcoma, or a soft tissue sarcoma. In some cases, the sarcoma expressing MUC16 is an epithelioid sarcoma or a rhabdoid tumor of the kidney and soft tissue.

[0013] In some embodiments, the MUC16-expressing cancer (eg, a sarcoma) is deficient in the expression of a functional Integrase Interactor 1 protein.

[0014] In some embodiments, the subject has previously been treated with anticancer therapy. In some cases, the subject is resistant to the previous therapy, responds insufficiently to the previous therapy, or relapses after the previous therapy. In some cases, the subject has previously been treated with chemotherapy drugs, radiotherapy, surgery, or immunotherapy drugs. 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 cancer expressing MUC16 is resistant to the previous therapy or responds insufficiently to the previous therapy, or relapses after the previous therapy.

[0015] In some embodiments, the bispecific antibody comprises a first antigen binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 1; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 2. In some cases, the first antigen binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 8, a HCDR2 having an amino acid sequence of SEQ ID NO: 9, and a HCDR3 having an amino acid sequence of SEQ ID NO: 10. In some cases, the first antigen binding domain comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 11, a LCDR2 having an amino acid sequence of SEQ ID NO: 12, and a LCDR3 having an amino acid sequence of SEQ ID NO: 13. In some cases, the first antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO:1 and a LCVR having the amino acid sequence of SEQ ID NO:2.

[0016] In some embodiments, the bispecific antibody, including those discussed above in which the first antigen binding domain is included, comprises a second antigen binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 3; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 2. In some cases, the second antigen binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 14, a HCDR2 having an amino acid sequence of SEQ ID NO: 15, and a HCDR3 having an amino acid sequence of SEQ ID NO: 16. In some cases, the second antigen binding domain comprises a LCDR1 having the amino acid sequence of SEQ ID NO: 11, a LCDR2 having the amino acid sequence of SEQ ID NO: 12, and a LCDR3 having the amino acid sequence of SEQ ID NO: 13. In some cases, the second antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO: 3 and a LCVR having the amino acid sequence of SEQ ID NO: 2.

[0017] 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 of isotype IgG1. In some cases, the human IgG heavy chain constant region is of isotype IgG4.

[0018] In some embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0019] In some embodiments, the first heavy chain or the second heavy chain (but not both) of the bispecific antibody comprises a CH3 domain comprising a H435R (EU numbering) modification and a Y436F (EU numbering) modification.

[0020] 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 having the amino acid sequence of SEQ ID NO: 29, a second heavy chain having the amino acid sequence of SEQ ID NO: 31, and a common light chain having the amino acid sequence of SEQ ID NO: 30.

[0021] In some embodiments, the subject has an elevated serum CA-125 level. In some embodiments, the subject has a serum CA-125 level that is at least twice the upper limit of normal. In some embodiments, the subject has a serum CA-125 level greater than 92 U / ml.

[0022] In some embodiments, the method further comprises administering a second therapeutic agent or treatment regimen. In some cases, the second therapeutic agent or treatment regimen comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 antibody is cemiplimab.

[0023] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 33; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 34. In some cases, the anti-PD-1 antibody or antigen-binding fragment comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 35, a HCDR2 having an amino acid sequence of SEQ ID NO: 36, and a HCDR3 having an amino acid sequence of SEQ ID NO: 37. In some cases, the anti-PD-1 antibody or antigen-binding fragment comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 38, a LCDR2 having an amino acid sequence of SEQ ID NO: 39, and a LCDR3 having an amino acid sequence of SEQ ID NO: 40. In some instances, the anti-PD-1 antibody or antigen-binding fragment comprises a HCVR having an amino acid sequence of SEQ ID NO: 33 and a LCVR having an amino acid sequence of SEQ ID NO: 34. In some instances, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42.

[0024] In some embodiments, the bispecific antibody is administered with a dosing regimen that includes a divided initial dose (e.g., an initial dose of 1 mg is divided into two equal 0.5 mg portions administered on two consecutive days). In some embodiments, the bispecific antibody is administered to a subject at a dose of 1 mg to 1000 mg per week. In some cases, the bispecific antibody is administered to a subject at a dose of 250 mg per week. In some cases, the bispecific antibody is administered to a subject at a frequency of once every 3 weeks. In some cases, the bispecific antibody is administered to a subject by intravenous administration. In some cases, the bispecific antibody is administered to a subject by subcutaneous administration. In some cases, the anti-PD-1 antibody is administered to a subject before, simultaneously with, or after the bispecific antibody.

[0025] In some embodiments of the method, the subject has stable disease, partial remission, or complete remission after at least one week of administration of the bispecific antibody at a dose of 1-250 mg. In some cases, the size of an epithelioid sarcoma in breast tissue of the subject is reduced after at least one week of administration of the bispecific antibody at a dose of 1-250 mg.

[0026] In some embodiments of the method, the bispecific antibody is administered with a dosing regimen comprising: administering an initial dose of 1 mg of the bispecific antibody in the first week of the dosing regimen; administering a transitional dose of 20 mg of the bispecific antibody in the second week of the dosing regimen; and administering a full dose of 250 mg of the bispecific antibody in the third week of the dosing regimen. In some cases, the initial dose is divided into two equal parts administered on consecutive days. In some cases, the transitional dose is divided into two equal parts administered on consecutive days. In some cases, the full dose is divided into two equal parts administered on consecutive days. In some embodiments, the two parts of the full dose include a 50 mg part and a 250 mg part. In some embodiments, the dosing regimen also includes administering a maintenance dose of 250 mg of the bispecific antibody during the fourth week of the dosing regimen. In some cases, the maintenance dose is administered weekly during the subsequent weeks of the dosing regimen. In some cases, the maintenance dose is administered every other week (Q2W) during the subsequent weeks of the dosing regimen.

[0027] Other embodiments of the present invention will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Binding of different concentrations of anti-MUC16 clone 3A5 and BSMUC16 / CD3-001 to CA125 as determined by ELISA (as described in Example 2 herein) is shown. BSMUC16 / CD3-001 and its MUC16 parent antibody showed significantly reduced binding signals at all tested concentrations compared to anti-MUC16 clone 3A5, which binds to the repeat region of MUC16.

[0029] Figure 2 Shown are the average tumor growth curves of groups of mice (5 per group) treated with CD3 binding control + isotype control (Δ), BSMUC16 / CD3-005 + isotype control (□), CD3 binding control + anti-PD-1 (▲), and BSMUC16 / CD3-005 + anti-PD-1 (■) (as described in Example 3 herein). The combination of anti-PD-1 antibody and anti-CD3×MUC16 bispecific antibody synergistically inhibited tumor growth.

[0030] Figure 3 Shown is the effect of incubation of T cells with BSMUC16 / CD3-001 on the percentage of PD-1 positive T cells. DETAILED DESCRIPTION

[0031] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not intended to be limited, because the scope of the present invention will only be limited by the appended claims. The features of any embodiment or embodiment can be combined with each other, and such combinations are clearly included in the scope of the present invention. Any specified value discussed above or herein can be combined with another related value discussed above or herein to list a range, and the value represents the upper and lower limits of the range, and such ranges are included in the scope of the present disclosure.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited numerical value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0033] 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 entirety.

[0034] Methods for treating or inhibiting the growth of cancer

[0035] The present invention includes methods for treating, improving or reducing the severity of at least one symptom or indication of a subject, or inhibiting the growth of cancer (e.g., recurrent epithelioid sarcoma). The method according to this aspect of the invention includes administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody against MUC16 and CD3, alone or in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1. The term "treatment" and the like as used herein means alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, delaying or inhibiting tumor growth, reducing tumor cell load or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and / or disappearance, preventing tumor recurrence, and / or increasing the duration of survival of the subject.

[0036] As used herein, the expression "subject in need" means a human or non-human mammal that exhibits one or more symptoms or signs of cancer, and / or has been diagnosed with cancer (including epithelioid sarcoma) and is in need of treatment for the cancer. In many embodiments, the term "subject" can be used interchangeably with the term "patient". For example, a human subject may be diagnosed with a primary or metastatic tumor and / or one or more symptoms or signs, including, but not limited to, enlarged lymph nodes, abdominal swelling, chest pain / tightness, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, splenomegaly, itching. The expression includes subjects with primary epithelioid sarcoma or confirmed epithelioid sarcoma. In specific embodiments, the expression includes human subjects with epithelioid sarcoma or another tumor expressing MUC16 and in need of treatment. In other specific embodiments, the expression includes subjects with MUC16+ tumors (e.g., tumors with MUC16 expression as determined by flow cytometry). In certain embodiments, the expression "subject in need" includes patients with epithelioid sarcoma that is resistant to or difficult to treat with previous therapies (e.g., treatment with conventional anticancer agents), or that is not adequately controlled by previous therapies. For example, the expression includes subjects who have been treated with chemotherapy, such as chemotherapeutic agents (e.g., tazemetostat) or paclitaxel compounds (e.g., docetaxel). The expression also includes subjects with epithelioid sarcoma for whom conventional anticancer therapy is undesirable, 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 "subject in need" includes patients with epithelioid sarcoma who have been treated but subsequently relapsed or metastasized. For example, a patient with epithelioid sarcoma may have been treated with one or more anticancer agents resulting in tumor regression; however, a cancer resistant to one or more anticancer agents (e.g., chemotherapy-resistant cancer) is subsequently treated with the methods of the present invention. The expression "subject in need thereof" also includes subjects at risk of developing epithelioid sarcoma, such as those with a family history of epithelioid sarcoma, those with a past history of infection associated with epithelioid sarcoma, those with a SMARCB1 gene mutation, or those with a compromised immune system due to HIV infection or immunosuppressive drugs.

[0037] In certain embodiments, the methods of the present invention can be used to treat patients who exhibit elevated levels of one or more cancer-associated biomarkers (e.g., programmed death ligand 1 (PD-L1), CA125, human epididymis protein 4 (HE4), and / or carcinoembryonic antigen (CEA)). For example, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody to a patient with elevated levels of PD-L1 and / or CA125.

[0038] In certain embodiments, the methods of the present invention are used in patients with epithelioid sarcoma. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "epithelioid sarcoma" refers to a tumor of soft tissue (e.g., under the skin of the fingers, hands, forearms, lower legs, or feet); however, tumors can also begin to grow in other parts of the body.

[0039] According to certain embodiments, the present invention includes methods for treating or delaying or inhibiting tumor growth. In certain embodiments, the present invention includes methods for promoting tumor regression. In certain embodiments, the present invention includes methods for reducing tumor cell load or reducing tumor load. In certain embodiments, the present invention includes methods for preventing tumor recurrence. According to this aspect of the invention, the method includes 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, wherein each antibody is administered to the subject in multiple doses, for example, as part of a specific therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of an anti-MUC16×CD3 antibody to a 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, one or more doses of an 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.

[0040] In certain embodiments, each dose of anti-MUC16 / anti-CD3 antibody is administered in more than one portion, e.g., in 2-5 portions ("fractionated dosing"), within a given dosing cycle. Anti-MUC16 / anti-CD3 bispecific antibodies can be administered in divided doses to reduce or eliminate cytokine "spikes" induced in response to antibody administration. Cytokine spikes refer to clinical symptoms of 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 an 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 within a given dosing period, a certain dose of the bispecific antibody is administered in divided doses, or in more than one portion, e.g., as 2 portions, as 3 portions, as 4 portions, or as 5 portions. In certain embodiments, a certain dose of a bispecific antibody is divided into 2 or more portions, wherein each portion contains an equal amount of antibody to the other portions. For example, a dose comprising 1000 micrograms of an anti-MUC16 / anti-CD3 antibody may be administered once a week, wherein the dose is administered in 2 portions within a week, 500 micrograms per portion. In certain embodiments, a dose of a bispecific antibody is administered in 2 or more portions, wherein the portions contain unequal amounts of antibodies, such as more or less than the first portion. For example, a dose comprising 1000 micrograms of an anti-MUC16 / anti-CD3 antibody may be administered once a week, wherein the dose is administered in 2 portions within a week, wherein the first portion contains 700 micrograms, and the second portion contains 300 micrograms. As another example, a dose comprising 1000 micrograms of an anti-MUC16 / anti-CD3 antibody may be administered once within 2 weeks, wherein the dose is administered in 3 portions within 2 weeks, wherein the first portion contains 400 micrograms, the second portion contains 300 micrograms, and the third portion contains 300 micrograms.

[0041] In certain embodiments, the invention includes methods of inhibiting, delaying or stopping tumor metastasis or tumor infiltration into peripheral organs. According to this aspect, the method comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody alone or in combination with an anti-PD-1 antibody.

[0042] In specific embodiments, the present invention provides methods for increasing anti-tumor efficacy or increasing tumor inhibition. According to this aspect of the invention, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody to a subject with epithelioid sarcoma 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 method provides increased tumor inhibition, such as an increase of about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80%, compared to a subject to whom the bispecific antibody is administered prior to administration of the anti-PD-1 antibody.

[0043] In certain embodiments, the methods of the present invention include administering a therapeutically effective amount of a bispecific anti-CD3×MUC16 antibody alone or in combination with an anti-PD-1 antibody to a subject with epithelioid sarcoma. In additional embodiments, epithelioid sarcoma is inert or aggressive. In certain embodiments, the subject has no response to a previous therapy or relapses after a previous therapy. In some embodiments, the subject has a CA-125 level equal to or greater than 2 times the upper limit of normal (ULN) (e.g., greater than about 92U / ml). In multiple embodiments, the subject's serum CA-125 level (before treatment) is equal to or greater than 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200U / ml. In certain embodiments, the methods of the present invention also include administering an additional therapeutic agent to the subject.

[0044] In certain embodiments, the methods of the present invention include administering a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody to a subject with MUC16+ cancer. In specific embodiments, the cancer is an epithelioid sarcoma. In other embodiments, the epithelioid sarcoma is inert or aggressive. In some embodiments, the epithelioid sarcoma is present in the patient's chest wall, lung, and / or breast tissue. In some embodiments, the serum CA-125 level of an epithelioid sarcoma patient is elevated (e.g., at least 2×ULN). In some embodiments, cancer is resistant to chemotherapeutic drugs. In certain embodiments, the subject has no response to a previous therapy or has relapsed after a previous therapy (e.g., chemotherapy).

[0045] In certain embodiments, the methods of the present invention include administering an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody to a subject in need as a "first-line" treatment (e.g., initial treatment). In other embodiments, an anti-PD-1 antibody is administered in combination with a bispecific anti-MUC16 / anti-CD3 antibody as a "second-line" treatment (e.g., after a prior therapy). For example, an anti-PD-1 antibody is administered in combination with a bispecific anti-MUC16 / anti-CD3 antibody as a "second-line" treatment to a subject who has relapsed after a prior therapy, such as chemotherapy.

[0046] In certain embodiments, the methods of the present invention are used to treat patients with MRD-positive diseases. Minimal residual disease (MRD) refers to a small amount of cancer cells remaining in the patient's body 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, often lead to recurrence of the disease. The present invention includes methods for inhibiting and / or eliminating residual cancer cells in a patient's body during MRD testing. MRD can be determined according to methods known in the art (e.g., MRD flow cytometry). According to this aspect of the invention, the method comprises administering to a subject in need thereof a bispecific anti-MUC16 / anti-CD3 antibody alone or in combination with an anti-PD-1 antibody.

[0047] According to certain embodiments, the methods of the invention comprise administering to a subject a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody and, optionally, a third therapeutic agent. The third therapeutic agent can be an agent selected from the group consisting of, for example, radiation, chemotherapy, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), LAG3 inhibitors (e.g., anti-LAG3 antibodies), CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, Ang2 inhibitors, transforming growth factor β (TGF.β.) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies to tumor-specific antigens (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), vaccines (e.g., Bacillus Calmette-Guérin, Calmette-Guerin), granulocyte-macrophage colony stimulating factor, cytotoxins, chemotherapeutic agents, IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, cytokines (such as IL-2, IL-7, IL-21 and IL-15), anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs) and dietary supplements (such as antioxidants). In certain embodiments, the antibody can be administered in combination with a therapy including a chemotherapeutic agent (e.g., tazemetostat, paclitaxel, carboplatin, doxorubicin, cyclophosphamide, cisplatin, gemcitabine or docetaxel), radiation and surgery. The phrase "in combination with..." used herein means that the antibody is administered to the subject at the same time, just before or just after the administration of a third therapeutic agent. In certain embodiments, the third therapeutic agent is administered as a co-formulation with the antibody.

[0048] In certain embodiments, the methods of the present invention include administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody. When the combination is administered, the administration of the antibody increases the inhibition of tumor growth. In certain embodiments, tumor growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, compared to an untreated subject or a subject administered either antibody as a monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody results in increased tumor regression, tumor shrinkage, and / or disappearance. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody results in a delay in tumor growth and development, for example, tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years, compared to an untreated subject or a subject treated with either antibody as a monotherapy. In certain embodiments, the anti-PD-1 antibody is administered in combination with a bispecific anti-MUC16 / anti-CD3 antibody to prevent tumor recurrence and / or prolong the survival duration of the subject, for example, to prolong the survival duration by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months, compared to an untreated subject or a subject administered with either antibody as a monotherapy. In certain embodiments, administration of the combined antibody increases progression-free survival or overall survival. In certain embodiments, the administration of an anti-PD-1 antibody in combination with a bispecific anti-MUC16 / anti-CD3 antibody increases the subject's response and duration of response by, for example, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50%, compared to an untreated subject or a subject receiving either antibody as a monotherapy. In certain embodiments, the administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a subject with epithelioid sarcoma results in the complete disappearance of all signs of tumor cells ("complete response"). In certain embodiments, the administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a subject with epithelioid sarcoma results in a reduction of at least 30% or more 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 having epithelioid sarcoma results in complete or partial disappearance of tumor cells / lesions, including new measurable lesions.Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody produces a synergistic anti-tumor effect that exceeds the combined effect of the two agents when administered alone.

[0049] In certain embodiments, the combination of antibodies administered is safe and well tolerated by patients, with no increase in adverse side effects (e.g., increased cytokine release ("cytokine storm") or increased T cell activation) compared to patients administered the bispecific antibody as a monotherapy.

[0050] In certain cases, a subject's response to therapy is classified as a complete response (CR), partial response (PR), progressive disease (PD), or stable disease (SD). CR is defined as the disappearance of all target lesions and a reduction in the short axis of any pathological lymph node (whether target or non-target) to <10mm (<1cm). PR is defined as a reduction of at least 30% in the sum of diameters of target lesions, with reference to the baseline sum diameter. PD is defined as an increase of at least 20% in the sum of diameters of target lesions, with reference to the minimum sum studied (including the baseline sum if it is the minimum for the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5mm (0.5cm). (Note: The appearance of one or more new lesions is also considered progression). SD is defined as a reduction that is not sufficient to meet the requirements of PR, and an increase that is not sufficient to meet the requirements of PD, with reference to the minimum sum diameter during the study.

[0051] Anti-PD-1 antibodies and antigen-binding fragments thereof

[0052] According to certain exemplary embodiments of the present invention, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds. In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region contains three domains: C H 1. C H 2 and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain (C L 1). V HArea and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-IL-4R antibody (or antigen-binding portion thereof) may be identical to 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.

[0053] The term "antibody" as used herein also includes the antigen binding fragment of a complete antibody molecule. The terms "antigen binding portion of an antibody", "antigen binding fragment of an antibody", etc. as used herein include any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen binding fragments of antibodies can be derived from full antibody molecules, for example, using any suitable standard technique (such as proteolytic digestion or the recombinant genetic engineering techniques involving the operation and expression of DNA encoding antibody variable domains and optionally antibody constant domains). Such DNA is known, and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA sequencing can be performed, and chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to import codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0054] 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 amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides), or binding FR3-CDR3-FR4 peptides. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, microbodies, 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.

[0055] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can have any size or amino acid composition and will generally include at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V L In the antigen-binding fragment associated with the structural domain, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or V L Structural domain.

[0056] In certain embodiments, the 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 the antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L-C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected through a full-length or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of the antibody of the present invention may contain any of the variable domain and constant domain configurations listed above, which are connected to each other and / or to one or more monomer V H or V L The domains are in non-covalent association (eg, through disulfide bonds) as homodimers or heterodimers (or other multimers).

[0057] The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies generally include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format can be adapted to the context of the antibodies or antigen-binding fragments of the present invention. For example, the present invention includes methods comprising the use of bispecific antibodies, wherein one arm of the immunoglobulin is specific for PD-1 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (for a review of the foregoing formats, see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that are then self-assembled into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Electronic version: Dec. 4, 2012]).

[0058] The antibody used in the inventive method can be a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. However, the human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies that have been implanted into human framework sequences using CDR sequences derived from the germline of another mammalian species (such as mice).

[0059] The antibody used in the method of the present invention can be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or separated by recombinant means, such as antibodies expressed by recombinant expression vectors transfected into host cells (described further below), antibodies separated from recombinant human antibody combinatorial libraries (described further below), antibodies separated from transgenic animals (e.g., mice) relative to human immunoglobulin genes (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, produced or separated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using transgenic animals with respect to human Ig sequences, undergo in vivo somatic cell mutagenesis) and thus the V H and V L Although the amino acid sequence of the region is derived from human germline V H and V L Sequences that are related to but that may not naturally occur within the human antibody germline repertoire in vivo.

[0060] According to certain embodiments, the antibody used in the methods of the present invention specifically binds to PD-1. The term "specifically binds" and the like means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" to PD-1 as used in the context of the present invention includes an antibody that binds to PD-1 or a portion thereof, and whose K DLess 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 analysis. However, an isolated antibody that specifically binds to human PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from other (non-human) species.

[0061] 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), a light chain variable region (LCVR), and / or a complementarity determining region (CDR) comprising any amino acid sequence of the anti-PD-1 antibodies listed in U.S. Patent Publication No. 20150203579. In certain exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof useful in the context of the methods of the present invention comprises a heavy chain complementarity determining region (HCDR) of a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 33 and a light chain complementarity determining region (LCDR) of a light chain variable region (LCVR) having an 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 HCDR1 comprises the amino acid sequence of SEQ ID NO: 35; HCDR2 comprises the amino acid sequence of SEQ ID NO: 36; HCDR3 comprises the amino acid sequence of SEQ ID NO: 37; LCDR1 comprises the amino acid sequence of SEQ ID NO: 38; LCDR2 comprises the amino acid sequence of SEQ ID NO: 39; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 40. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR having SEQ ID NO: 33 and a LCVR having 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 having 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 having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42 is a fully human anti-PD-1 antibody called REGN2810 (also known as cemiplimab). According to certain exemplary embodiments, the methods of the present invention include the use of REGN2810 or a bioequivalent thereof. The term "bioequivalent" as used herein means an anti-PD-1 antibody or PD-1 binding protein or a fragment thereof as a drug equivalent or drug substitute, which, when administered at the same molar dose (whether single or multiple doses) under similar experimental conditions, does not show a significant difference in its absorption rate and / or extent from that of REGN2810. In the context of the present invention, the term refers to antigen binding proteins that bind to PD-1, and their safety, purity and / or efficacy are not clinically significant compared to REGN2810.

[0062] Other anti-PD-1 antibodies that can be used in the context of the methods of the present invention include, for example, antibodies mentioned and known in the art, such 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 listed in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0063] The anti-PD-1 antibodies used in the context of the methods of the present invention may have pH-dependent binding characteristics. For example, the anti-PD-1 antibodies used in the methods of the present invention may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, the anti-PD-1 antibodies of the present invention may exhibit enhanced binding to their antigens at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values ​​less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. The expression "neutral pH" used herein refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0064] In certain cases, "reduced binding to PD-1 at acidic pH compared to neutral pH" refers to the K of the antibody binding to PD-1 at acidic pH. D The K value of the antibody binding to PD-1 at neutral pH D For example, for the purposes of the present invention, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K D 0 or greater, then the antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to PD-1 at acidic pH compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K of an antibody or antigen-binding fragment of the invention is DThe ratio may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0065] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening antibody populations for reduced (or enhanced) binding to specific antigens at acidic pH compared to neutral pH. In addition, antibodies with pH-dependent characteristics can be produced by modifying the antigen-binding domain at the amino acid level. For example, by replacing one or more amino acids of the antigen-binding domain (e.g., inside the CDR) with histidine residues, antibodies with reduced antigen binding at acidic pH relative to neutral pH can be obtained. The expression "acidic pH" used herein refers to a pH of 6.0 or less.

[0066] Bispecific anti-MUC16 / anti-CD3 antibody

[0067] According to certain exemplary embodiments of the present invention, the method comprises administering a therapeutically effective amount of a bispecific antibody that specifically binds to CD3 and MUC16. Such antibodies may be referred to herein as, for example, "anti-MUC16 / anti-CD3" or "anti-MUC16×CD3" or "MUC16×CD3" bispecific antibodies or other similar terms.

[0068] As used herein, the expression "bispecific antibody" refers to an immunoglobulin comprising at least a first antigen binding domain and a second antigen binding domain. In the context of the present invention, the first antigen binding domain specifically binds to a first antigen (e.g., MUC16), and the second antigen binding domain specifically binds to a second different antigen (e.g., CD3). Each antigen binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each domain comprising three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen binding domain may be represented by the prefix "A", and the CDRs of the second antigen binding domain may be represented by the prefix "B". Therefore, 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.

[0069] The first antigen binding domain and the second antigen binding domain are each connected to a separate multimerization domain. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or configuration. In the context of the present invention, the multimerization component is the Fc portion of an immunoglobulin (comprising C H2 -C H3 domain), such as an Fc domain selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and IgG of any allotype within each isotype group.

[0070] The bispecific antibodies of the present invention generally comprise two multimerization domains, such as two Fc domains, which are respectively parts of a single antibody heavy chain. The first multimerization domain and the second multimerization domain can have the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain can have different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0071] Any bispecific antibody form or technology can be used to prepare 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 connected (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) with 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 forms that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or dual-body bispecific forms, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-in-holes, common light chains (e.g., common light chains with knobs-in-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab2 bispecific forms (for a review of the aforementioned forms, see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein).

[0072] In the context of the bispecific antibodies of the invention, the Fc domain may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that result in the modified Fc domain having a modified binding interaction (e.g., enhanced or weakened) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises one or more modifications in the Fc domain. H2 or C H3 The Fc domain comprises a modification in the region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome at a pH range of from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in U.S. Patent Publication No. 20150266966, which is incorporated herein in its entirety.

[0073] The present disclosure also includes a first C H 3 domain and the second Ig C H 3-domain bispecific antigen-binding molecule, wherein the first and second Ig C H At least one amino acid in the first Ig C domain and the second Ig C domain differs from each other, and wherein at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. H 3 domains bind protein A and the second Ig C H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may also contain a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Pat. No. 8,586,713. H Other modifications within 3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 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 IgG4 antibodies.

[0074] In certain embodiments, the Fc domain can be chimeric, combining Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise Fc sequences from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence, and C derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule described herein include, from N-terminus to C-terminus: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any antigen-binding molecule described herein comprises, from N-terminus to C-terminus: [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any antigen-binding molecule of the present invention are described in U.S. Patent Publication No. 20140243504, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0075] According to certain exemplary embodiments of the present invention, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (A-HCVR and B-HCVR), a light chain variable region (A-LCVR and B-LCVR) and / or a complementarity determining region (CDR) comprising any amino acid sequence of the bispecific anti-MUC16 / anti-CD3 antibody listed in U.S. Patent Publication No. 20180112001. In certain exemplary embodiments, a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof useful in the context of the methods of the invention comprises: (a) a first antigen-binding arm comprising heavy chain complementarity determining regions (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO:1, and light chain complementarity determining regions (A-LCDR1, A-LCDR2, and A-LCDR3) comprising the amino acid sequence of SEQ ID NO:2; and (b) a second antigen-binding arm comprising heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a LCVR (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO:8; A-HCDR2 comprises the amino acid sequence of SEQ ID NO:9; A-HCDR3 comprises the amino acid sequence of SEQ ID NO:10; A-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; A-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; A-LCDR3 comprises the amino acid sequence of SEQ ID NO:13; B-HCDR1 comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:26; B-HCDR2 comprises the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, or SEQ ID NO:27; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28; and B-LCDR1 comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:26; In some embodiments, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding arm comprising a HCVR comprising SEQ ID NO: 1 (A-HCVR) and a LCVR comprising SEQ ID NO: 2 (A-LCVR); and (b) a second antigen-binding arm comprising a HCVR comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 (B-HCVR) and a LCVR comprising SEQ ID NO: 2 (B-LCVR). In certain exemplary embodiments, the bispecific anti-CD3×MUC16 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-CD3×MUC16 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.

[0076] In certain embodiments, the anti-tumor activity of the bispecific anti-CD3×MUC16 antibodies of the invention is not substantially hampered by the presence of high levels (e.g., up to 10,000 U / ml) of circulating CA125. Serum levels of CA125 are elevated in the serum of most ovarian cancer patients (published median levels are about 656 U / ml). As shown in Example 2 below, high levels of CA125 in serum or ascites do not significantly interfere with the anti-tumor spectrum of the bispecific antibodies of the invention.

[0077] Other bispecific anti-MUC16 / anti-CD3 antibodies that can be used in the context of the methods of the invention include, for example, any of the antibodies described in U.S. Patent Publication No. WO 20180112001.

[0078] Combination therapy

[0079] According to certain embodiments, the methods of the present invention include administering an anti-MUC16 / anti-CD3 bispecific antibody in combination with an anti-PD-1 antibody to a subject. In certain embodiments, the methods of the present invention include administering an antibody with additive or synergistic activity to treat cancer, preferably epithelioid sarcoma. As used herein, the expression "in combination with" refers to administering an anti-MUC16 / anti-CD3 bispecific antibody before, after, or simultaneously with the administration of an anti-PD-1 antibody. The term "in combination with" also includes sequential or concomitant administration of an 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 can be administered more than 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes prior to administration of the bispecific anti-MUC16 / anti-CD3 antibody. When administered "after" the bispecific anti-MUC16 / anti-CD3 antibody, the anti-PD-1 antibody can 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 "simultaneously" with the bispecific anti-MUC16 / anti-CD3 antibody refers to administration of the anti-PD-1 antibody to the subject in a separate dosage form within less than 5 minutes after (before, after, or simultaneously with) administration of the bispecific anti-MUC16 / anti-CD3 antibody, or administration to the subject in a single combined dosage form comprising both the anti-PD-1 antibody and the bispecific anti-MUC16 / anti-CD3 antibody.

[0080] In certain embodiments, the methods of the present invention include administering a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer drug. In certain embodiments, the methods of the present invention include administering an anti-PD-1 antibody and an anti-MUC16 / anti-CD3 bispecific antibody in combination with radiation therapy to produce a long-lasting anti-tumor response and / or increase the survival of patients with cancer.

[0081] In some embodiments, the methods of the present invention include administering radiation therapy before, simultaneously with, or after administering an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a patient with cancer. For example, radiation therapy may be administered to a tumor lesion in one or more doses after administering one or more doses of the antibody. In some embodiments, after systemic administration of an anti-PD-1 antibody and / or a bispecific anti-MUC16 / anti-CD3 antibody, radiation therapy may be administered locally to the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablative radiation).

[0082] Pharmaceutical compositions and administration

[0083] The present invention includes methods comprising administering to a subject a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody, wherein one or more of the antibodies is contained in a pharmaceutical composition, alone or in combination (single). The pharmaceutical compositions of the present invention can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, etc. A wide variety of suitable formulations can be found in the formulary known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semisolid gels and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) JPharmSci Technol 52: 238-311.

[0084] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endosomes (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 compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other biologically active agents.

[0085] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge becomes empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In fact, the disposable pen delivery device is pre-filled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0086] A variety of reusable pen delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany) etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM(Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM pen (Abbott Labs, Abbott Park IL), etc.

[0087] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a small portion 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 of Langer, 1990, Science 249: 1527-1533.

[0088] The injectable preparation can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by known methods. For example, the injectable preparation can be prepared by, for example, dissolving, suspending or emulsifying the antibody described above or its salt in a sterile aqueous medium or an oily medium conventionally used for injection. As aqueous media for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers (such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc.). As an oily medium, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

[0089] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared into a dosage form suitable for a unit dose that conforms to the dosage of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0090] Administration regimen

[0091] The invention includes methods comprising administering to a subject a bispecific anti-MUC16×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, as long as a therapeutic response is achieved.

[0092] According to certain embodiments of the present invention, multiple doses of bispecific anti-MUC16 / anti-CD3 antibodies, alone or in combination with anti-PD-1 antibodies, may be administered to a subject over a specified time course. The method according to this aspect of the present invention comprises sequentially administering to a subject one or more doses of bispecific anti-MUC16 / anti-CD3 antibodies, alone or in combination with one or more doses of anti-PD-1 antibodies. As used herein, "sequential administration" refers to administering each dose of the antibody to the subject at different time points, such as on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising administering a single initial dose of the antibody, followed by one or more second doses of the antibody, and optionally followed by one or more third doses of the antibody in sequence to the patient.

[0093] The terms "initial dose", "second dose" and "third dose" indicate the time sequence of administration. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial dose, the second dose, and the third dose may all contain the same amount of antibody (anti-PD-1 antibody or bispecific antibody). However, in certain embodiments, the amounts contained in the initial dose, the second dose, and / or the third dose are different from each other during the treatment (e.g., adjusted upward or downward when appropriate). 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 a "loading dose", followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency. For example, an anti-PD-1 antibody may be administered to a patient with 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.

[0094] In an exemplary embodiment of the invention, each second and / or third dose is administered 1 / 2 to 14 weeks (e.g., 1 / 2, 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2 or longer) after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means that in a sequence of multiple administrations, a dose of the bispecific anti-MUC16 / anti-CD3 (and / or anti-PD-1 antibody) is administered to the patient prior to administration of the immediately next dose in the sequence, without an intervening dose.

[0095] The method according to this aspect of the invention may include administering any number of second and / or third doses of a bispecific anti-MUC16 / anti-CD3 antibody (and / or anti-PD-1 antibody) to a patient. For example, in certain embodiments, only a single second dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to a patient. Similarly, in certain embodiments, only a single third dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0096] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, during the course of the treatment regimen, the frequency of the second and / or third dose administered to the patient can vary. The physician can also adjust the frequency of administration during the course of treatment, depending on the needs of each patient after the clinical examination.

[0097] In certain embodiments, one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody (e.g., anti-PD-1 antibody) are administered more frequently (twice a week, once a week, or once every 2 weeks) at the beginning of a treatment regimen as an "induction dose," followed by less frequent administration of subsequent doses ("consolidation doses" or "maintenance doses") (e.g., once every 4-12 weeks).

[0098] The present invention includes methods comprising sequentially administering to a patient a bispecific anti-MUC16 / anti-CD3 antibody, alone or in combination with an anti-PD-1 antibody, to treat epithelioid sarcoma. In some embodiments, the methods of the present invention 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 method comprises 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 a bispecific antibody to inhibit tumor growth and / or prevent tumor recurrence in patients with epithelioid sarcoma. In some embodiments, the anti-PD-1 antibody is administered in 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 and increased prevention of tumor recurrence compared to untreated subjects or subjects administered either antibody as a monotherapy). Alternative embodiments of the present invention relate to the concomitant administration of anti-PD-1 antibodies and bispecific antibodies, which are administered in separate doses at a frequency similar to or different from that of the anti-PD-1 antibody. In some embodiments, the bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. In certain embodiments, the bispecific antibody is administered as a single-dose formulation with the anti-PD-1 antibody.

[0099] Dosage The amount of bispecific anti-MUC16 / anti-CD3 antibody, and optionally anti-PD-1 antibody, administered to a subject according to the methods of the invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of an antibody (anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody) that results in one or more of the following: (a) reducing the severity or duration of symptoms of cancer (e.g., epithelioid sarcoma); (b) inhibiting tumor growth, or increasing tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delaying tumor growth and development; (d) inhibiting or delaying or stopping tumor metastasis; (e) preventing tumor recurrence; (f) increasing the survival of a subject with cancer (e.g., epithelioid sarcoma); and / or (g) reducing the use or need for conventional anti-cancer therapy (e.g., reducing or eliminating the use of chemotherapeutic agents or cytotoxic agents) compared to untreated subjects or subjects administered either antibody as a monotherapy.

[0100] 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.

[0101] In the case of an anti-PD-1 antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg In some embodiments, the anti-PD-1 antibody is administered at 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. In some embodiments, 350 mg of anti-PD-1 antibody is administered.

[0102] The amount of bispecific anti-MUC16 / anti-CD3 antibody and optionally anti-PD-1 antibody contained in a single dose can be expressed in milligrams of antibody per kilogram of subject body weight (i.e., mg / kg). In certain embodiments, the bispecific anti-MUC16 / anti-CD3 antibody used in the methods of the present invention, and optionally anti-PD-1 antibody, can 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 can be administered at a dose of about 0.1 mg / kg to about 20 mg / kg of patient body weight, and optionally the anti-PD-1 antibody can be administered at a dose of about 0.1 mg / kg to about 20 mg / kg of patient body weight.

[0103] A summary of the sequences cited herein and the corresponding SEQ ID NOs is shown in Table 1 below.

[0104] Table 1: Sequence summary

[0105]

[0106]

[0107] Example

[0108] The following examples are provided to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0109] Example 1: Generation of bispecific antibodies that bind ovarian cell specificity (MUC16) and CD3

[0110] The present invention provides bispecific antigen binding molecules that bind to CD3 and MUC16; such bispecific antigen binding molecules are also referred to herein as "anti-MUC16 / anti-CD3 or anti-MUC16×CD3 bispecific molecules". The anti-MUC16 portion of the anti-MUC16 / anti-CD3 bispecific molecule is used to target tumor cells expressing MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is used to activate T cells. The binding of MUC16 to tumor cells and CD3 to T cells promotes the directed killing (cell lysis) of targeted tumor cells by activated T cells.

[0111] Bispecific antibodies comprising an anti-MUC16 specific binding domain and an anti-CD3 specific binding domain are constructed using standard methods, wherein the anti-MUC16 antigen binding domain and the anti-CD3 antigen binding domain each comprise a different, unique pair of HCVRs with a common LCVR. In an exemplary bispecific antibody, the molecule is constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a common light chain from an anti-MUC16 antibody. In other cases, bispecific antibodies can be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody, or an antibody light chain that is known to be promiscuous or effectively paired with a number of heavy chain arms.

[0112] As described in U.S. Patent Application Publication No. US20140243504A1, published on August 28, 2014, exemplary bispecific antibodies having an IgG1 Fc domain (BSMUC16 / CD3-001, -002, -003, and -004) or a modified (chimeric) IgG4 Fc domain (BSMUC16 / CD3-005) were made.

[0113] A summary of the components of the antigen-binding domains of the various anti-MUC16×CD3 bispecific antibodies constructed is shown in Table 2 .

[0114] Table 2: Summary of components of anti-MUC16×CD3 bispecific antibodies

[0115]

[0116] Example 2: CA-125 does not interfere with anti-MUC16×CD3 antibody activity in vitro

[0117] FACS binding and cytotoxicity assays were used to evaluate the effect of soluble CA-125 (a shed form of MUC16) on the activity of BSMUC16 / CD3-001 in the presence of high levels of CA-125 purified from the ascites of ovarian cancer patients. CA-125 levels are elevated in the serum of most ovarian cancer patients, and its circulating levels could affect any therapy targeting MUC16 by acting as an antigen silencer. The CA-125 level used in the assay (10,000 U / ml) greatly exceeds the published median level of 656.6 U / mL in ovarian cancer patients. The ability of BSMUC16 / CD3-001 to kill OVCAR-3 cells expressing MUC16 was assessed in the presence of soluble CA-125 enriched from human ascites (creative Biomart, NY, USA) or a membrane proximal construct expressing the five carboxyl-terminal SEA domains and the membrane-proximal region of MUC16 (MUC16Δ) at a 4:1 effector / target ratio and a fixed concentration of BSMUC16 / CD3-001 or CD3-binding control antibody (100 pM) and serial dilutions of MUC16-1H or MUC16Δ at 37°C for 72 h. To monitor specific killing of target cells carrying MUC16, OVCAR-3 cells were labeled with 1 μM purple cell tracer. After labeling, cells were plated overnight at 37°C. In addition, human PBMCs were plated at 1 × 10 6 Cells / mL were plated in supplemented RPMI medium and incubated overnight at 37°C to enrich lymphocytes by eliminating adherent cells. The next day, target cells were incubated at 37°C for 72 hours with initial PBMCs (effective / target cell ratio 4:1) eliminated from adherent cells and serially diluted BSMUC16 / CD3-001 or CD3 binding controls. Cells were removed from the cell culture plate using trypsin and analyzed by FACS. For FACS analysis, cells were stained with dead / live far-red cell tracers (Invitrogen). To assess the specificity of killing, cells were gated on a population labeled with a purple cell tracer. The percentage of live target cells used to calculate adjusted survival is reported as follows: adjusted survival = (R1 / R2) * 100, where R1 =% live target cells in the presence of antibody, R2 =% live target cells in the absence of test antibodies. T cell activation was assessed by incubating cells with directly conjugated antibodies to CD2, CD69, and CD25 and reporting the percentage of activated (CD69+) T cells or (CD25+) T cells over total T cells (CD2+).

[0118] Binding of BSMUC16 / CD3-001 and antibodies known to bind CA-125 (clone 3A5) to CA125 obtained from human ascites was determined 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 96-well microtiter plates overnight at 4°C. The plates were then washed with PBST and blocked for 1 hour with 0.5% BSA in PBS. Biotinylated BSMUC16 / CD3-001, MUC16 parental antibody, α-MUC16 3A5, and non-binding controls (BSMUC16 / CD3-001 isotype control and α-MUC16 3A5 isotype control) were added to the plate at a concentration of 10, 1, 0.3, or 0.1 nM in 0.5% BSA in PBS within 1 hour, followed by washing with PBST. Streptavidin conjugated to horseradish peroxidase (SA-HRP) (ThermoFisher Scientific, Waltham, MA, USA) was added to the wells at a 1:10000 dilution in a 1.0 mg / mL stock solution 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 to the manufacturer's instructions. The absorbance of each well at 450 nm was recorded using a Victor multilabel plate reader (Perkin Elmer; Melville, NY). Data analysis was performed using GraphPad Prism software.

[0119] Excess CA-125 had minimal effect on the binding of BSMUC16 / CD3-0001 to OVCAR-3 cells, indicating that minimal binding to CA-125 ( Figure 1 In contrast, CA-125 greatly inhibited the ability of a comparable antibody that could bind to the repeat region of MUC16 (internal form of antibody clone 3A) ( Figure 1). In addition, a soluble MUC16 construct containing the membrane proximal region up to the 5th SEA domain of MUC16 (MUC16Δ) significantly inhibited the binding of BSMUC16 / CD3-001, indicating that BSMUC16 / CD3-001 binds to the membrane proximal region, as discussed in more detail in WO 2018 / 067331, which is incorporated herein by reference. Consistent with the binding studies, BSMUC16 / CD3-001 was also able to induce T cell-mediated killing in the presence of CA-125, but not in the presence of high concentrations of MUC16Δ (data not shown). Therefore, BSMUC16 / CD3-001 can bind to MUC16 and induce T cell redirected killing even in the presence of high concentrations of CA-125.

[0120] Example 3: PD-1 blockade enhances the anti-tumor activity of anti-MUC16×CD3 bispecific antibodies in xenogeneic and syngeneic tumor models

[0121] The in vivo efficacy of blockade of anti-MUC16 / anti-CD3 bispecific antibodies in combination with PD-1 was evaluated in xenogeneic and syngeneic tumor models.

[0122] A. Xenogeneic Model – OVCAR-3 / Luc

[0123] For xenogeneic models, thirteen days after implantation of human PBMC, immunodeficient NSG mice were intraperitoneally (IP) injected with OVCAR-3 / Luc cells previously passaged in vivo (day 0). Mice were treated intraperitoneally with 12.5 μg / mouse BSMUC16 / CD3-001, or 12.5 μg CD3 alone or in combination with 100 μg REGN2810 was administered on days 5 and 8 in combination with controls. Tumor burden was assessed by BLI on days 4, 8, 12, 15, 20, and 25 after tumor transplantation. As determined by BLI measurements on day 25, treatment with 12.5 μg BSMUC16 / CD3-001 can produce significant anti-tumor efficacy determined by BLI measurements, and the combination with REGN2810 (anti-PD-1) can further enhance anti-tumor efficacy. Before dosing began, the tumor burden of all groups was similar to that assessed by BLI. There was no significant difference in tumor burden between the groups.

[0124] BSMUC16 / CD3-001 significantly reduced tumor burden at 12.5 μg, and the addition of anti-PD-1 enhanced anti-tumor efficacy compared to BSMUC16 / CD3-001 alone. Human OVCAR-3 / Luc cells were transplanted into NSG mice transplanted with human T cells. On days 5 and 8, mice were treated with 12.5 μg BSMUC16 / CD3-001 administered intravenously, or with CD3 binding controls or non-binding controls (12.5 μg IV). The data shown in Table 3 below are tumor burdens assessed by BLI on day 25 after tumor transplantation. Unpaired nonparametric Mann-Whitney t test was used to determine statistical significance. Treatment with BSMUC16 / CD3-001+ / -REGN2810 was compared to CD3 binding control (*p<0.05 for BSMUC16 / CD3-001, **p<0.01 for BSMUC16 / CD3-001 and REGN2810), and treatment with BSMUC16 / CD3-001 alone was compared to the combination of REGN2810 (#p<0.05).

[0125] Table 3: Bioluminescence on day 25 after tumor transplantation

[0126]

[0127] B. Syngeneic Model – ID8-VEGF / huMUC16

[0128] To examine the efficacy of the immunocompetent model, the mouse CD3 gene was replaced with human CD3, and a portion of the mouse MUC16 gene was replaced with a human sequence. This replacement resulted in mice whose T cells expressed human CD3 and a chimeric MUC16 molecule containing a portion of human MUC16 to which the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies bound.

[0129] For this first syngeneic tumor model, an ID8-VEGF cell line engineered to express a portion of human MUC16 was used. ID8-VEGF / huMUC16 cells were transplanted intraperitoneally into mice and treated three days after transplantation with 5 mg / kg BSMUC16 / CD3-001 or a combination of a CD3 binding control and an isotype control or with anti-PD-1 (5 mg / kg IV). Treatment with BSMUC16 / CD3-001 prolonged median survival compared to the group receiving the CD3 binding control, but the addition of anti-PD-1 blockade also resulted in 50% survival of the mice.

[0130] BSMUC16 / CD3-001 significantly increased the median survival time in the ID8-VEGF ascites model, and the addition of PD-1 (REGN2810) blockade allowed several mice to survive. Mice expressing human CD3 instead of mouse CD3 and chimeric MUC16 molecules were transplanted with a murine ovarian tumor line expressing a portion of human MUC16. On day 3 after transplantation, mice were administered BSMUC16 / CD3-001 (5 mg / kg IV), or a CD3 binding control (5 mg / kg IV) with an isotype control or anti-PD-1. Mice were treated on days 3, 7, 10, 14, and 17 after tumor transplantation. Data shown are median survival rates. Mice were sacrificed when they gained more than 20% body weight due to abdominal distension caused by ascites. The Mantel-Cox method was used to determine statistical significance. Both BSMUC16 / CD3-001 and BSMUC16 / CD3-001+anti-PD-1 treatments increased median survival time, and the combination of BSMUC16 / CD3-001+anti-PD-1 produced 50% survival, indicating a synergistic effect between the MUC16×CD3 bispecific antibody and the anti-PD-1 antibody. The results are shown in Table 4 below.

[0131] Table 4: Median survival of the ID8-VEGF / huMUC16 model

[0132]

[0133] Similar results were also observed when BSMUC16 / CD3-001 was administered at 1 mg / kg in combination with anti-PD-1 antibody.

[0134] C. Syngeneic Model – MC38 / huMUC16

[0135] As discussed above, the mice used in this experiment were engineered so that the mouse CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with a human sequence. This replacement resulted in mice whose T cells expressed human CD3 and expressed chimeric MUC16 molecules containing a portion of human MUC16 to which the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies bound.

[0136] For this second homologous tumor model, an MC38 line engineered to express a portion of human MUC16 was used. MC38 / huMUC16 cells were transplanted subcutaneously into mice and treated with a combination of BSMUC16 / CD3-005 or CD3 binding control with an isotype control (1 mg / kg IV) or with anti-PD-1 (5 mg / kg IV) on day 7 post-transplant. 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.

[0137] In the MC38 subcutaneous model, the combination of BSMUC16 / CD3-005 and anti-PD-1 blockade produced better anti-tumor efficacy than BSMUC16 / CD3-005 alone. Mice expressing human CD3 instead of mouse CD3 and chimeric MUC16 molecules were transplanted with the murine tumor line MC38, which expresses a portion of human MUC16. On day 7 after transplantation, mice were administered BSMUC16 / CD3-005, or a CD3 binding control (1 mg / kg IV) with an isotype control or anti-PD-1 antibody (5 mg / kg IV). Mice were treated on days 7, 11, and 14 after tumor transplantation. The results are shown in Figure 2 Two-way ANOVA and Tukey's multiple comparison test were used to determine statistical significance. BSMUC16 / CD3-005 plus anti-PD-1 significantly and synergistically inhibited tumor growth over CD3-bound controls.

[0138] Example 4: Immuno-PET imaging of engineered mice shows localization of anti-MUC16×CD3 bispecific antibodies to T cell-rich organs

[0139] PET imaging was used to assess the in vivo localization of BSMUC16 / CD3-001 and BSMUC16 / CD3-005 and the expression of MUC16 protein in wild-type and genetically humanized mice. 89 The biodistribution of Zr-labeled anti-MUC16 antibody (a bivalent anti-MUC16 antibody generated using the same anti-MUC16 heavy and light chains as the bispecific antibody, referred to herein as "parent") in both wild-type and humanized mice was similar, suggesting that humanized MUC16 protein expression / availability for the antibody was low. In contrast, when mice were administered therapeutically relevant doses of 89 When the Zr-labeled BSMUC16 / CD3-001 bispecific antibody was used, the bispecific antibody was significantly distributed to the spleen and lymph nodes due to recognition by CD3-positive T cells in these lymphoid organs (data not shown). Ex vivo biodistribution analysis in separate tissues confirmed localization in the lymph nodes and spleen (data not shown).89 The Zr-labeled BSMUC16 / CD3-005 bispecific antibody has a lower affinity for CD3, so the absorption of this bispecific antibody in lymphatic tissues is greatly reduced compared with BSMUC16 / CD3-001. To evaluate whether BSMUC16 / CD3-001 and BSMUC16 / CD3-005 can accumulate in tumors expressing MUC16, mice bearing ID8-VEGF-huMUC16 tumors were administered 89 Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005. ΔAlthough lymphatic uptake of BSMUC16 / CD3-001 was higher, there was no significant difference in tumor uptake between the bispecific antibodies (data not shown).

[0140] Preparation of immunoconjugates and small animal PET : After deglycosylation of the antibody with N-glycoprotein deglycosylase (PNGase F), BSMUC16 / CD3-001 and control antibodies were conjugated to the glutamine residue at position 295 by DFO through transamidation by microbial transglutaminase. The DFO-conjugated antibody was then conjugated to zirconium-89 ( 89 The mice received a final dose of 0.5 mg / kg of the antibody via tail vein injection. PET imaging was then performed to assess the in vivo localization of the radioimmunoconjugate on day 6 after administration, followed by ex vivo biodistribution studies. For experiments with tumor-bearing mice, 10×10 6 ID8-VEGF-huMUC16Δ tumor cells. When the average tumor size reached 150mm 3 At 20 days after transplantation, tumor-bearing mice received 89 Zr radiolabeled antibodies.

[0141] PET and CT images were acquired using a pre-calibrated Sofie Biosciences G8 PET / CT instrument (Sofie Biosciences (Culver city, CA) and Perkin Elmer). The energy window was in the range of 150 to 650 keV, and the reconstruction resolution at the center of the field of view was 1.4 mm. On the 6th day after administration, mice were induced anesthetized using isoflurane and kept under a continuous flow of isoflurane during a 10-minute static PET acquisition. CT images were obtained after PET acquisition. PET images were subsequently reconstructed using preconfigured settings. Attenuation-corrected PET data and CT data were processed into pseudo-color-aligned PET-CT maximum intensity projections on a color scale using VivoQuant software (inviCRO Imaging Services) to represent a signal range of 0 to 30% of the injected dose per volume (expressed as %ID / g). For in vitro biodistribution analysis, mice were euthanized after imaging on the 6th day after administration. Blood was collected in a counting tube by cardiac puncture. Then normal tissues (inguinal and axillary lymph nodes, thymus, spleen, heart, lung, stomach, small intestine, liver, kidney, bone and ovary) are excised and placed in counting tubes. Similarly, tumors are collected in counting tubes. All tubes have been weighed in advance and then weighed again to determine the weight of blood and tissue. The γ-emission radioactivity of all samples is then calculated on an automatic gamma counter (Wizard 2470, Perkin Elmer), and the results are reported in counts per minute (cpm). The %ID of each sample is determined using sample counts relative to the dose standard counts prepared from the original injection material. Subsequently, a single %ID / g value is obtained by dividing the respective weight of appropriate blood, tissue or tumor sample with %ID values.

[0142] 89 Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005 showed specific localization to MUC16+ tumors and CD3+ lymphoid tissues, with lymphoid distribution correlating with relative CD3 affinity. In the presence of CD3+ tissues, both MUC16×CD3 bispecific antibodies showed equivalent tumor localization.

[0143] Example 5: Toxicology studies in cynomolgus monkeys showed that the anti-MUC16×CD3 bispecific antibody had no obvious toxicity

[0144] BSMUC16 / CD3-001 cross-reacts with monkey MUC16 and CD3. In order to determine safety and tolerability and characterize the pharmacokinetics of the bispecific antibody, a multiple-dose toxicity study was conducted in cynomolgus macaques. Six monkeys of each sex in each group received weekly administration of BSMUC16 / CD3-001, receiving a total of five doses of 0.01, 0.1 or 1 mg / kg. At the end of the dosing period, three animals of each sex in each group were euthanized and examined by microscopy, while the remaining three animals of each sex in each group were subjected to a 12-week treatment-free recovery period to assess the reversibility or persistence of any BSMUC16 / CD3-001-related effects. BSMUC16 / CD3-001 was well tolerated, and all animals survived until the scheduled autopsy time. Toxicokinetic analysis showed that the exposure of each dose group was proportional to the dose, and the kinetics were linear, and no gender differences were observed (data not shown). Continuous exposure to BSMUC16 / CD3-001 was observed throughout the dosing period, and in the 0.1 and 1.0 mg / kg groups, BSMUC16 / CD3-001 exposure was maintained until the end of the recovery period in all (n=6) and 50% of the animals, respectively. After the 8th week of the recovery period, BSMUC16 / CD3-001 was not detected in the serum of any animal in the 0.01 mg / kg group. The elimination half-life of BSMUC16 / CD3-001 is approximately 10 days.

[0145] No BSMUC16 / CD3-001-related clinical observations were found during the dosing or recovery period, and there were no changes in urinalysis parameters, peripheral blood immunophenotyping, food consumption or body weight. Importantly, BSMUC16 / CD3-001 administration did not result in any changes in respiratory, neurological or cardiovascular safety pharmacology evaluations, including no changes in ECG parameters. No BSMUC16 / CD3-001-related organ weight changes were found in terminal or recovery autopsies, and no macroscopic changes were recorded. Dose-related, reversible increases in circulating inflammatory markers (C-reactive protein (CRP) and IL-6) were observed within 1 day after the initial dose of 1.0 or 0.1 mg / kg, but these increases were not obvious after subsequent doses (data not shown). Based on minimal increases in serum cytokines, no T cell redistribution was detected after BSMUC16 / CD3-001 administration (data not shown), which is contrary to the description of several CD3 bispecific molecules for hematological tumors.

[0146] The cynomolgus macaque study was conducted according to the guidelines of IACUC. A control (diluted placebo) or BSMUC16 / CD3-001 (0.01, 0.1 or 1 mg / kg) was administered to cynomolgus macaques (6 animals per sex per group) once a week by 30-minute intravenous infusion. The control was 10 mM histidine, 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 different time points for clinical pathology and histopathology. BSMUC16 / CD3-001 concentration was determined by ELISA, and WinNonLin software was used for toxicokinetic analysis. CRP was analyzed on a Roche Modular P 800 system. Cytokines were measured by MSD (Meso Scale Diagnostics, Rockville, MD). T cells were quantified using flow cytometry. Briefly, blood was collected in EDTA tubes, lysed, stained for CD3, CD4, and CD8 (BD Biosciences), and the relative values ​​of each phenotype were determined using FACSCanto II. These values ​​were then multiplied by the absolute lymphocyte values ​​(obtained by hematological analysis) to calculate the absolute cell counts for each phenotype.

[0147] Immunohistochemical staining for MUC16 was present in the expected tissues: pancreas (mesothelium, ductal epithelium), heart and ovary (data not shown), as well as salivary glands (goblet cells), liver (mesothelium, bile ducts), lungs (mesothelium, bronchiolar / bronchial epithelium), small intestine (mesothelium), testes (mesothelium, rete testis / ductus efferentus), and tonsils (epithelium, mucous glands) (not shown). BSMUC16 / CD3-001-associated microscopic changes, assessed by hematoxylin and eosin (H&E) histological staining, included inflammation (leukocyte infiltration) and increased mesothelial cell size and cellularity, which resulted in non-adverse thickening of the serosal lining and / or submesothelial connective tissue of multiple thoracic and peritoneal organs. These changes were generally focal or multifocal in nature, minimal to mild in severity, and were believed to be a hit target for BSMUC16 / CD3-001, a result of engagement of MUC16 expressed on serosal epithelial (mesothelial) cells and activation of T cells. Importantly, at the end of the recovery period, serosal changes were reversed or tended to be reversed (data not shown).

[0148] Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after BSMUC16 / CD3-001 administration, but no overt toxicity.

[0149] Example 6: Assessment of serum cytokine induction in tumor-bearing mice

[0150] Since cytokine release syndrome (CRS) is a common and severe side effect of CD3 bispecific and CAR T-cell therapies, studies were performed to monitor serum cytokines in relevant models following BSMUC16 / CD3-001 treatment. In tumor-free genetically humanized MUC16 / CD3 mice, no serum cytokine response was seen following BSMUC16 / CD3-001 administration.

[0151] In order to evaluate the in vivo T cell activation carried out by BSMUC16 / CD3-001, the serum cytokine levels of tumor-bearing mice were measured. In 0.5mg / kg BSMUC16 / CD3-001, CD3 binding control and non-binding control group, serum samples were collected 4 hours after the first antibody dose. Compared with non-binding control and CD3 binding control, the treatment of BSMUC16 / CD3-001 activates T cells, as determined by the induction of IFNγ, TNFα, IL-2, IL-6, IL-8 and IL-10 (data not shown). The cytokine response induced by BSMUC16 / CD3-001 requires the presence of both T cells and OVCAR-3 / Luc cells, because the mice carrying only OVCAR3 / Luc cells do not have detectable human IFNγ in serum, and the mice without tumor cells provided for cross-linked MUC16 do not show the increase (data not shown) of serum IFNγ in response to BSMUC16 / CD3-001.

[0152] Measurement of serum cytokine levels: Four hours after the first 0.5 mg / kg dose, T cell activation in response to treatment with BSMUC16 / CD3-001 was assessed by measuring serum concentrations of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-2 (IL-2), IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and IL-1B. Cytokine levels were analyzed using the V-plex human proinflammatory-10Plex ​​kit according to the manufacturer's instructions (Meso Scale Diagnostics, Rockville, MA). Cytokines were measured in two independent studies of 4-6 mice per group.

[0153] Example 7: MUC16 expression in humanized mice and the effects of anti-MUC16×CD3 bispecific antibodies on MUC16-positive tissues

[0154] In order to study the anti-tumor efficacy of BSMUC16 / CD3-001 in mice with a complete immune system, mice were genetically engineered to express human CD3 on T cells and MUC16 regions covering antibody binding regions, both of which are in endogenous mouse loci (knock-in mice). In order to verify these mice, MUC16 expression was checked by both RT-PCR and IHC. RNA expression was detected in the trachea, and low levels of RNA expression were also detected in the lungs, heart, ovaries, pancreas and bladder (data not shown), which is similar to the published mouse MUC16 expression data. In order to evaluate MUC16 protein expression, anti-human MUC16 antibodies that recognize the proximal region of the membrane of MUC16 were used to perform IHC on selected tissues. MUC16 protein expression was confirmed in the surface epithelium of the ovaries and stomachs of these mice. MUC16 was also observed in the tracheal lining / epithelium and submucosal glands, as described in humans (data not shown).

[0155] Histological Analysis of Mouse Tissues: Tissues from humanized or wild-type mice were harvested and stained by IHC with an anti-MUC16 antibody that binds to the membrane proximal domain of MUC16 using Ventana Discovery XT (Ventana; Tucson, AZ). 5 μm paraffin sections were cut onto Superfrost PLUS slides and baked at 60°C for one hour. Immunohistochemical staining was performed on the Discovery XT automated IHC staining system using the Ventana DAB Map detection kit. Dewaxing was performed at 75°C for 8 minutes using EZ Prep solution. Mild antigen retrieval (95°C, 8 minutes, then 100°C, 24 minutes) was performed using Tris-EDTA buffer pH 9 (CC1) from Ventana. Multiple blocking steps were then performed. Tissue sections were incubated with anti-MUC16 antibody (2 μg / ml) at room temperature for 8 hours. An isotype control antibody that recognizes an irrelevant non-binding antibody was used as a negative control. Primary antibodies and negative controls were applied manually. Biotinylated goat anti-human IgG (Jackson ImmunoResearch) was used as a secondary antibody (1 μg / ml), and samples were incubated for one hour at room temperature. The chromogenic signal was generated using the Ventana DAB MAP kit. Slides were manually counterstained with hematoxylin (2 minutes), dehydrated, and coverslipped. Images were acquired on an Aperio AT 2 slide scanner (Leica Biosystems; Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs; Corrales, NM). H&E staining was performed by Histoserv, Inc (Germantown, MD, USA).

[0156] As assessed by T cell receptor (TCR) Vβ use, T cells in these mice are polyclonal, express human CD3, and are similar in number to wild-type mice (data not shown). To determine whether BSMUC16 / CD3-001 induces any T cell activation or has an effect on normal tissues in these animals, non-tumor-bearing mice were injected with a high dose of BSMUC16 / CD3-001 (10 mg / kg), and then the number of T cells in the blood, serum cytokines, and tissue pathology were examined. Although T cells can be activated by anti-human CD3 antibodies (OKT3), as measured by the marginalization of T cells from the blood and an increase in serum cytokine levels (data not shown), BSMUC16 / CD3-001 does not induce any such effects, indicating that the accessibility of the MUC16 target is limited (data not shown). To determine whether BSMUC16 / CD3-001 induces any microscopic changes in MUC16-expressing tissues, MUC16 and CD3 humanized mice received two doses of 10 mg / kg of BSMUC16 / CD3-001 on days 0 and 3. On day 5, several MUC16-expressing tissues (trachea, stomach, and ovaries) were examined, and no cellular infiltration or necrosis was observed in these tissues after BSMUC16 / CD3-001 administration (data not shown).

[0157] Histopathological examination revealed no inflammation or infiltration into MUC16-expressing tissues in mice following BSMUC16 / CD3-001 administration at the time of examination.

[0158] The results of this study, as well as the cynomolgus macaque study discussed in Example 5, demonstrate the safety profile of BSMUC16 / CD3-001. BSMUC16 / CD3-001 only minimally induced serum cytokines, and although there was focal induction of inflammation and thickening of the serosal lining in MUC16 expression, suggesting on-target activity, these effects disappeared by the end of the recovery period and were consistent with increased inflammation and cellularity indicative of repair. The observed serosal changes were not associated with any clinical observations, clinical pathology (other than the inflammatory response), or microscopic changes in the underlying parenchyma. Thus, studies in both genetically humanized mice and cynomolgus macaques indicate that BSMUC16 / CD3-001 is well tolerated.

[0159] Example 8: Monitoring PD-1 expression in a FACS-based cytotoxicity assay using naive human effector cells

[0160] To monitor specific killing of target cells carrying Muc16 by flow cytometry, the ovarian cell line OVCAR-3 was labeled with 1 μM purple cell tracer. After labeling, cells were plated overnight at 37°C. In addition, human PBMCs were plated at 1×10 6 Cells / mL were plated in supplemented RPMI medium and incubated overnight at 37°C to enrich lymphocytes by eliminating adherent macrophages, dendritic cells and some monocytes. The next day, target cells were incubated at 37°C for 72 hours with initial PBMCs (effect / target cells 4:1) eliminated from adherent cells and serially diluted BSMUC16 / CD3-001 or CD3 binding controls. Cells were removed from the cell culture plates using trypsin and analyzed by FACS. For FACS analysis, cells were stained with dead / live far-red cell tracers (Invitrogen). To assess the specificity of killing, cells were gated on the population labeled with purple cell tracers.

[0161] PD-1 expression was assessed by incubating cells with directly conjugated antibodies to CD2, CD4, CD8, and PD-1, reporting the percentage of PD-1 / CD4 positive T cells or PD-1 / CD8 positive T cells to 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 presented in Figure 3 Shown in.

[0162] Example 9: Methods of treating epithelioid sarcoma using an anti-MUC16×anti-CD3 bispecific antibody alone or in combination with an anti-PD-1 antibody

[0163] A patient with relapsed epithelioid sarcoma who had exhausted all available treatment options was treated with REGN4018 (anti-MUC16 × anti-CD3 bispecific antibody).

[0164] In this study, treatment assessment was based on tumor response. Tumor response assessment was based on levels of the tumor marker CA-125 in patients with and without measurable disease. In addition, biomarker analysis included peripheral T cell phenotyping, as REGN4018 treatment is expected to transiently reduce the population of peripheral CD3 T cells. Further analyses will include biomarkers, such as tumor expression of proteins such as MUC16 and PD-L1, and may include genetic analysis of tumors (RNA and somatic DNA sequencing) of ctDNA, variants that influence the clinical course of the underlying disease or modulate treatment side effects.

[0165] Objectives: Exploratory objectives will be explored.

[0166] Research Exploratory goals yes:

[0167] Collection of blood for cytokine profiling, pharmacokinetics, and tumor biopsy is optional. If available, these samples will be used for the following exploratory objectives:

[0168] (1) Evaluate biomarkers that may be associated with mechanism of action, improved understanding of the disease / target, observed toxicity, and potential anti-tumor activity, including but not limited to:

[0169] - Circulating proteins, including cytokines

[0170] - Changes in gene expression in tumors

[0171] - Tumor expression levels of proteins such as MUC16.

[0172] Patient Demographic Information:

[0173] -Age: 20

[0174] -Gender: Female

[0175] Diagnosis: The patient had multiple recurrent metastatic epithelioid sarcomas with chest wall and lung lesions. Immunohistochemistry showed that tumor specimens expressed MUC16, while CA-125 (the shed extracellular portion of MUC16) was elevated in serum samples.

[0176] History of disease progression to date: The patient was initially diagnosed with epithelioid sarcoma of the right radius. She received radiation therapy to the right upper extremity (50.4 Gy) and local surgery with negative margins. The first recurrence was associated with mid-arm lymph node recurrence, which was treated with surgical resection and further radiation therapy (54 Gy). A second recurrence of PET-high-absorption lymph node disease was detected in the axilla and was treated surgically. A second recurrence of disease was present in the right elbow within the previously irradiated area. The patient was treated with tazemetostat in an earlier trial, but treatment was discontinued due to tolerability and compliance issues. There was evidence of further disease progression in the arm and the development of pulmonary nodules, so the patient was treated with pembrolizumab. She had a prolonged period of stable disease before treatment was discontinued due to disease progression. The patient was subsequently treated with nivolumab / ipilimumab and underwent palliative amputation of the right forelimb due to worsening tumor-related ulceration. Disease evaluation showed stable chest disease, but immune checkpoint inhibitor therapy was discontinued due to immune-related pulmonary inflammation. The patient was treated again with tazemetostat, but treatment was discontinued due to disease progression. She was subsequently treated with CLR-131 (molecular targeted radiation therapy); however, the disease progressed further. Recently, the patient received radiation therapy to the right breast (5 fractions, 20 Gy each) to relieve the ulcerative skin lesions.

[0177] The patient currently has evidence of disease in the chest wall / breast tissue bilaterally and multiple measurable lesions in both lungs. Her clinical status is stable with a Karnofsky score of 70 (primarily due to amputation). In this setting, there are currently no available clinical trial options or alternative 'standard' therapies for her disease.

[0178] Current Study Design and Treatment Programs: REGN4018 is an investigational agent currently being evaluated in a Phase 1 / 2 study in adults with ovarian, fallopian tube, or primary peritoneal cancer who are platinum-treated and / or platinum-intolerant.

[0179] REGN4018 will be administered weekly by intravenous infusion over 4 hours (including washout). Treatment will continue (once a week) for 6 weeks (42 days) in a cycle. During the 1st cycle, patients receive increasing doses of REGN4018 until the full dose shown in the table below is reached. The initial dose of the drug is given in divided doses on consecutive days (see below). Any dose can be divided at any time point to improve tolerability.

[0180] Table 5: Dosing regimen for REGN4018 therapy

[0181]

[0182] Rationale for dose selection – REGN4018 is an investigational drug currently being evaluated in a Phase 1 / 2 study in adults. Patients with this SPS are being treated at a dose level one dose lower (i.e., 250 mg, which is the highest dose level that has been demonstrated to be tolerated) than the dose currently being evaluated in the monotherapy cohort in the Phase 1 / 2 study (450 mg). The dose can subsequently be adjusted at the physician’s discretion based on new information in the hope of providing clinical benefit to the patient.

[0183] REGN4018 Administration procedures – REGN4018 drug product is provided as a lyophilized single-use product at 5 mg / vial and / or 50 mg / vial for administration by intravenous infusion. REGN4018 will be administered intravenously weekly. REGN4018 will be administered by intravenous infusion over 4 hours (including washout). Patients must be observed in a monitored setting for at least 24 hours for the following doses / conditions:

[0184] a) Cycle 1

[0185] - Cycle 1 Day 1 Initial Dose

[0186] - Cycle 1 Day 8 / 9 split transition dose

[0187] - First full dose in divided doses on Day 15 / 16 of Cycle 1

[0188] - Cycle 1 Day 22 250 mg first dose

[0189] b) Escalate to the new highest tolerated dose level.

[0190] c) Observation of ≥Grade 2 cytokine release syndrome (CRS) adverse events occurring after any subsequent dose of REGN4018

[0191] d) Restarting study drug after a prolonged period of discontinuation (e.g., stopping study drug for more than 21 days).

[0192] During the inpatient observation period, hospitalization alone is not sufficient to classify an adverse event as serious. Prolonged hospitalization (beyond the protocol-specified 24 hours) due to an adverse event will be considered a serious condition.

[0193] If divided doses are given, the second day of monitoring should follow the same guidelines as the first day of infusion. For Cycle 1 Day 9, Cycle 1 Day 16, Cycle 1 Day 23 (if applicable), it should be 24 hours.

[0194] After the patient tolerates the first full dose, the patient may subsequently receive study therapy in an outpatient infusion unit, and the investigator may elect to shorten the duration of the REGN4018 infusion (including washout) to 3 hours, followed by well-tolerated weekly REGN4018 infusions shortened to 2 hours, 1 hour, and 30 minutes, respectively. REGN4018 will be administered intravenously in an inpatient or day hospital setting, with immediate access to an acute care hospital unit.

[0195] No premedication was required and was at the investigator's discretion.

[0196] Dose escalation - Based on updated information from ongoing Phase 1 / 2 studies, dose escalation / decrease may be implemented at the investigator's discretion. 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.

[0197] Dosage Considerations - Dose adjustments / reductions of REGN4018 may be implemented at the investigator's discretion and in the best interest of the patient. Adverse events (AEs) will be treated symptomatically.

[0198] Duration of Therapy: In REGN4018 therapy, each cycle will be 6 weeks (42 days). Treatment will continue until unacceptable toxicity, lack of clinical benefit, patient withdrawal of consent, physician discretion (e.g., based on intolerable adverse reactions, quality of life, new treatment options), or lack of drug supply. Due to disease progression, if the patient is able to tolerate treatment and the disease has not progressed, and in the opinion of the investigator, the patient has a clinical benefit from continued study treatment; treatment may be continued at the discretion of the physician. Clinical benefit will be objectively assessed at the end of the study or by planned imaging reassessments (and compared to baseline) as clinically indicated at regular intervals as outlined in this protocol.

[0199] If a clinical or radiological response occurs after at least 24 weeks of treatment, patients can choose to switch to a Q2W (every two weeks) dosing regimen of REGN4018 and continue all relevant study assessments. When disease progression subsequently occurs, patients can continue treatment at a dose level (including new dose levels, higher dose levels) selected by the investigator based on the patient's clinical benefit. Post-treatment follow-up will be performed within 30 days of the last dose and again at 90 days (±10 days) after the last dose to complete safety assessments at the end of the study.

[0200] Available safety data for REGN4018 therapy: A Phase 1 / 2 study of REGN4018 in adults with ovarian cancer is ongoing and patients are currently enrolled at a dose of 450 mg (full dose; level 8a). The previous dose level of 250 mg (dose level 7a) is the dose recommended in this SPS and has been shown to be tolerable. The currently available safety data (including treatment-emergent adverse events (TEAEs) and treatment-related TEAEs) are as follows:

[0201] Treatment-emergent adverse events – The most commonly reported TEAEs (all grades, ≥10% of patients) were: cytokine release syndrome (n=20; 69%); abdominal pain (n=18; 62.1%); nausea (n=13; 44.8%); back pain (n=10; 34.5%); anemia and fatigue (n=9 each; 31% each); vomiting (n=8; 27.6%); diarrhea and cough (n=7 each; 24.1% each) ); pyrexia and hypomagnesemia (n=5; 17.2% each); constipation, dyspnea, dry eyes, ocular discharge, loss of appetite, headache, hypotension, infusion-related reactions and dyspepsia, pleuritic pain (n=4 each; 13.8% each); fatigue, back pain, non-cardiac chest pain, musculoskeletal pain, ocular pruritus, ocular congestion, conjunctivitis, sinus tachycardia, and chills (n=3 each; 10.3% each). The incidence of TEAEs was highest during the first week of treatment and decreased in subsequent weeks.

[0202] Treatment-related TEAEs – Twenty-eight of 29 patients (96.6%) experienced at least one treatment-related TEAE assessed by the investigator during treatment. The most commonly reported treatment-related TEAEs assessed by the investigator (all grades, ≥10% of patients) 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 reactions, and pyrexia (n=4 each; 13.8% each); diarrhea, dyspepsia, cough, dry eyes, 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). No grade 4 or 5 treatment-related TEAEs occurred.

[0203] Potential for REGN4018 efficacy in the patient considered in this SPS: Immunohistochemistry confirmed that this patient's cancer expressed MUC16, a relevant target for REGN4018. Additionally, CA-125 (the shed extracellular portion of MUC16) was elevated in serum samples. A potential beneficial effect is that the study drug may cause the patient's cancer to stop growing or shrink for a period of time. It may reduce symptoms (such as pain) caused by the cancer.

[0204] Identified and Potential Risks: The following are important and potential risks of therapy based on the drug's safety profile and the patient's medical history:

[0205] Important risks identified: Infusion-related reactions and cytokine release syndrome – Infusion-related reactions (IRR) and cytokine release syndrome (CRS) are associated with typical signs and symptoms, which include but are not limited to flushing, tachycardia, hypotension, dyspnea, bronchospasm, back pain, fever, urticaria, edema, nausea, and rash.

[0206] Infusion-related reactions are common adverse drug reactions (ADRs) to monoclonal antibodies. Symptoms are temporally related to drug administration and can range from symptomatic discomfort to fatal events. Many reporting terms are used to identify IRRs, and although there is considerable overlap in symptoms, the etiology of each symptom is very different. These terms include anaphylaxis, anaphylactoid reactions, CRS, and complement activation-associated pseudoallergy (CARPA). The main reason for attempting to identify the etiology of symptoms and the actual diagnosis is to ensure that reactions that may recur or even worsen with subsequent exposure to the drug (e.g., anaphylaxis) are identified rather than reactions that may improve with repeated exposure (anaphylaxis and CRS).

[0207] IRR was defined as an adverse reaction occurring during the infusion and within 2 hours thereafter. In the context of this study, signs and symptoms of IRR occurring more than 2 hours after completion of the infusion were defined as CRS.

[0208] Clinically, CRS is a syndrome that includes symptoms of fever, hypotension, tachycardia, and hypoxia. In addition, neurological findings may occur, including delirium, encephalopathy, aphasia, lethargy, agitation, tremor, and seizures, which reflect the immune effector cell-associated neurotoxicity syndrome (ICANS).

[0209] Risk Mitigation Measures: IRR and CRS

[0210] 1) Use initial doses and transition doses that gradually approach full dose levels;

[0211] 2) inpatient monitoring for at least 24 hours during the initial dose increase and at other designated times;

[0212] 3) Infusion-related reactions and cytokine release syndrome should be managed with symptomatic treatment and supportive care according to institutional standards

[0213] Assessment of Drug Response: The safety and tolerability of REGN4018 in patients will be monitored by clinical assessment of adverse events and repeated measurements of clinical assessments including vital signs (temperature, blood pressure, pulse, oxygen saturation, and respiration), physical examination (full and limited), 12-lead electrocardiogram (ECG), echocardiogram, chest X-ray, and laboratory evaluations (including standard hematology, blood chemistry, and urinalysis).

[0214] Because cytokine release following initial dosing (first and / or subsequent doses) has been observed in bispecific antibodies and similar molecules, specific measures were implemented in this study. These measures included: 1 mg initial dose (day 1 of cycle 1), 20 mg transition dose (day 8 of cycle 1), split-dose options, the need for monitoring during dose selection, and the use of anti-IL-6 pathway therapies (e.g., tocilizumab) and corticosteroids for IRR / CRS management.

[0215] Tumor assessments will include all known or suspected sites of disease. Tumor assessments will be performed prior to the start of Cycle 1 (baseline), then prior to Cycle 2 (±7 days), and then prior to each even-numbered cycle (±7 days). Additional tumor assessments may be performed as clinically indicated. Tumor assessments should be repeated at the end of treatment (if applicable) if more than 2 cycles have passed since the last evaluation. Assessments will be performed according to institutional standards and will be compared to baseline measurements performed within 28 days of starting study therapy.

[0216] Due to MUC16 expression on the corneal and conjunctival epithelium, a baseline ocular examination is required.

[0217] Blood samples will be collected for drug concentration measurements and ADA assessments.

[0218] A brief neurological examination was required within 24 hours of study drug administration.

[0219] Serum and plasma samples will be collected for additional biomarker analysis. Exploratory predictive and pharmacodynamic biomarkers associated with REGN4018 treatment exposure, clinical activity, or underlying disease will be investigated from collected serum, plasma, whole blood, body fluids, archived tumor tissue, in-study tumor biopsies, tumor DNA (including circulating tumor DNA), and tumor RNA samples.

[0220] Antitumor activity will be evaluated by CT or MRI or PET-CT, and performance status and serum CA-125 levels will be monitored.

[0221] Permitted and prohibited concomitant therapies and rationales:

[0222] Prohibited and Discouraged Drugs – During participation in the current study, patients must not receive any standard or investigational oncology treatments other than REGN4018 monotherapy, but local palliative external beam radiation therapy is permitted.

[0223] Patients were advised not to receive systemic corticosteroids such as hydrocortisone, prednisone, or prednisolone at any time during the study. or dexamethasone Inhaled, topical, ophthalmic, or intranasal steroids are permitted except in the case of a life-threatening emergency and / or treatment of IRR / CRS that is unresponsive or expected to be unresponsive to anti-IL-6 pathway therapy (e.g., tocilizumab) or irAEs severe enough to require corticosteroids.

[0224] Permitted drugs- Local palliative treatment (e.g., radiation therapy) may be permitted. Pre-infusion medications such as antihistamines, acetaminophen, nonsteroidal anti-inflammatory drugs (e.g., ketorolac or ibuprofen), and / or oral opioids (e.g., hydromorphone) were at the discretion of the investigator. No specific pre-medication was mandated by the protocol.

[0225] Physiologic replacement doses of systemic corticosteroids are permitted, even if >10 mg / day of prednisone equivalents. Short-term use of corticosteroids is permitted for prevention (eg, contrast dye allergy) or treatment of nonautoimmune conditions (eg, delayed hypersensitivity reactions to contact allergens).

[0226] Any other medications considered necessary for the patient's health and not expected to interfere with the evaluation of the study drug may be administered at the investigator's discretion. Treatment of bone metastases (bisphosphonates, denosumab) and hormonal therapy were permitted.

[0227] Study endpoints:

[0228] The primary endpoints of the study were dose-limiting toxicities, treatment-emergent adverse events (TEAEs; including immune-related adverse events [irAEs]), serious adverse events (SAEs), deaths, laboratory abnormalities (grade 3 or higher according to CTCAE), and pharmacokinetics of therapy.

[0229] The secondary endpoint of the study was ORR based on response evaluation criteria in solid tumors (RECIST).

[0230] Preliminary Results: Safety and efficacy data from a single patient study of REGN4018 monotherapy in relapsed epithelioid sarcoma are presented below.

[0231] REGN4018 was administered weekly intravenously (IV) to patients with recurrent epithelioid sarcoma and elevated cancer antigen (CA)-125 levels at a dose range of 1–250 mg. An escalating dose of the first two doses was used to mitigate the risk of cytokine release syndrome (CRS) through a gradual increase in drug exposure. Primary endpoints included safety and pharmacokinetics. Secondary endpoints included efficacy as determined by objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.

[0232] Patients experienced grade 2 cytokine release syndrome on C1D2 and C1D9. Other treatment-emergent adverse events included grade 1 cough and grade 2 intermittent hypoxia, myalgia, constipation, pericardial and pleural effusions.

[0233] After treatment, the patient's CA-125 level decreased and the target lesions on CT scan according to RECIST v1.1 showed a partial response that lasted for 60 weeks, decreasing from a baseline summed diameter of 89 mm to 60 mm at cycle 4 imaging. In addition, the patient had a skin cancer ulcer on the right breast that decreased in size. These lesions improved significantly after REGN4018 treatment, resulting in almost complete wound healing, and the pain caused by such lesions was also significantly improved.

[0234] In this single-patient study, the patient tolerated REGN4018 and had signs of clinical activity as determined by ORR according to Response Evaluation Criteria in Solid Tumors (RECIST). Data from this analysis support the use of REGN4018 (anti-MUC16 × anti-CD3) bispecific antibody for the treatment of relapsed epithelioid sarcoma.

[0235] Treatment of Epithelioid Sarcoma by Combination of REGN4018 and Cemiplimab: It is expected that patients with recurrent epithelioid sarcoma can be administered a combination of an anti-PD-1 antibody (e.g., cemiplimab) and the REGN4018 bispecific antibody (i.e., anti-MUC16 x anti-CD3 antibody) to treat tumors.

[0236] Example 10: Semi-quantitative MUC16 immunohistochemical expression in different subtypes of childhood sarcomas

[0237] Semiquantitative MUC16 immunohistochemistry (IHC) expression in different subtypes of pediatric sarcomas was investigated. Seventy-two samples were collected from patients <25 years of age at Children's Hospital between 2015 and 2021, including epithelioid sarcoma (n=6), rhabdoid tumors of the kidney and soft tissue (n=4), rhabdomyosarcoma (n=23), Ewing's sarcoma (n=19), and other soft tissue sarcomas (n=19). IHC was performed on 4 μm thick formalin-fixed paraffin-embedded tissue sections of representative tissue blocks for each case or sample. The assay was performed on the DAKO Omnis staining platform using an anti-CA125 antibody (clone M11, monoclonal mouse, ready-to-use, DAKO, catalog number GA701) and the Envision Flex detection kit (DAKO, catalog number GV800). Appropriate positive and negative controls were used. Immunoreactivity was assessed using the H-score method.

[0238] Of the six epithelioid sarcoma samples selected for MUC16 IHC, three showed diffuse strong staining reactions (H-score of 300), one showed heterogeneous staining (H-score of 110), and the remaining two were negative (H-score of 0). Among the remaining tumor entities, MUC16 expression was present in two of the four kidney and soft tissue rhabdoid tumor samples (H-scores of 300 and 170, respectively), but was absent in the remaining 62 cases (H-score of 0).

[0239] MUC16 expression is a common feature of epithelioid sarcomas and may be seen in other INI-1 (SMARCB1)-deficient malignancies.

[0240] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, except 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.

[0241] Table 6: Sequences excluded from ST.26 format sequence listing

[0242] SEQ ID NO: sequence 12 TAS 39 AAS

[0243] *******

Claims

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

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, the method comprising administering to the subject a bispecific antibody comprising a first antigen binding domain that specifically binds to mucin 16 (MUC16) on a target tumor cell, and a second antigen binding domain that specifically binds to human CD3 on a T cell.

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

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

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

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

8. The method of any one of claims 1 to 7, wherein the subject is resistant to or has had an inadequate response to or has relapsed following a previous therapy.

9. The method of any one of claims 1 to 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) having the amino acid sequence of SEQ ID NO: 1; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

2.

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

10.

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

13.

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

2.

13. The method of any one of claims 1 to 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) having the amino acid sequence of SEQ ID NO: 3; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

2.

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

16.

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

13.

16. The method of any one of claims 13 to 15, wherein the second antigen binding domain comprises a HCVR having the amino acid sequence of SEQ ID NO: 3 and a LCVR having the amino acid sequence of SEQ ID NO:

2.

17. The method of any one of claims 1 to 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 of isotype IgGl.

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

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

21. The method of any one of claims 17 to 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 to 16, wherein the bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:

29.

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

31.

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

30.

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

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

27. The method of claim 26, wherein the second therapeutic agent or treatment regimen comprises an anti-PD-1 antibody or an 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) having the amino acid sequence of SEQ ID NO: 33; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein the three light chain complementarity determining regions are contained in a light chain variable region (LCVR) having 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 having an amino acid sequence of SEQ ID NO: 35, a HCDR2 having an amino acid sequence of SEQ ID NO: 36, and a HCDR3 having an amino acid sequence of SEQ ID NO:

37.

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

40.

31. The method of any one of claims 28 to 30, wherein the anti-PD-1 antibody or antigen-binding fragment comprises a HCVR having the amino acid sequence of SEQ ID NO: 33 and a LCVR having 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 having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO:

42.

33. The method of any one of claims 1 to 32, wherein the bispecific antibody is administered in a dosing regimen comprising divided initial doses.

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

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

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

37. The method of any one of claims 1 to 36, wherein the subject has previously been treated with chemotherapeutic drugs, radiation therapy, surgery, or immunotherapeutic drugs.

38. The method of claim 37, wherein the chemotherapeutic 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 to 36, wherein the subject has stable disease, partial remission, or complete remission after administration of the bispecific antibody at a dose of 1-250 mg for at least one week.

42. The method of any one of claims 1 to 36, wherein the size of the epithelioid sarcoma of breast tissue of the subject is reduced after at least one week of administration of the bispecific antibody at a dose of 1-250 mg.

43. The method of any one of claims 1 to 36, wherein the antibody is administered to the subject intravenously or subcutaneously.

44. The method of any one of claims 1 to 43, wherein the bispecific antibody is administered in a dosing regimen comprising: An initial dose of 1 mg of the bispecific antibody was administered in week 1 of the dosing regimen; a transition dose of 20 mg of the bispecific antibody was administered in week 2 of the dosing regimen; and a full dose of 250 mg of the bispecific antibody was administered in week 3 of the dosing regimen.

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

46. ​​The method of claim 44 or claim 45, wherein the transition dose is divided into two equal portions administered on consecutive days.

47. The method of any one of claims 44 to 46, wherein the full dose is divided into two parts administered on consecutive days.

48. The method of claim 47, wherein said two portions of said complete dose comprise a 50 mg portion and a 250 mg portion.

49. The method of any one of claims 44 to 48, wherein the dosing regimen further comprises administering a maintenance dose of 250 mg of the bispecific antibody 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.

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