Methods for treating cancer using bispecific anti-CD3xMUC16 antibodies and anti-PD-1 antibodies

Abstract

The method of the present invention comprises administering to an individual in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to a programmed death 1 (PD-1) receptor and a therapeutically effective amount of a bispecific antibody that specifically binds to mucin 16 (MUC16) and CD3.

Description

[0001] References to sequence listings

[0002] This application incorporates by reference a Sequence Listing filed in computer readable form as file 10469WO01-Sequence.txt, created on June 12, 2019, and containing 33,567 bytes. Technical Field

[0003] The present invention relates to a method for treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody that specifically binds to a programmed death 1 (PD-1) receptor and a bispecific antibody that binds to mucin 16 (MUC16) and CD3. Background Art

[0004] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125 or CA-125, is a single transmembrane domain highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer. MUC16 consists of three major domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with sea urchin sperm, enterokinase and agrin (SEA) domains, and a carboxyl-terminal domain containing a segment of the transmembrane region and a short cytoplasmic tail. Proteolytic cleavage causes the extracellular portion of MUC16 to fall into the bloodstream. MUC16 is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma-mass forming type, cervical adenocarcinoma and gastrointestinal 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). Expression on cancer cells has been shown to protect tumor cells from the immune system (Felder, M. et al., 2014, Molecular Cancer, 13:129). Anti-MUC16 antibodies have been explored for the treatment of ovarian cancer. Oregovomab and abgovomab are anti-MUC16 antibodies that have had limited success. (Felder, supra, Das, S. and Batra, SK 2015, Cancer Res. 75:4660-4674).

[0005] CD3 is a homodimeric or heterodimeric 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 dimeric association of two of the following four different chains: ε, ζ, δ and γ. CD3 dimer arrangements include γ / ε, δ / ε and ζ / ζ. Anti-CD3 antibodies have been shown to aggregate CD3 on T cells, thereby causing T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes to participate in T cell activation. In addition, bispecific antibodies capable of binding CD3 and target antigens have been proposed for therapeutic use to participate in targeting T cell immune responses to tissues and cells expressing the target antigen.

[0006] The programmed death-1 (PD-1) receptor, which signals in the tumor microenvironment, plays a key role in enabling tumor cells to escape immune surveillance by the host immune system. Blockade of the PD-1 signaling pathway has demonstrated clinical activity in patients with a variety of tumors, 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 clinical activity of PD-1 blockade in patients with aggressive NHL and Hodgkin's lymphoma (Lesokhin, et al. 2014, Abstract 291, 56th ASH Annual Meeting and Exposition, San Francisco, Calif.; Ansell et al. 2015, N. Engl. J. Med. 372(4):311-9).

[0007] Ovarian cancer is the most lethal of gynecological malignancies; although the estimated number of new cases of ovarian cancer in women in the United States is much lower than some other cancers, the mortality-to-morbidity ratio of ovarian cancer is considerably higher (Siegal et al., CA: Journal of Clinical Oncology (CA Cancer J Clin) 66:7-30, 2016). Ovarian cancer is often diagnosed at an advanced stage, contributing to its lethality. The current standard treatment for ovarian cancer is surgery followed by chemotherapy, that is, a combination of a platinum agent and a taxane. Although most patients respond to initial treatment, the majority experience disease recurrence, leading to cycles of repeated surgery and additional rounds of chemotherapy. Although recurrent ovarian cancer may respond to further treatment, almost all of them eventually become resistant to currently available therapies. Even with recent advances in therapy, such as PARP inhibitors for patients carrying BRCA or other homologous recombination deficiency (HRD) mutations, advanced ovarian cancer remains a disease with high unmet needs.

[0008] Evidence suggests that ovarian cancer may be amenable to some forms of immunotherapy (Kandalaft et al., J. Clin. Oncol., 29:925-933, 2011). + For patients with T lymphocyte infiltration, the tumor was intraepithelial CD8 + Ovarian cancer patients with positive T lymphocyte infiltration have significantly better overall and progression-free survival (Hamanishi et al., PNAS, 104:3360-65, 2007; and Zhang et al., New England Journal of Medicine, 348:203-213, 2003). In addition, some patients have shown spontaneous immune responses to their tumors, as confirmed by tumor-reactive T cells and antibody detection in the blood, tumors or ascites of patients with advanced disease (Schliengar et al., Clin Cancer Res, 9:1517-1527, 2003). Blockade of the PD-1 / PD-L1 checkpoint pathway has shown some benefits in ovarian cancer; in early clinical trials, PD-1 blockade monotherapy produced an overall response rate (ORR) of about 10% to 15% (Hamanishi et al., supra). However, blocking this pathway alone is clearly not enough.

[0009] Given the high unmet need for effective therapies for ovarian cancer, combination therapy with agents that enhance T cell function (e.g., PD-1 inhibitors, such as anti-PD-1 antibodies) and agents directed against target antigens (bispecific anti-MUC16 / anti-CD3 antibodies) may be useful, as shown herein. Summary of the invention

[0010] According to certain embodiments, the present invention provides a method for treating, ameliorating at least one symptom or indication of cancer in an individual, or inhibiting the development of cancer. The method according to this aspect of the invention comprises administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) and a therapeutically effective amount of a bispecific antibody that specifically binds to MUC16 and CD3.

[0011] In certain embodiments of the present invention, methods for treating, ameliorating at least one symptom or indication of cancer in an individual, or inhibiting the development of cancer are provided. In certain embodiments of the present invention, methods for delaying tumor growth or preventing tumor recurrence are provided. According to this aspect and other aspects of the present invention, the method comprises sequentially administering to an individual in need thereof one or more doses of a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1 and one or more doses of a therapeutically effective amount of a bispecific antibody that specifically binds to MUC16 and CD3.

[0012] In one aspect, the present invention provides a method for treating a tumor or inhibiting tumor growth, the method comprising administering to an individual in need (a) a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody, the bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3. In some cases, the anti-PD-1 antibody is administered before, at the same time, or after the bispecific antibody. In some cases, the anti-PD-1 antibody is administered before the bispecific antibody. In some cases, the anti-PD-1 antibody is administered at least 1 week before the bispecific antibody. In some cases, one or more doses of the anti-PD-1 antibody are administered in combination with one or more doses of the bispecific antibody. In some cases, the anti-PD-1 antibody is administered at a dose between 0.1 mg / kg and 20 mg / kg of individual body weight. In some cases, each dose of the anti-PD-1 antibody is included between 10 and 8000 micrograms. In some cases, the bispecific antibody is administered at a dose between 0.1 mg / kg and 20 mg / kg of individual body weight. In some cases, each dose of the bispecific antibody is comprised between 10 and 8000 micrograms. In some cases, each dose of the anti-PD-1 antibody is administered 0.5 to 12 weeks after the previous dose. In some cases, each dose of the bispecific antibody is administered 0.5 to 12 weeks after the previous dose. In various embodiments, the antibody is administered intravenously, subcutaneously, or intraperitoneally.

[0013] In some embodiments, the tumor comprises ovarian cancer.In some embodiments, the individual is resistant or inadequately responsive to or has relapsed following prior therapy.

[0014] In some cases, the method further comprises administering to the individual a third therapeutic agent or therapy. In some embodiments, the third therapeutic agent or therapy is selected from the group consisting of: radiation, surgery, chemotherapeutic agents, cancer vaccines, PD-L1 inhibitors, LAG-3 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor β (TGF.β.) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies to tumor-specific antigens, Bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony stimulating factor, cytotoxins, interleukin 6 receptor (IL-6R) inhibitors, interleukin 4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs and dietary supplements.

[0015] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34. In some cases, 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 some cases, the HCVR comprises the amino acid sequence of SEQ ID NO: 33, and the LCVR comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:42.

[0016] In some embodiments, the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 three light chain CDRs (A-LCDR1, A-LCDR2, and A-LCDR3) of a light chain variable region (A-LCVR) comprising the amino acid sequence of SEQ ID NO: 2. In some cases, 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; and A-LCDR3 comprises the amino acid sequence of SEQ ID NO: 13. In some cases, A-HCVR comprises the amino acid sequence of SEQ ID NO: 1 and A-LCVR comprises the amino acid sequence of SEQ ID NO: 2.

[0017] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, and 7, and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising an amino acid sequence of SEQ ID NO: 2. In some cases, B-HCDR1, B-HCDR2, and B-HCDR3 comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-15-16, 17-18-19, 20-21-22, 23-24-25, and 26-27-28, respectively; and B-LCDR1, B-LCDR2, and B-LCDR3 comprise an amino acid sequence of SEQ ID NOs: 11-12-13, respectively. In some cases, the B-HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 4, 5, 6, and 7, and the B-LCVR comprises the amino acid sequence of SEQ ID NO:2.

[0018] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0019] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:4 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0020] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:5 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0021] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:6 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0022] In some embodiments, the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0023] In some embodiments, the anti-PD-1 antibody, the bispecific antibody, or both comprises a human IgG1 or IgG4 heavy chain constant region.

[0024] In another aspect, the present invention provides a method for treating a tumor or inhibiting tumor growth, the method comprising administering to an individual in need thereof (a) a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, wherein: (a) the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (b) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 a second antigen-binding arm comprising the amino acid sequence of SEQ ID NO: 35. NO:2; and (c) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0025] In some embodiments of the methods, the anti-PD-1 antibody and the bispecific antibody respectively comprise the following: (a) 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; (b) 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; and A-LCDR3 comprises the amino acid sequence of SEQ ID NO:13; and (c) B-HCDR1 comprises the amino acid sequence of SEQ ID NO:14; B-HCDR2 comprises the amino acid sequence of SEQ ID NO:15 NO:15; B-HCDR3 comprises the amino acid sequence of SEQ ID NO:16; B-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; B-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; and B-LCDR3 comprises the amino acid sequence of SEQ ID NO:13.

[0026] In some embodiments of the methods, the anti-PD-1 antibody and the bispecific antibody respectively comprise the following: (a) the HCVR comprises the amino acid sequence of SEQ ID NO:33, and the LCVR comprises the amino acid sequence of SEQ ID NO:34; (b) the A-HCVR comprises the amino acid sequence of SEQ ID NO:1 and the A-LCVR comprises the amino acid sequence of SEQ ID NO:2; and (c) the B-HCVR comprises the amino acid sequence of SEQ ID NO:3, and the B-LCVR comprises the amino acid sequence of SEQ ID NO:2.

[0027] In some embodiments of the method, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:42; the first antigen-binding arm of the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:30; and the second antigen-binding arm of the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:30.

[0028] In some embodiments of the methods, the tumor comprises ovarian cancer.

[0029] In another aspect, the present invention provides a method for treating a tumor or inhibiting tumor growth, the method comprising administering to an individual in need thereof (a) a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, wherein: (a) the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (b) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 a second antigen-binding arm comprising the amino acid sequence of SEQ ID NO: 2 NO:2; and (c) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0030] In some embodiments of the methods, the anti-PD-1 antibody and the bispecific antibody respectively comprise the following: (a) 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; (b) 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; and A-LCDR3 comprises the amino acid sequence of SEQ ID NO:13; and (c) B-HCDR1 comprises the amino acid sequence of SEQ ID NO:26; B-HCDR2 comprises the amino acid sequence of SEQ ID NO: NO:27; B-HCDR3 comprises the amino acid sequence of SEQ ID NO:28; B-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; B-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; and B-LCDR3 comprises the amino acid sequence of SEQ ID NO:13.

[0031] In some embodiments of the methods, the anti-PD-1 antibody and the bispecific antibody comprise the following, respectively: (a) the HCVR comprises the amino acid sequence of SEQ ID NO:33 and the LCVR comprises the amino acid sequence of SEQ ID NO:34; (b) the A-HCVR comprises the amino acid sequence of SEQ ID NO:1 and the A-LCVR comprises the amino acid sequence of SEQ ID NO:2; and (c) the B-HCVR comprises the amino acid sequence of SEQ ID NO:7 and the B-LCVR comprises the amino acid sequence of SEQ ID NO:2.

[0032] In some embodiments of the method, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:42; the first antigen-binding arm of the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:30; and the second antigen-binding arm of the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a light chain comprising the amino acid sequence of SEQ ID NO:30.

[0033] In some embodiments of the methods, the tumor comprises ovarian cancer.

[0034] In various embodiments of any of the methods discussed above or herein, the anti-tumor activity of the bispecific antibody is not significantly hindered by circulating CA-125 at concentrations up to 10kU / ml. In various embodiments of any of the methods discussed above or herein, the individual has been diagnosed with ovarian cancer and the individual has a CA-125 circulating content up to 10kU / ml. In some embodiments of the methods discussed above or herein, the individual has a high CA-125 serum content before starting treatment. In some embodiments of the methods discussed above or herein, the individual has a CA-125 serum content greater than or equal to 2 times the upper limit of the normal CA-125 serum content before starting treatment. Some embodiments of the methods discussed above or herein include monitoring CA-125 serum content, such as by comparing the CA-125 serum content at various points during or after treatment with the baseline serum CA-125 content in a specific patient or the baseline serum CA-125 content in the total patient population to estimate the effectiveness of the treatment.

[0035] In some embodiments, the antibodies discussed herein are used to make a medicament for use in any of the methods discussed above or herein. In some embodiments, the antibodies discussed herein are used in a medicament or for treating a cancer as discussed above or herein. For example, the present invention includes:

[0036] (A) Use of a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, for the manufacture of a medicament for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1);

[0037] (B) Use of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) for the manufacture of a medicament for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3;

[0038] (C) Use of a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, for the manufacture of an agent for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 three light chain CDRs (A-HCDR2, A-HCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 35. NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0039] (D) Use of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) for the manufacture of an agent for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 a second antigen-binding arm comprising the amino acid sequence of SEQ ID NO: 2 NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0040] (E) Use of a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, for the manufacture of an agent for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 three light chain CDRs (A-HCDR2, A-HCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 35. NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0041] (F) Use of an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) for the manufacture of an agent for treating a tumor in an individual in need thereof or inhibiting tumor growth in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 a second antigen-binding arm comprising the amino acid sequence of SEQ ID NO: 2 NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0042] (G) a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, for use in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) to treat a tumor or inhibit tumor growth in an individual in need thereof;

[0043] (H) an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), for use in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3 to treat a tumor in an individual in need thereof or inhibit tumor growth;

[0044] (I) A bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, which is used in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) to treat tumors in individuals in need thereof or inhibit tumor growth, wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 three light chain CDRs (A-HCDR2, A-HCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 35; NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0045] (J) An antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), for use in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3 to treat a tumor in an individual in need thereof or inhibit tumor growth, wherein: (i) the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 a second antigen-binding arm comprising the amino acid sequence of SEQ ID NO: 2 NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:3 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2;

[0046] (K) A bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3, for use in combination with an antibody or an antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1) to treat a tumor in an individual in need thereof or inhibit tumor growth, wherein: (i) the anti-PD-1 antibody or the antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (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 three light chain CDRs (A-HCDR2, A-HCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 35; NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2, and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2; and

[0047] (L) An antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), for use in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds to MUC16 and a second antigen-binding arm that specifically binds to CD3 to treat a tumor in an individual in need thereof or inhibit tumor growth, wherein: (i) the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33 and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen-binding arm of the bispecific antibody comprises a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 1 and three heavy chain CDRs (A-HCDR1, A-HCDR2, and A-HCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 35; NO:2; and (iii) the second antigen-binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and B-HCDR3) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO:7 and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0048] Other embodiments of the present invention will become apparent from a review of the ensuing embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Binding of various concentrations of anti-MUC16 clone 3A5 and BSMUC16 / CD3-001 to CA125 as determined by ELISA (described in Example 2 herein) is illustrated. BSMUC16 / CD3-001 and its MUC16 parent antibody exhibited significantly reduced binding signals at all tested concentrations compared to anti-MUC16 clone 3A5, which binds to the repeat region of MUC16.

[0050] Figure 2Illustrate 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-CD3xMUC16 bispecific antibody synergistically inhibited tumor growth.

[0051] Figure 3 The effect of culturing T cells with BSMUC16 / CD3-001 on the percentage of PD-1 positive T cells is shown. DETAILED DESCRIPTION

[0052] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and thus methods and conditions can be changed. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, because the scope of the present invention will only be limited by the appended claims. Any embodiment or embodiment features can be combined with each other, and the combination is clearly included in the scope of the present invention. Any specific value discussed above or herein can be combined with another related value discussed above or herein to describe the range of values ​​with the upper and lower ends of the representation range, and the range is included in the scope of the present invention.

[0053] 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, when used with respect to a particular stated value, the term "about" means that the value may vary within a range of no more than 1% from the stated 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.).

[0054] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, 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.

[0055] Method for treating cancer or inhibiting the development of cancer

[0056] The present invention includes methods for treating, ameliorating at least one symptom or indication of cancer in an individual, or reducing its severity or inhibiting the development of cancer. The method according to this aspect of the invention comprises administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1 and a therapeutically effective amount of a bispecific anti-MUC16 and CD3 antibody. As used herein, the term "treat" or similar terms means alleviating symptoms, eliminating the cause of symptoms on a temporary or permanent basis, 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 prolonging the duration of survival of an individual.

[0057] As used herein, the expression "individual in need" means a human or non-human mammal that exhibits one or more symptoms or indications of cancer and / or has been diagnosed with cancer (including ovarian cancer) and is in need of one or more symptoms or indications of cancer or cancer treatment. In multiple embodiments, the term "individual" can be used interchangeably with the term "patient". For example, a human individual may be diagnosed with a primary or metastatic tumor and / or suffer from one or more symptoms or indications, including but not limited to one or more swollen lymph nodes, abdominal swelling, chest pain / chest pressure, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, itching. The expression includes individuals with primary or established ovarian tumors. In specific embodiments, the expression includes human individuals with ovarian cancer or another tumor expressing MUC16 and in need of its treatment. In other specific embodiments, the expression includes individuals with MUC16+ tumors (e.g., tumors with MUC16 expression as determined by flow cytometry). In certain embodiments, the expression "individuals in need" includes patients with ovarian cancer who are resistant to, difficult to treat with, or cannot be adequately controlled by previous therapies (e.g., treatment with conventional anticancer agents). For example, the expression includes individuals who have been treated with chemotherapy, such as platinum-based chemotherapeutic agents (e.g., cisplatin) or paclitaxel compounds (e.g., docetaxel). The expression also includes individuals with ovarian tumors for whom conventional anticancer therapy cannot be taken, 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 "individuals in need" includes patients with ovarian tumors who have been treated but subsequently relapsed or metastasized. For example, patients with ovarian tumors who may have received treatment with one or more anticancer agents, resulting in tumor regression; however, patients with ovarian tumors who subsequently relapsed with cancer resistant to one or more anticancer agents (e.g., chemotherapy-resistant cancer) are treated by the methods of the present invention.

[0058] The expression "individual in need thereof" also includes individuals at risk of developing ovarian cancer, such as those with a family history of ovarian cancer, those with a past history of ovarian cancer-related infections, those with BRCA1 / 2 gene mutations, or those with a compromised immune system due to HIV infection or due to immunosuppressive medications.

[0059] In certain embodiments, the methods of the present invention can be used to treat patients who display high levels of one or more cancer-associated biomarkers, such as programmed death ligand 1 (PD-L1), CA125, human epididymis protein 4 (HE4), and / or carcinoembryonic antigen (CEA). For example, the methods of the present invention comprise administering a therapeutically effective amount of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a patient with high levels of PD-L1 and / or CA125.

[0060] In certain embodiments, the methods of the present invention are used in individuals with ovarian cancer. The terms "tumor," "cancer," and "malignancy" are used interchangeably herein. As used herein, the term "ovarian cancer" refers to ovarian and fallopian tube tumors, and includes serous carcinoma, endometrioid carcinoma, clear cell carcinoma, and mucinous carcinoma.

[0061] According to certain embodiments, the present invention includes methods for treating tumors 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 present invention, the method comprises sequentially administering a therapeutically effective amount of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual in need, wherein each antibody is administered to the individual 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-PD-1 antibody to an individual 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 bispecific antibody are administered to the individual 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.

[0062] In certain embodiments, each dose of anti-MUC16 / anti-CD3 antibody is administered in more than 1 portion, such as 2 to 5 portions ("split dosing") within a given dosing period. Anti-MUC16 / anti-CD3 bispecific antibodies can be administered in split doses to reduce or eliminate cytokine "spikes" induced in response to antibody administration. Cytokine spikes refer to clinical symptoms and infusion-related reactions of cytokine release syndrome ("cytokine storm"). In certain embodiments, the methods of the present invention comprise administering one or more doses of an anti-PD-1 antibody and one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody to an individual in need thereof, wherein a dose of the bispecific antibody is administered in split doses or in more than 1 portion, such as 2, 3, 4, or 5 portions within a given dosing period. In certain embodiments, a dose of a bispecific antibody is split into 2 or more portions, wherein each portion contains an amount of antibody equal to another portion. For example, an anti-MUC16 / anti-CD3 antibody comprising a dose of 1000 micrograms can be administered once a week, wherein the dose is divided into 2 portions administered within the weeks, each portion comprising 500 micrograms. In certain embodiments, a dose of a bispecific antibody is split into 2 or more portions for administration, wherein each portion comprises an unequal amount of antibody, such as more or less than the first portion. For example, an anti-MUC16 / anti-CD3 antibody comprising a dose of 1000 micrograms can be administered once a week, wherein the dose is divided into 2 portions administered within the weeks, wherein the first portion comprises 700 micrograms and the second portion comprises 300 micrograms. As another example, an anti-MUC16 / anti-CD3 antibody comprising a dose of 1000 micrograms can be administered once every 2 weeks, wherein the dose is divided into 3 portions administered within a 2-week period, wherein the first portion comprises 400 micrograms, the second portion comprises 300 micrograms and the third portion comprises 300 micrograms.

[0063] In certain embodiments, the present invention includes methods for inhibiting, preventing or stopping tumor metastasis or tumor infiltration into peripheral organs. According to this aspect, the method comprises administering to an individual in need thereof a therapeutically effective amount of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody.

[0064] In specific embodiments, the present invention provides methods for improving anti-tumor efficacy or enhancing tumor suppression. According to this aspect of the invention, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody to an individual with ovarian cancer, followed by 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 before the bispecific antibody. In certain embodiments, the method provides an enhancement of, for example, about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% tumor suppression compared to an individual administered with a bispecific antibody before the anti-PD-1 antibody.

[0065] In certain embodiments, the methods of the present invention comprise administering to an individual with ovarian cancer a therapeutically effective amount of an anti-PD-1 antibody and a therapeutically effective amount of a bispecific anti-CD3xMUC16 antibody. In specific embodiments, the ovarian cancer is a serous carcinoma. In further embodiments, the ovarian cancer is indolent or aggressive. In certain embodiments, the individual has not responded to prior therapy or has relapsed after prior therapy. In certain embodiments, the methods of the present invention further comprise administering to the individual an additional therapeutic agent.

[0066] In certain embodiments, the methods of the present invention comprise administering a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody to an individual with MUC16+ cancer. In specific embodiments, the cancer is ovarian cancer. In further embodiments, the ovarian cancer is indolent or aggressive. In some embodiments, the cancer is platinum-resistant ovarian cancer. In some embodiments, the cancer is paclitaxel-resistant ovarian cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is primary peritoneal cancer, optionally wherein the patient has a high level of serum CA-125. In specific embodiments, the cancer is pancreatic cancer (e.g., pancreatic adenocarcinoma). In certain embodiments, the individual has not responded to a previous therapy or has relapsed after a previous therapy (e.g., chemotherapy).

[0067] In certain embodiments, the methods of the present invention comprise administering an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual in need thereof as a "first line" therapy (e.g., initial therapy). In other embodiments, an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody are administered as a "second line" therapy (e.g., after a prior therapy). For example, an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody are administered as a "second line" therapy to an individual who has relapsed after a prior therapy, such as chemotherapy.

[0068] 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 a patient during or after treatment, wherein the patient may or may not show symptoms or signs of the disease. If the residual cancer cells are not eliminated, then it usually leads to disease recurrence. The present invention includes methods for inhibiting and / or eliminating residual cancer cells in a patient during MRD testing. MRD can be analyzed according to methods known in the art (e.g., MRD flow cytometry). According to this aspect of the invention, the method comprises administering an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual in need.

[0069] According to certain embodiments, the method of the present invention comprises administering to an individual a therapeutically effective amount of each of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody and 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, tumor Antibodies to 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., BCG), granulocyte-macrophage colony stimulating factor, cytotoxins, chemotherapeutic agents, IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), and dietary supplements (e.g., antioxidants). In certain embodiments, the antibodies can be administered in combination with therapies including: chemotherapeutic agents (e.g., paclitaxel, carboplatin, doxorubicin, cyclophosphamide, cisplatin, gemcitabine, or docetaxel), radiation, and surgery. As used herein, the phrase "and / in combination with" means that the antibody is administered to an individual simultaneously with, 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. In related embodiments, the present invention includes a method comprising administering a therapeutically effective amount of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual using a background anti-cancer treatment regimen. The background anti-cancer treatment regimen may include a process of administering, for example, a chemotherapeutic agent or radiation. The anti-PD-1 antibody and the bispecific anti-MUC16 / anti-CD3 antibody may be added to the background anti-cancer treatment regimen. In some embodiments, the antibody is added as part of a "background de-escalation" regimen, in which the background anti-cancer therapy is gradually (e.g., in a stepwise manner) withdrawn from the individual over time, and the antibody is administered to the individual at a constant dose, or at an increasing dose, or at a decreasing dose over time.

[0070] In certain embodiments, the methods of the present invention comprise administering to an individual in need thereof a therapeutically effective amount of an anti-PD-1 antibody and a therapeutically effective amount of a bispecific anti-MUC16 / anti-CD3 antibody, wherein the antibody administration results in enhanced 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 individual or an individual administered with 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 enhanced 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 progression, 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 individual or an individual treated with either antibody as a monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody prevents tumor recurrence and / or prolongs the duration of survival of an individual, for example, the duration of survival is extended 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 individual or an individual administered with either antibody as a monotherapy. In certain embodiments, combined administration of the antibodies prolongs progression-free survival or overall survival. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody enhances the response in an individual and prolongs the duration of the response, e.g., by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to an untreated individual or an individual who has received either antibody as a monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual with ovarian cancer results in a complete disappearance of all signs of tumor cells (a "complete response"). In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual with ovarian cancer results in a reduction in tumor cells or tumor size of at least 30% or more (a "partial response"). In certain embodiments, administration of an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to an individual with ovarian cancer 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.

[0071] In certain embodiments, the combination of administered antibodies is safe and well tolerated by patients, with no increase in deleterious side effects (e.g., increased cytokine release ("cytokine storm") or increased T cell activation) compared to patients administered the bispecific antibody as a monotherapy.

[0072] Anti-PD-1 antibodies and antigen-binding fragments thereof

[0073] 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 comprising four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V 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 H and V L The V region can be further subdivided into hypervariable regions called complementarity determining regions (CDR) interspersed with more conserved regions called framework regions (FR). 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 its antigen-binding portion) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0074] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody and similar terms thereof include any naturally occurring, enzymatically obtainable, synthesized or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optional constant domains. The DNA is known and / or is easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries) or can be synthesized. DNA can be sequenced and manipulated 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 introduce codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0075] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide) or a hypervariable region of a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0076] The antigen-binding fragment of an antibody will generally contain at least one variable domain. The variable domain can be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the 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 dimeric and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or VL domain.

[0077] 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 and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or may be connected through a complete or partial hinge or linker region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the invention may contain binding sites to each other and / or to one or more monomeric V H or V LThe domains are non-covalently associated (eg, via one or more disulfide bonds) homo- or heterodimers (or other multimers) having any of the variable and constant domain configurations listed above.

[0078] As used herein, the term "antibody" also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or an antigen-binding fragment of an antibody will generally comprise 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. Any multispecific antibody format can be adapted for use in the context of an antibody or antigen-binding fragment of an antibody of the present invention using conventional techniques available in the art. 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 coupled 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 bifunctional antibody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, hole-in-knob, common light chain (e.g., common light chain with hole-in-knob, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab.sup.2 bispecific formats (for reviews of the aforementioned 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 coupling, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0079] The antibody used in the method for the present invention 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. The human antibody of the present invention can still include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), for example, in CDR and, more precisely, CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies wherein derived from another mammalian species, for example, the CDR sequences of the germline of mice have been transplanted to human framework sequences.

[0080] 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 using recombinant expression vectors transfected into host cells (further described below), antibodies separated from recombinant combinatorial human antibody libraries (further described below), antibodies separated from transgenic animals (e.g., mice) for 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 human immunoglobulin gene sequences to other DNA sequences. The recombinant human antibody has a variable region and a constant region derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibody is subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using transgenic animals for human Ig sequences), and therefore 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 and sequences related thereto but which may not naturally exist within the human antibody germline repertoire in vivo.

[0081] According to certain embodiments, the antibodies used in the methods of the present invention specifically bind to PD-1. The term "specifically binds" or its analogous terms means that the antibody or its antigen-binding fragment forms a complex with the antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, as used in the context of the present invention, an antibody that "specifically binds" to PD-1 includes an antibody that binds to PD-1 or a portion thereof, wherein K is greater than or equal to 1, as measured in a surface plasmon resonance analysis. D Less than about 500nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, less than about 50nM, less than about 40nM, less than about 30nM, less than about 20nM, less than about 10nM, less than about 5nM, less than about 4nM, less than about 3nM, less than about 2nM, less than about 1nM, or less than about 0.5nM. However, an isolated antibody that specifically binds to human PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0082] 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 antibody as described in U.S. Patent Publication No. 20150203579. In certain exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof that can be used in the context of the method of the present invention comprises a heavy chain complementarity determining region (HCDR) of a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 33 and a light chain complementarity determining region (LCDR) of a light chain variable region (LCVR) comprising 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 yet other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 33 and a LCVR comprising SEQ ID NO: 34. In certain embodiments, the methods of the present invention comprise the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42. An exemplary antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain comprising the amino acid sequence of SEQ ID NO: 42 is a fully human anti-PD-1 antibody called REGN2810 (also known as cemiplimab). According to certain exemplary embodiments, the methods of the present invention comprise the use of REGN2810 or a bioequivalent thereof. As used herein, the term "bioequivalent" refers to an anti-PD-1 antibody or a PD-1 binding protein or fragment thereof that is a pharmaceutical equivalent or pharmaceutical substitute that does not show a significant difference in absorption rate and / or extent from that of REGN2810 when administered at the same molar dose in a single dose or multiple doses under similar experimental conditions. In the context of the present invention, the term refers to an antigen binding protein that binds to PD-1 that does not have a clinically meaningful difference from REGN2810 in terms of its safety, purity and / or efficacy.

[0083] Other anti-PD-1 antibodies that can be used in the context of the methods of the invention include, for example, those referred to and known in the art as nivolumab (U.S. Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (U.S. Pat. No. 8,609,089), pidilizumab (U.S. Pat. No. 8,686,119), or any anti-PD-1 antibody as described in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587.

[0084] The anti-PD-1 antibodies used in the context of the methods of the present invention may have a pH-dependent binding profile. For example, the anti-PD-1 antibodies used in the methods of the present invention may exhibit a weakened binding to PD-1 at an acidic pH compared to a neutral pH. Alternatively, the anti-PD-1 antibodies of the present invention may exhibit an enhanced binding to their antigen at an acidic pH compared to a neutral pH. The expression "acidic pH" includes a pH value of less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH value" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0085] In some cases, "reduced binding to PD-1 at acidic pH compared to neutral pH" means that the binding K of the antibody to PD-1 at acidic pH is D The value is similar to the K value of antibody binding to PD-1 at neutral pH. D For example, for purposes of the present invention, an antibody or antigen-binding fragment thereof is considered to be a ratio of about 3.0 to about 5.5 (or vice versa). D , then the antibody or antigen-binding fragment thereof can 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 the 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.

[0086] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening for an antibody population whose binding to a specific antigen is weakened (or enhanced) at an acidic pH compared to a neutral pH. In addition, modifications of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, antibodies whose antigen binding is weakened at an acidic pH relative to a neutral pH can be obtained by replacing one or more amino acids of the antigen-binding domain (e.g., within a CDR) with histidine residues. As used herein, the expression "acidic pH" means a pH of 6.0 or less.

[0087] Bispecific anti-MUC16 / anti-CD3 antibody

[0088] According to certain exemplary embodiments of the present invention, the method comprises administering a therapeutically effective amount of a bispecific antibody that specifically binds CD3 and MUC 16. The antibody may be referred to herein as, for example, an "anti-MUC16 / anti-CD3" or "anti-MUC16xCD3" or "MUC16xCD3" bispecific antibody or other similar terms.

[0089] 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), and each domain comprises three CDRs. In the case of a bispecific antibody, the CDRs of the first antigen binding domain may be designated by the prefix "A" and the CDRs of the second antigen binding domain may be designated 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.

[0090] 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 is capable of associating with a second multimerization domain having 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 a C H2 -C H3 domain), such as an Fc domain of an IgG of the isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype within each isotype group.

[0091] The bispecific antibodies of the present invention generally comprise two multimerization domains, e.g., two Fc domains that are each individually part of a single antibody heavy chain. The first multimerization domain and the second multimerization domain may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0092] Any bispecific antibody format or technology can be used to make the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or bifunctional antibody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, mortise-in-knob, common light chain (e.g., common light chain with mortise-in-knob, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab 2 Bispecific formats (for review of the aforementioned formats, see, eg, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0093] In the case 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 the wild-type naturally occurring form of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain, wherein the one or more modifications result in a modified Fc domain having a modified binding interaction (e.g., enhanced or weakened) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises CH2 or C H3 The invention relates to a modification in a region, wherein the modification enhances the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where the pH is in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in U.S. Patent Publication No. 20150266966, which is incorporated herein in its entirety.

[0094] The present invention 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 The first Ig C H Domain 3 binds protein A and the second Ig C H The 3 domain contains a mutation that weakens or abolishes protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Pat. No. 8,586,713. H Additional modifications within 3 include in the case of IgG1 antibodies: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU); in the case of IgG2 antibodies: N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU); and in the case of IgG4 antibodies: Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU).

[0095] In certain embodiments, the Fc domain may be a chimeric combined Fc sequence derived from more than one immunoglobulin isotype. For example, the chimeric Fc domain may comprise a sequence derived from human IgG1, human IgG2, or human IgG4. H Part or all of Zone 2 C H 2 sequences and part or all of the C sequences derived from human IgG1, human IgG2 or human IgG4 H3 sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region and a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule described herein include from the N-terminus to the C-terminus: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any antigen-binding molecule described herein comprises from N-terminus to C-terminus: [IgG1C 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.

[0096] 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), which comprises any amino acid sequence of the bispecific anti-MUC16 / anti-CD3 antibody as described in U.S. Patent Publication No. 20180112001. In certain exemplary embodiments, a bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof that can be used in the context of the methods of the present invention comprises: (a) a first antigen-binding arm comprising heavy chain complementarity determining regions (A-HCDR1, A-HCDR2, and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO:1 and light chain complementarity determining regions (A-LCDR1, A-LCDR2, and A-LCDR3) of a light chain variable region (A-LCVR) comprising the amino acid sequence of SEQ ID NO:2; and (b) a second antigen-binding arm comprising heavy chain CDRs (B-HCDR1, B-HCDR2, and B-HCDR3) of a heavy chain variable region (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-HCDR1, B-HCDR2, and B-HCDR3) of a light chain variable region (B-LCVR) comprising the amino acid sequence of SEQ ID NO:8. Light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) of the amino acid sequence of NO:2.According to certain embodiments, A-HCDR1 comprises the amino acid sequence of SEQ ID NO:8; A-HCDR2 comprises the amino acid sequence of SEQ ID NO:9; A-HCDR3 comprises the amino acid sequence of SEQ ID NO:10; A-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; A-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; A-LCDR3 comprises the amino acid sequence of SEQ ID NO:13; B-HCDR1 comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:26; B-HCDR2 comprises the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, or SEQ ID NO:27; and B-HCDR3 comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28; and B-LCDR1 comprises the amino acid sequence of SEQ ID NO:11; A-LCDR2 comprises the amino acid sequence of SEQ ID NO:12; A-LCDR3 comprises the amino acid sequence of SEQ ID NO:13; In yet other embodiments, the bispecific anti-MUC16 / anti-CD3 antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding arm comprising a HCVR comprising SEQ ID NO: 1 (A-HCVR) and a LCVR comprising SEQ ID NO: 2 (A-LCVR); and (b) a second antigen-binding arm comprising a HCVR comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 (B-HCVR) and a LCVR comprising SEQ ID NO: 2 (B-LCVR). In certain exemplary embodiments, the bispecific anti-CD3xMUC16 antibody comprises a MUC16 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:30, and the CD3 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:30.In certain exemplary embodiments, the bispecific anti-CD3xMUC16 antibody comprises a MUC16 binding arm and a CD3 binding arm, wherein the MUC16 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:30, and the CD3 binding arm comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a light chain comprising the amino acid sequence of SEQ ID NO:30.

[0097] In certain embodiments, the anti-tumor activity of the bispecific anti-CD3xMUC16 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. CA125 serum levels are increased in the serum of most ovarian cancer patients (median published levels are about 656 U / ml). As demonstrated in Example 2 below, high levels of CA125 in serum or ascites will not significantly interfere with the anti-tumor profile of the bispecific antibodies of the invention.

[0098] 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. 20180112001.

[0099] Combination therapy

[0100] According to certain embodiments, the methods of the present invention comprise administering an anti-MUC16 / anti-CD3 bispecific antibody and an anti-PD-1 antibody to an individual. In certain embodiments, the methods of the present invention comprise administering antibodies to obtain additive or synergistic activity to treat cancer, preferably ovarian cancer. As used herein, the expression "and / in combination with" means that the anti-MUC16 / anti-CD3 bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. The term "and / in combination with" also includes sequential or simultaneous 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 means that the anti-PD-1 antibody is administered to the individual in a separate dosage form within less than 5 minutes (before, after, or simultaneously) of the administration of the bispecific anti-MUC16 / anti-CD3 antibody, or in a single combined dosage formulation comprising the anti-PD-1 antibody and the bispecific anti-MUC16 / anti-CD3 antibody.

[0101] In certain embodiments, the methods of the present invention comprise administering a third therapeutic agent, wherein the third therapeutic agent is an anticancer agent. As used herein, "anticancer agent" means any agent suitable for treating cancer, including but not limited to cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P'-(DDD)), biologics (e.g., antibodies and interferons), and radiopharmaceuticals. As used herein, "cytotoxin or cytotoxic agent" also refers to a chemotherapeutic agent and means any agent that is harmful to cells. Examples include (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, cranberries, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.

[0102] In certain embodiments, the methods of the invention comprise administering a third therapeutic agent selected from the group consisting of: radiation; surgery; a cancer vaccine; a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody); a LAG-3 inhibitor; a CTLA-4 inhibitor (e.g., ipilimumab); a TIM3 inhibitor; a BTLA inhibitor; a TIGIT inhibitor; a CD47 inhibitor; an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA); an indoleamine-2,3-dioxygenase (IDO) inhibitor; a vascular endothelial growth factor (VEGF); GF) antagonists [e.g., "VEGF traps", such as aflibercept or other VEGF inhibitory fusion proteins described in U.S. Pat. No. 7,087,411; or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab); or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)]; Ang2 inhibitors (e.g., nesvacumab); transforming growth factor β (TGF.β.) inhibitors; epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab); agonists of co-stimulatory receptors (e.g., agonists of glucocorticoid-induced TNFR-related protein); antibodies against 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., BCG, cancer vaccines); adjuvants for enhancing antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor cytotoxins; chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, cytoxan, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine); radiation therapy; IL-6R inhibitors (e.g., sarilumab); IL-4R inhibitors (e.g., dupilumab); IL-10 inhibitors; cytokines, such as IL-2, IL-7, IL-21, and IL-15; antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADCs and anti-DS6-DM4 ADCs); chimeric antigen receptor T cells (e.g., CD19-targeted T cells), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs); and dietary supplements, such as antioxidants. .

[0103] In certain embodiments, the methods of the present invention comprise administering an anti-PD-1 antibody and an anti-MUC16 / anti-CD3 bispecific antibody and radiation therapy to generate a long-term durable anti-tumor response and / or prolong the survival of a patient suffering from cancer.

[0104] 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 cancer patient. For example, one or more doses of radiation therapy may be administered after one or more doses of the antibody are administered to the tumor lesion. In some embodiments, radiation therapy may be administered locally after systemic administration of an anti-PD-1 antibody and / or a bispecific anti-MUC16 / anti-CD3 antibody to the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablative radiation). In certain embodiments, the antibody may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., carboplatin and / or paclitaxel) or a VEGF antagonist (e.g., afalibre).

[0105] Pharmaceutical compositions and administration

[0106] The present invention includes methods comprising administering to an individual an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody, wherein the antibodies are contained in a separate or combined (single) pharmaceutical composition. The pharmaceutical compositions of the present invention can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. Numerous suitable formulations can be found in all formulary books known to pharmaceutical chemists: Remington's Pharmaceutical Sciences Pharmaceutical Sciences , Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol with various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al., Compendium of excipients for parenteral formulations, PDA (1998), J Pharm Sci Technol 52: 238-311.

[0107] 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 endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any suitable route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa, etc.), and can be administered together with other biologically active agents.

[0108] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily applicable to deliver the pharmaceutical composition of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable sleeve containing the pharmaceutical composition. Once all of the pharmaceutical composition in the sleeve has been administered and the sleeve is empty, the empty sleeve can be easily discarded and replaced with a new sleeve containing the pharmaceutical composition. Subsequently, the pen delivery device can be reused. In a disposable pen delivery device, there is no replaceable sleeve. Specifically, the disposable pen delivery device is pre-filled with a pharmaceutical composition stored in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0109] 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 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 close to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science, 249: 1527-1533.

[0110] Injectable preparations 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, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody described above or its salt in a conventional sterile aqueous medium or oily medium for injection. There are physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with a suitable solubilizing agent, as aqueous media for injection, and the solubilizing agent is, for example, alcohol (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyethylene oxide (50 moles) adducts of hydrogenated castor oil)], etc. Using, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent, as an oily medium, the solubilizing agent is, for example, benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

[0111] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a dosage form in a unit dosage form suitable for the dosage of the active ingredient. The dosage form in the unit dosage form includes, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0112] Administration regimen

[0113] The present invention includes methods comprising administering an anti-PD-1 antibody to an individual 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 a less frequent dosing frequency, as long as a therapeutic response is achieved. In certain embodiments, the present invention includes methods comprising administering a bispecific anti-MUC16 / anti-CD3 antibody to an individual 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 a less frequent dosing frequency, as long as a therapeutic response is achieved. In certain embodiments, the methods involve administering an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 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 a less frequent dosing frequency, as long as a therapeutic response is achieved.

[0114] According to certain embodiments of the present invention, multiple doses of anti-PD-1 antibodies and bispecific anti-MUC16 / anti-CD3 antibodies can be administered to an individual within a defined time course. The method according to this aspect of the present invention comprises sequentially administering one or more doses of anti-PD-1 antibodies and one or more doses of bispecific anti-MUC16 / anti-CD3 antibodies to an individual. As used herein, "sequential administration" means that each dose of the antibody is administered to an individual at a different time point, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising sequentially administering to a patient a single initial dose of an anti-PD-1 antibody, followed by one or more second doses of an anti-PD-1 antibody, and optionally followed by one or more third doses of an anti-PD-1 antibody. In certain embodiments, the method further comprises sequentially administering to a patient a single initial dose of a bispecific anti-MUC16 / anti-CD3 antibody, followed by one or more second doses of a bispecific antibody, and optionally followed by one or more third doses of a bispecific antibody.

[0115] According to certain embodiments of the present invention, multiple doses of anti-PD-1 antibody and bispecific anti-MUC16 / anti-CD3 antibody can be administered to an individual within a defined time course. The method according to this aspect of the present invention comprises sequentially administering multiple doses of anti-PD-1 antibody and bispecific anti-MUC16 / anti-CD3 antibody to an individual. As used herein, "sequential administration" means that each dose of anti-PD-1 antibody and bispecific antibody is administered to an individual at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months).

[0116] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the temporal order 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 "secondary dose" is the dose administered after the initial dose; and the "tertiary dose" is the dose administered after the second dose. The initial, second and third doses 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, second and / or third doses differ from one another during the course of treatment (e.g., adjusted upward or downward as needed). 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 "rapid-acting dose", followed by subsequent doses (e.g., "maintenance doses") on a less frequent basis. For example, a rapid-acting dose of about 1 to 3 mg / kg of patient body weight, followed by one or more maintenance doses of about 0.1 to about 20 mg / kg of anti-PD-1 antibody, may be administered to a patient with ovarian cancer.

[0117] In an exemplary embodiment of the invention, each second dose and / or third dose is administered 1 / 2 to 14 (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 more) weeks after the previous dose. As used herein, the phrase "previous dose" means a dose of an anti-PD-1 antibody (and / or bispecific anti-MUC16 / anti-CD3 antibody) administered to a patient in a sequence of multiple administrations prior to administration of the next dose in an order without an intervening dose.

[0118] The methods according to this aspect of the invention may comprise administering any number of second and / or third doses of an anti-PD-1 antibody (and / or bispecific anti-MUC16 / anti-CD3 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. Likewise, 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) third doses are administered to a patient.

[0119] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as the 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 the other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administering the second dose and / or the third dose to the patient can vary during the course of the treatment regimen. The frequency of administration can also be adjusted by the physician during the course of treatment after clinical examination depending on the needs of the individual patient.

[0120] In certain embodiments, one or more doses of an anti-PD-1 antibody and / or a bispecific anti-MUC16 / anti-CD3 antibody are administered at the beginning of a treatment regimen on a more frequent basis (twice a week, once a week, or once every 2 weeks) as an "induction dose," followed by subsequent doses ("boost doses" or "maintenance doses") administered on a less frequent basis (e.g., once every 4 to 12 weeks).

[0121] The present invention includes methods comprising sequentially administering an anti-PD-1 antibody and a bispecific anti-MUC16 / anti-CD3 antibody to a patient to treat ovarian cancer (e.g., serous carcinoma). In some embodiments, the methods of the present invention comprise administering one or more doses of an anti-PD-1 antibody, followed by one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody. In certain embodiments, the methods of the present invention comprise administering a single dose of an anti-PD-1 antibody, followed by one or more doses of a bispecific anti-MUC16 / anti-CD3 antibody. In some embodiments, one or more doses of an anti-PD-1 antibody of about 0.1 mg / kg to about 20 mg / kg, followed by one or more doses of a bispecific antibody of about 0.1 mg / kg to about 20 mg / kg may be administered to inhibit tumor growth and / or prevent tumor recurrence in individuals with ovarian cancer. In some embodiments, administration of one or more doses of an anti-PD-1 antibody followed by one or more doses of a bispecific antibody results in enhanced anti-tumor efficacy (e.g., greater inhibition of tumor growth and enhanced prevention of tumor recurrence compared to untreated individuals or individuals administered either antibody as a monotherapy). Alternative embodiments of the present invention relate to simultaneous administration of an anti-PD-1 antibody and a bispecific antibody, the bispecific antibody being administered at a separate dose at a similar or different frequency relative to the anti-PD-1 antibody. In some embodiments, the bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. In certain embodiments, the bispecific antibody is administered in a single dosage form with the anti-PD-1 antibody.

[0122] dose

[0123] The amount of anti-PD-1 antibody and / or bispecific anti-MUC16 / anti-CD3 antibody administered to an individual according to the methods of the present invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of an antibody (anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody) that causes one or more of the following: (a) reducing the severity or duration of symptoms of cancer (e.g., ovarian cancer); (b) inhibiting tumor growth or enhancing tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delaying tumor growth and development; (d) inhibiting or preventing or stopping tumor metastasis; (e) preventing recurrence of tumor growth; (f) prolonging the survival of an individual with cancer (e.g., ovarian cancer); 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 an untreated individual or an individual administered with either antibody as a monotherapy.

[0124] In the case of an anti-PD-1 antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, such as 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 amount of anti-PD-1 antibody administered is about 250 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. In some embodiments, 250 mg of anti-PD-1 antibody is administered.

[0125] In the case of a bispecific anti-MUC16 / anti-CD3 antibody, a therapeutically effective amount can be from about 10 micrograms (mcg) to about 8000 mcg, such as about 10 mcg, about 20 mcg, about 50 mcg, about 70 mcg, about 100 mcg, about 120 mcg, about 150 mcg, about 200 mcg, about 250 mcg, about 300 mcg, about 350 mcg, about 400 mcg, about 450 mcg, about 500 mcg, about 550 mcg, about 600 mcg, about 700 mcg, about 800 mcg, about about 900mcg, about 1000mcg, about 1050mcg, about 1100mcg, about 1500mcg, about 1700mcg, about 2000mcg, about 2050mcg, about 2100mcg, about 2200mcg, about 2500mcg, about 2700mcg, about 2800mcg, about 2900mcg, about 3000mcg, about 4000mcg, about 5000mcg, about 6000mcg, about 7000mcg or about 8000mcg of a bispecific anti-MUC16 / anti-CD3 antibody.

[0126] The amount of anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody contained in a single dose can be expressed in terms of milligrams of antibody per kilogram of individual body weight (i.e., mg / kg). In certain embodiments, the anti-PD-1 antibody or bispecific anti-MUC16 / anti-CD3 antibody used in the methods of the present invention can be administered to an individual at a dose of about 0.0001 to about 100 mg / kg of individual body weight. For example, 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. 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.

[0127] A summary of the sequences and corresponding SEQ ID NOs referred to herein is shown in Table 1 below.

[0128] Table 1: Sequence overview

[0129]

[0130]

[0131] Examples

[0132] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0133] Example 1: Generation of a bispecific antibody that binds ovarian cell specificity (MUC16) and CD3

[0134] The present invention provides bispecific antigen binding molecules that bind CD3 and MUC16; the bispecific antigen binding molecules are also referred to herein as "anti-MUC16 / anti-CD3 or anti-MUC16xCD3 bispecific molecules". The anti-MUC16 portion of the anti-MUC16 / anti-CD3 bispecific molecule is suitable for targeting tumor cells expressing MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is suitable for activating T cells. The simultaneous binding of MUC16 on tumor cells and CD3 on T cells facilitates the directed killing (cell lysis) of target tumor cells by activated T cells.

[0135] 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 distinct HCVR paired 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 known to be effectively promiscuous or paired with multiple heavy chain arms.

[0136] As described in U.S. Patent Application Publication No. US20140243504A1 published on August 28, 2014, exemplary bispecific antibodies were made having an IgG1 Fc domain (BSMUC16 / CD3-001, -002, -003, and -004) or a modified (chimeric) IgG4 Fc domain (BSMUC16 / CD3-005).

[0137] A summary of the components of the antigen binding domains of the various anti-MUC16xCD3 bispecific antibodies constructed is set forth in Table 2.

[0138] Table 2: Overview of components of the anti-MUC16xCD3 bispecific antibody

[0139]

[0140] Example 2: CA-125 does not interfere with the in vitro activity of anti-MUC16xCD3 antibodies

[0141] The effect of soluble CA-125 (the shunt form of MUC16) on BSMUC16 / CD3-001 activity was evaluated using FACS binding and cytotoxicity assays in the presence of high levels of CA-125 purified from the ascites of ovarian cancer patients. CA-125 levels are increased in the serum of most ovarian cancer patients and circulating levels could affect any therapy targeting MUC16 by acting as an antigen silencer. The levels of CA-125 used in the analysis (10,000 U / ml) far exceed the median published level of 656.6 U / mL in ovarian cancer patients. The ability of BSMUC16 / CD3-001 to kill OVCAR-3 cells expressing MUC16 in the presence of enriched soluble CA-125 from human ascites (creative Biomart, NY, USA) or a membrane-proximal construct expressing five carboxyl-terminal SEA domains and the membrane-proximal region of MUC16 (MUC16Δ) was tested at 37°C for 72 hours at a 4:1 effector / target ratio with fixed concentrations of BSMUC16 / CD3-001 or CD3-binding control antibody (100 pM) and serial dilutions of MUC16-1H or MUC16Δ. To monitor specific killing of target cells carrying MUC16, OVCAR-3 cells were labeled with 1 μM Violet Cell Tracker. After labeling, cells were plated overnight at 37°C. Separately, human PBMCs were plated at 1×10 6 Cells / ml were plated in medium supplemented with RPMI and incubated overnight at 37°C to enrich lymphocytes by depleting adherent cells. The next day, the target cells were co-cultured with the initial PBMCs (4:1 effector cell / target cell ratio) depleted of adherent cells and serial dilutions of BSMUC16 / CD3-001 or CD3 binding control at 37°C for 72 hours. Cells were removed from the cell culture plate using trypsin and analyzed by FACS. For FACS analysis, cells were stained with a dead / live far-red cell tracer (Invitrogen). To assess killing specificity, cells were gated on a purple cell tracer-labeled population. The percentage of live target cells was reported to calculate the adjusted survival rate as follows: adjusted survival rate = (R1 / R2) × 100, where R1 =% of live target cells in the presence of antibody, and R2 =% of live target cells in the absence of the test antibody. T cell activation was assessed by incubating cells with antibodies directly conjugated to CD2, CD69 and CD25 and by reporting the percentage of activated (CD69+) T cells or (CD25+) T cells among total T cells (CD2+).

[0142] Binding of BSMUC16 / CD3-001 to an antibody known to bind CA-125 (clone 3A5) to CA125 obtained from human ascites fluid was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, soluble CA-125 (creative Biomart, NY, USA) at a concentration of 4000 units / ml in PBS was passively adsorbed to 96-well microtiter plates overnight at 4°C. Subsequently, the plates were washed with PBST and blocked with 0.5% BSA / PBS for 1 hour. Biotinylated BSMUC16 / CD3-001, MUC16 parental antibody, a-MUC16 3A5, and non-binding controls (BSMUC16 / CD3-001 isotype control and a-MUC16 3A5 isotype control) were added to the plates at concentrations of 10, 1, 0.3, or 0.1 nM in 0.5% BSA / PBS for 1 hour, followed by washing with PBST. Streptavidin coupled to horseradish peroxidase (SA-HRP) (ThermoFisher Scientific, Waltham, MA, USA) was added to the wells at a 1:10000 dilution of a 1.0 mg / mL stock solution and incubated for 1 hour to detect plate-bound biotinylated antibodies. The plates were washed according to the manufacturer's instructions and developed with 3-3'-tetramethylbenzidine, 5-5'-tetramethylbenzidine (BD Biosciences, Franklin Lakes, NJ, USA) substrates. The absorbance of each well at 450 nm was recorded on a Victor Multilabel Plate Reader (PerkinElmer, Melville, NY). Data were analyzed using GraphPad Prism software.

[0143] Excess CA-125 had minimal effect on BSMUC16 / CD3-0001 cells binding to OVCAR-3, indicating minimal binding to CA-125 ( Figure 1 In contrast, CA-125 greatly inhibited the ability of a comparable antibody (antibody clone 3A internal version) that could bind to the repeat region of MUC16 ( Figure 1). In addition, as discussed in more detail in WO 2018 / 067331, which is incorporated herein by reference, a soluble MUC16 construct containing a membrane-proximal region reaching 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. In contrast to the binding studies, BSMUC16 / CD3-001 can also induce T cell-mediated killing in the presence of CA-125 but not in the presence of high concentrations of MUC16Δ (data not shown). Therefore, even in the presence of high concentrations of CA-125, BSMUC16 / CD3-001 can bind to MUC16 and induce T cell redirected killing.

[0144] Example 3: PD-1 blockade enhances the anti-tumor activity of the anti-MUC16xCD3 bispecific antibody in allogeneic and syngeneic tumor models

[0145] The in vivo efficacy of anti-MUC16 / anti-CD3 bispecific antibodies and PD-1 blockade was evaluated in allogeneic and syngeneic tumor models.

[0146] A. Allogeneic Model - OVCAR-3 / Luc

[0147] For allogeneic models, 13 days after transplanting human PBMC, OVCAR-3 / Luc cells (day 0) that were previously passaged in vivo were injected intraperitoneally (IP) to immune-deficient NSG mice. Mice were treated with IP with 12.5 μg / mouse BSMUC16 / CD3-001, and 12.5 μg CD3 binding controls were applied alone or in combination with 100 μg REGN2810 on the 5th and 8th days. Tumor burden was assessed by BLI on the 4th, 8th, 12th, 15th, 20th and 25th days after tumor implantation. As measured by BLI measurement on the 25th day, the treatment performed with 12.5 μg BSMUC16 / CD3-001 produced significant anti-tumor efficacy as measured by BLI measurement and further enhanced anti-tumor efficacy with REGN2810 (anti-PD-1). As assessed by BLI before administration began, all groups had similar tumor burden. There was no significant difference in tumor burden between the groups.

[0148] 12.5 μg BSMUC16 / CD3-001 significantly reduced tumor burden and the addition of anti-PD-1 enhanced anti-tumor efficacy compared to the addition of BSMUC16 / CD3-001 alone. Human OVCAR-3 / Luc cells were implanted in NSG mice transplanted with human T cells. Mice were treated with BSMUC16 / CD3-001 administered with 12.5 μg IV or treated with CD3 binding control or non-binding control (12.5 μg IV) on the 5th and 8th days. The data shown in Table 3 below are tumor burdens as assessed by BLI on the 25th day after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t test. Treatment with BSMUC16 / CD3-001+ / -REGN2810 was compared to CD3 binding control (*p<0.05 for BSMUC16 / CD3-001, **p<0.01 for BSMUC16 / CD3-001 and REGN2810), and treatment with BSMUC16 / CD3-001 alone and in combination with REGN2810 were compared (#p<0.05).

[0149] Table 3: Bioluminescence on day 25 after tumor implantation

[0150]

[0151] B. Syngeneic Model - ID8-VEGF / huMUC16

[0152] To examine efficacy in an immune competent model, the murine CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with a human sequence. The replacement generated mice whose T cells expressed human CD3 and, in the context of the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies, a chimeric MUC16 molecule containing a portion of human MUC16.

[0153] For this first syngeneic tumor model, an ID8-VEGF cell line engineered to express a portion of human MUC16 was used. Three days after implantation, mice were implanted IP with ID8-VEGF / huMUC16 cells and treated with 5 mg / kg BSMUC16 / CD3-001 or CD3 binding control and isotype control or in combination with anti-PD-1 (5 mg / kg IV). Treatment with BSMUC16 / CD3-001 extended median survival compared to the group receiving CD3 binding control, but the addition of anti-PD-1 blockade also resulted in 50% mouse survival.

[0154] BSMUC16 / CD3-001 significantly prolonged the median survival time in the ID8-VEGF ascites model and the addition of PD-1 (REGN2810) blockade allowed several mice to survive. A murine ovarian tumor line expressing a portion of human MUC16 was implanted into mice expressing human CD3 but not mouse CD3 and chimeric MUC16 molecules. Mice were administered BSMUC16 / CD3-001 (5 mg / kg IV) or CD3 binding control (5 mg / kg IV) with isotype control or anti-PD-1 on day 3 after implantation. Mice were treated on days 3, 7, 10, 14, and 17 after tumor implantation. The data shown are median survival. Mice were sacrificed when they gained more than 20% body weight due to abdominal distension induced by ascites. Statistical significance was determined using the Mantel-Cox method. BSMUC16 / CD3-001 and BSMUC16 / CD3-001+anti-PD-1 treatments resulted in prolonged median survival, and the combination of BSMUC16 / CD3-001+anti-PD-1 produced a 50% survival rate, demonstrating a synergistic effect between the MUC16xCD3 bispecific antibody and the anti-PD-1 antibody. The results are shown in Table 4 below.

[0155] Table 4: Median survival in the ID8-VEGF / huMUC16 model

[0156] Antibody (mg / kg) Median survival time (days) CD3 Binding Control (5) + Isotype Control (5) 36 BSMUC16 / CD3-001(5)+isotype control(5) 46 CD3 binding control (5) + PD-1 (5) 32 BSMUC16 / CD3-001(5)+PD-1(5) 69.5

[0157] Similar results were observed when 1 mg / kg BSMUC16 / CD3-001 was administered together with anti-PD-1 antibody.

[0158] C. Syngeneic Model - MC38 / huMUC16

[0159] As discussed above, the mice used in this experiment were engineered so that the murine CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with a human sequence. The replacement produced mice whose T cells expressed human CD3 and, in the context of the BSMUC16 / CD3-001 and BSMUC16 / CD3-005 bispecific antibodies, expressed a chimeric MUC16 molecule containing a portion of human MUC16.

[0160] For this second syngeneic tumor model, the MC38 line engineered to express a portion of human MUC16 was used. Mice were implanted SC with MC38 / huMUC16 cells and treated with BSMUC16 / CD3-005 or CD3 binding control in combination with isotype control (1 mg / kg IV) or with anti-PD-1 (5 mg / kg IV) on day 7 after tumor implantation. The anti-PD-1 antibody used in this experiment was a commercially available murine antibody (clone RMP1-14, BioXCell). The combination of BSMUC16 / CD3-005 and anti-PD-1 showed a synergistic anti-tumor effect.

[0161] In the MC38 SC model, the combination of BSMUC16 / CD3-005 and anti-PD-1 blockade produced better anti-tumor efficacy than BSMUC16 / CD3-005 alone. The murine tumor line MC38 expressing a portion of human MUC16 was implanted into mice expressing human CD3 but not mouse CD3 and chimeric MUC16 molecules. Mice were administered BSMUC16 / CD3-005 or CD3 binding control (1 mg / kg IV) with isotype control or anti-PD-1 antibody (5 mg / kg IV) on day 7 after implantation. Mice were treated on days 7, 11, and 14 after tumor implantation. The results are shown in Figure 2 Statistical significance was determined using two-way ANOVA with Tukey's multiple comparison test. BSMUC16 / CD3-005 plus anti-PD-1 significantly and synergistically inhibited tumor growth compared to CD3-bound control.

[0162] Example 4: ImmunoPET imaging in engineered mice shows localization of anti-MUC16xCD3 bispecific antibodies to T cell-enriched organs

[0163] The in vivo localization of BSMUC16 / CD3-001 and BSMUC16 / CD3-005 and the expression of MUC16 protein were evaluated in wild-type and genetically humanized mice using PET imaging. 89 The biodistribution of Zr-labeled anti-MUC16 antibodies (bivalent anti-MUC16 antibodies generated using the same anti-MUC16 heavy and light chains as bispecifics, referred to herein as "parent") was similar, indicating low expression / availability of humanized MUC16 protein for the antibody. In contrast, when mice were administered therapeutically relevant doses of 89When the Zr-labeled BSMUC16 / CD3-001 bispecific antibody was used, distribution to the spleen and lymph nodes was evident due to recognition by CD3-positive T cells in these lymphoid organs (data not shown). Ex vivo biodistribution analysis in separate tissues confirmed localization to the lymph nodes and spleen (data not shown). 89 The uptake of the Zr-labeled BSMUC16 / CD3-005 bispecific antibody was greatly reduced due to its lower affinity for CD3. To evaluate whether BSMUC16 / CD3-001 and BSMUC16 / CD3-005 can accumulate in MUC16-expressing tumors, mice bearing ID8-VEGF-huMUC16Δ tumors were administered 89 Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005. Although lymphatic uptake of BSMUC16 / CD3-001 was higher, tumor uptake did not differ significantly between the bispecific antibodies (data not shown).

[0164] Preparation of immunoconjugates and small animal PET BSMUC16 / CD3-001 and control antibodies were conjugated with DFO to glutamine at position 295 by transamidation using microbial transglutaminase followed by antibody deglycosylation with PNGase F. Subsequently, the DFO-conjugated antibodies were conjugated with zirconium-89 ( 89 The mice received a final dose of 0.5 mg / kg of the antibody by tail vein injection. Subsequently, PET imaging was performed to assess the in vivo localization of the radioimmunoconjugate on day 6 after dosing, followed by ex vivo biodistribution studies. For experiments in tumor-bearing mice, mice were implanted subcutaneously with 10×10 6 ID8-VEGF-huMUC16Δ tumor cells. 20 days after implantation, the tumors were an average of 150 mm 3 When tumor-bearing mice were given 89 Zr radiolabeled antibodies.

[0165] PET and CT images were acquired using a precalibrated Sofie Biosciences G8 PET / CT instrument (Sofie Biosciences (Culver city, CA) and PerkinElmer). The energy window ranged from 150 to 650 keV with a reconstruction resolution of 1.4 mm at the center of the field of view. On day 6 after dosing, mice underwent induction anesthesia using isoflurane and were maintained under a continuous flow of isoflurane during a 10-minute static PET acquisition. CT images were obtained after PET acquisition. Subsequently, PET images were reconstructed using pre-arranged settings. Attenuation-corrected PET data and CT data were processed into false-color co-registered PET-CT maximum intensity projections using VivoQuant software (inviCRO Imaging Services) on a colorimeter calibrated to indicate a signal range of 0 to 30% per volume injected dose expressed as ID / g%. For ex vivo biodistribution analysis, mice were euthanized after imaging on day 6 after dosing. Blood was collected into counting tubes by cardiac puncture. Subsequently, normal tissues (inguinal and axillary lymph nodes, thymus, spleen, heart, lung, stomach, small intestine, liver, kidney, bone and ovary) were removed and placed in counting tubes. Tumors were collected in counting tubes in a similar manner. All test tubes were weighed in advance and then weighed again to measure the weight of blood and tissue. Subsequently, the γ-emitting radioactivity of all samples was counted on an automatic γ counter (Wizard 2470, Perkin Elmer), and the results were reported in units of counts / minute (cpm). The ID% of each sample was determined using sample counts relative to the dose standard counts prepared from the original injection material. Subsequently, a separate ID / g% value was obtained by dividing the ID% value by the corresponding weight of the appropriate blood, tissue or tumor sample.

[0166] through 89 Zr-labeled BSMUC16 / CD3-001 and 89 Zr-labeled BSMUC16 / CD3-005 exhibited specific localization to MUC16+ tumors and CD3+ lymphoid tissues, with lymphoid distribution correlating with relative CD3 affinity. Both MUC16xCD3 bispecifics exhibited equivalent tumor localization in the presence of CD3+ tissues.

[0167] Example 5: Toxicology studies in cynomolgus monkeys showed no significant toxicity of anti-MUC16xCD3 bispecific antibody

[0168] BSMUC16 / CD3-001 cross-reacts with monkey MUC16 and CD3. In order to determine the safety and tolerability of the bispecific antibody and characterize its pharmacokinetics, a multiple-dose toxicity study was performed in cynomolgus monkeys. Six monkeys / sex / groups received weekly administration of 0.01, 0.1 or 1 mg / kg BSMUC16 / CD3-001, for a total of five doses. At the end of the dosing period, 3 animals / sex / groups were euthanized and the microscopic findings of the tissues were examined, while the remaining three animals / sex / groups underwent 12 weeks of treatment-free recovery to assess the reversibility or persistence of any BSMUC16 / CD3-001-related effects. BSMUC16 / CD3-001 was well tolerated, and all animals survived to the pre-scheduled autopsy time. Toxicokinetic analysis confirmed dose-proportional exposure and linear kinetics throughout the dose group, and no gender differences were observed (data not shown). Continuous exposure to BSMUC16 / CD3-001 was observed throughout the dosing period, and BSMUC16 / CD3-001 exposure was maintained in all (n=6) animals and 50% of the animals in the 0.1 and 1.0 mg / kg groups, respectively, until the end of the recovery period. BSMUC16 / CD3-001 was not detected in the serum of any animal in the 0.01 mg / kg group after recovery week 8. The elimination half-life of BSMUC16 / CD3-001 was approximately 10 days.

[0169] There were no BSMUC16 / CD3-001-related clinical observations during the dosing or recovery phases, nor were there any changes in urinalysis parameters, peripheral blood immunophenotype, food consumption, or body weight. Importantly, BSMUC16 / CD3-001 administration did not produce any changes in respiratory, neurological, or cardiovascular safety pharmacological assessments, including no changes in ECG parameters. No BSMUC16 / CD3-001-related changes in organ weights were found, nor were any macroscopic changes noted at terminal or recovery autopsies. 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 evident 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), in contrast to what has been described for several CD3 bispecific molecules against hematological tumors.

[0170] The cynomolgus monkey study was conducted according to the criteria of IACUC. Control articles (diluted placebo) or BSMUC16 / CD3-001 (0.01, 0.1 or 1 mg / kg) were administered to cynomolgus monkeys (6 animals / sex / group) once a week by 30-minute IV infusion. The control article was 10 mM histidine diluted with 10% sucrose and 0.05% polysorbate 20, pH 6, 0.9% sodium chloride for injection, USP (sterile saline). Blood samples or tissues were collected at various time points for clinical pathology and pathological histology. BSMUC16 / CD3-001 concentration was determined by ELISA, and toxicokinetic analysis was performed using WinNonLin software. CRP was analyzed on a Roche Modular P 800 system. Cytokines were measured by MSD (Meso Scale Diagnostics, Rockville, MD). T cells were quantified using flow cytometry. Briefly, blood was collected in potassium EDTA tubes, lysed, stained for CD3, CD4, and CD8 (BD Biosciences), and the relative values ​​of each phenotype were determined using a FACS Canto II. Subsequently, these values ​​were multiplied by the absolute lymphocyte values ​​(by hematological analysis) to calculate the absolute cell counts for each phenotype.

[0171] Immunohistochemical staining for MUC16 was present in the following 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 / efferent ducts) and tonsils (epithelium, mucous glands) (not shown). BSMUC16 / CD3-001-related microscopic changes assessed by hematoxylin and eosin (H&E) histological staining included inflammation (leukocyte infiltration) and increased mesothelial cell size and cellularity, resulting in non-detrimental 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 and minimal to mild in severity and were considered on-target for BSMUC16 / CD3-001 due to engagement of MUC16 expressed on serosal epithelial (mesothelial) cells and activation of T cells. Importantly, serosal changes reversed or tended to reverse by the end of the recovery period (data not shown).

[0172] Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein and no overt toxicity following BSMUC16 / CD3-001 administration.

[0173] Example 6: Evaluation of serum cytokine induction in tumor-bearing mice

[0174] Because cytokine release syndrome (CRS) is a frequent severe side effect of CD3 bispecific and CAR T cell therapy, studies were performed to monitor serum cytokines in relevant models following treatment with BSMUC16 / CD3-001. In genetically humanized MUC16 / CD3 mice that do not bear tumors, there was no apparent serum cytokine response upon BSMUC16 / CD3-001 administration.

[0175] In order to evaluate the in vivo T cell activation performed by BSMUC16 / CD3-001, the serum cytokine content of mice carrying tumors was measured. Serum samples were collected 4 hours after 0.5mg / kg of the first antibody dose of BSMUC16 / CD3-001, CD3 binding control and non-binding control group. Compared with non-binding control and CD3 binding control, as determined by IFNγ, TNFα, IL-2, IL-6, IL-8 and IL-10 induction, the treatment activated T cells (data not shown) performed with BSMUC16 / CD3-001. The cytokine response induced by BSMUC16 / CD3-001 requires the presence of T cells and OVCAR-3 / Luc cells, because there is no detectable human IFNγ in the serum of mice carrying only OVCAR3 / Luc cells, and mice without tumor cells cross-linked for MUC16 do not show an increase in serum IFNγ in response to BSMUC16 / CD3-001 (data not shown).

[0176] Measurement of serum cytokine levels : T cell activation in response to treatment with BSMUC16 / CD3-001 was assessed by measuring serum concentrations of interferon gamma (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 four hours after the first 0.5 mg / kg dose. Cytokine content was analyzed using a V-plex human proinflammatory-10Plex ​​kit following the manufacturer's instructions (Meso Scale Diagnostics, Rockville, MA). Cytokines were measured in two separate studies with 4 to 6 mice per group.

[0177] Example 7: MUC16 expression in humanized mice and the effects of anti-MUC16xCD3 bispecific antibodies on MUC16-positive tissues

[0178] In order to investigate 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 in both endogenous murine loci (gene knock-in mice). In order to verify these mice, MUC16 expression was checked by RT-PCR and IHC. RNA expression was detected in the trachea and low RNA expression levels were detected in the lungs, heart, ovaries, pancreas and bladder (data not shown), which is similar to the published data on murine MUC16 expression. In order to assess MUC16 protein expression, IHC was performed on selected tissues using anti-human MUC16 antibodies that recognize the near-membrane region of MUC16. MUC16 protein expression was confirmed in the epithelial surfaces of the ovaries and stomachs in these mice. As described in humans, MUC16 was also observed in the tracheal lining / epithelium and submucosal glands (data not shown).

[0179] About Histology of Mouse TissuesTissues from humanized or WT mice were harvested and stained by IHC using Ventana Discovery XT (Ventana; Tucson, AZ) with an anti-MUC16 antibody that binds to the juxtamembrane domain of MUC16. 5 μm paraffin sections were cut onto Superfrost PLUS slides and baked at 60°C for one hour. Immunohistochemical staining was performed using the Ventana DAB MAP detection kit on the DiscoveryXT automated IHC staining system. Deparaffinization was performed at 75°C for 8 minutes using EZPrep solution. Mild antigen retrieval (95°C, 8 minutes, followed by 100°C, 24 minutes) was performed using pH 9 Tris-EDTA buffer (CC1) from Ventana. This was followed by multiple blocking steps. Tissue sections were incubated with anti-MUC16 antibody (2 μg / ml) at RT 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 manually coated. Biotinylated goat anti-human IgG (Jackson ImmunoResearch) was used as a secondary antibody (1 μg / ml), and samples were incubated for one hour at RT. The chromogenic signal was developed using the Ventana DABMAP kit. The slides were counterstained with hematoxylin (2 minutes) manually, dehydrated, and covered with a coverslip. Images were obtained on an Aperio AT 2 slide scanner (Leica Biosystems; Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs; Corrales, NM). H&E staining was performed by Histoserv (Germantown, MD, USA).

[0180] As assessed by T cell receptor (TCR) Vβ use, T cells in these mice were polyclonal, expressed human CD3 and were present in numbers similar to wild-type mice (data not shown). To determine whether BSMUC16 / CD3-001 induced any T cell activation or effects on normal tissues in these animals, non-tumor-bearing mice were injected with a high dose of BSMUC16 / CD3-001 (10 mg / kg), and T cell numbers in the blood, serum cytokines, and pathological histology were subsequently measured. Although T cells can be activated by anti-human CD3 antibodies (OKT3) as measured by T cell aggregation relative to blood and increased serum cytokine levels (data not shown), BSMUC16 / CD3-001 did not induce any of the effects, indicating limited accessibility of the MUC16 target (data not shown). To determine whether BSMUC16 / CD3-001 induced any microscopic changes in MUC16 expressing tissues, MUC16 and CD3 humanized mice received two doses of BSMUC16 / CD3-001 at 10 mg / kg on days 0 and 3. On day 5, several MUC16 expressing tissues (trachea, stomach, and ovaries) were examined, and no cellular infiltration or necrosis was seen in these tissues after BSMUC16 / CD3-001 administration (data not shown). Histopathological examination revealed no inflammation or infiltration into MUC16 expressing tissues in mice after BSMUC16 / CD3-001 administration at the time of examination.

[0181] The results of this study, as well as the cynomolgus monkey study discussed in Example 5, demonstrate the safety profile of BSMUC16 / CD3-001. BSMUC16 / CD3-001 induced only minimal serum cytokines, and although there were focal inductions of inflammation and thickening of the serosal lining indicating MUC16 expression on target activity, these effects resolved before the end of the recovery period and were consistent with inflammation and increased cellularity indicative of repair. The observed serosal changes were not associated with any clinical observations, clinical pathology (except for inflammatory responses), or microscopic changes in the underlying brain parenchyma. Therefore, studies in both genetically humanized mice and cynomolgus monkeys showed that BSMUC16 / CD3-001 was well tolerated.

[0182] Example 8: Monitoring PD-1 expression in a FACS-based cytotoxicity assay using naive human effector cells

[0183] To monitor specific killing of target cells carrying Muc16 by flow cytometry, the ovarian cell line OVCA-3 was labeled with 1 μM purple cell tracer. After labeling, cells were plated overnight at 37°C. Separately, 1 × 10 6Human PBMCs were plated at 100 cells / ml and cultured overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells and some monocytes. The next day, target cells were co-cultured with initial PBMCs (4:1 effector cells / target cells) depleted of adherent cells and serial dilutions of BSMUC16 / CD3-001 or CD3 binding controls at 37°C for 72 hours. Cells were removed from the cell culture plates using trypsin and analyzed by FACS. For FACS analysis, cells were stained with a dead / live far-erythrocyte tracer (Invitrogen). To assess killing specificity, cells were gated on a population labeled with a purple cell tracer.

[0184] PD-1 expression was assessed by reporting the percentage of PD-1 / CD4 positive T cells or PD-1 / CD8 positive T cells among total T cells (CD2+) by incubating cells with antibodies directly conjugated to CD2, CD4, CD8, and PD-1. 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 control. Results are shown in Figure 3 middle.

[0185] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to the modifications described herein, will become apparent to those skilled in the art from the foregoing description. The modifications are intended to fall within the scope of the appended claims.

Claims

1. Use of a combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and a bispecific anti-MUC16×CD3 antibody in the preparation of a medicament for treating an individual's ovarian cancer expressing MUC16 or inhibiting the growth of an individual's ovarian cancer expressing MUC16, wherein (a) an anti-PD-1 antibody or an antigen-binding fragment thereof specifically binds to programmed death receptor 1 (PD-1) and comprises three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 consist of the amino acid sequences of SEQ ID NOs: 35, 36, 37, 38, 39 and 40, respectively; (b) The bispecific anti-MUC16×CD3 antibody comprises a first antigen-binding arm that specifically binds to MUC16, and a second antigen-binding arm that specifically binds to CD3, wherein (i) the first antigen-binding arm comprises three heavy chain complementary determining regions A-HCDR1, A-HCDR2 and A-HCDR3 and three light chain complementary determining regions A-LCDR1, A-LCDR2 and A-LCDR3, wherein A-HCDR1, A-HCDR2, A-HCDR3, A-LCDR1, A-LCDR2 and A-LCDR3 consist of the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively; and (ii) the second antigen-binding arm comprises three heavy chain complementary determining regions B-HCDR1, B-HCDR2 and B-HCDR3 and three light chain complementary determining regions B-LCDR1, B-LCDR2 and B-LCDR3, wherein B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 consist of the amino acid sequences of SEQ ID NOs: 14, 15, 16, 11, 12 and 13, respectively, or consist of the amino acid sequences of SEQ ID NOs: 26, 27, 28, 11, 12 and 13.

2. The use according to claim 1, wherein the individual is resistant or has responded inadequately to previous therapy or has relapsed after previous therapy.

3. The use according to claim 1 or 2, wherein the first antigen-binding arm of the bispecific anti-MUC16×CD3 antibody comprises a heavy chain variable region HCVR consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region LCVR consisting of the amino acid sequence of SEQ ID NO:

2.

4. The use according to claim 1 or 2, wherein the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 of the second antigen-binding arm of the bispecific anti-MUC16×CD3 antibody consist of the amino acid sequences of SEQ ID NOs: 14, 15, 16, 11, 12 and 13, respectively. 5 . The use according to claim 4 , wherein the second antigen-binding arm of the bispecific anti-MUC16×CD3 antibody comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 3, and a LCVR consisting of the amino acid sequence of SEQ ID NO:

2.

6. The use according to claim 1 or 2, wherein the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 of the second antigen-binding arm of the bispecific anti-MUC16×CD3 antibody consist of the amino acid sequences of SEQ ID NOs: 26, 27, 28, 11, 12 and 13, respectively.

7. The use according to claim 6, wherein the second antigen-binding arm of the bispecific anti-MUC16×CD3 antibody comprises a HCVR consisting of the amino acid sequence of SEQ ID NO:7, and a LCVR consisting of the amino acid sequence of SEQ ID NO:

2.

8. The use according to claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 33, and a LCVR consisting of the amino acid sequence of SEQ ID NO:

34.

9. The use according to claim 1 or 2, wherein the bispecific anti-MUC16×CD3 antibody comprises a first heavy chain consisting of the amino acid sequence of SEQ ID NO: 29, which is paired with a light chain consisting of the amino acid sequence of SEQ ID NO: 30; and a second heavy chain consisting of the amino acid sequence of SEQ ID NO: 31, which is paired with a light chain consisting of the amino acid sequence of SEQ ID NO:

30.

10. The use according to claim 1 or 2, wherein the bispecific anti-MUC16×CD3 antibody comprises a first heavy chain consisting of the amino acid sequence of SEQ ID NO: 29, which is paired with a light chain consisting of the amino acid sequence of SEQ ID NO: 30; and a second heavy chain consisting of the amino acid sequence of SEQ ID NO: 32, which is paired with a light chain consisting of the amino acid sequence of SEQ ID NO:

30.

11. The use according to claim 1 or 2, wherein the anti-PD-1 antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 41, and a light chain consisting of the amino acid sequence of SEQ ID NO:

42.

12. The use according to claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment and the bispecific anti-MUC16 x CD3 antibody are formulated separately.

13. The use according to claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment and the bispecific anti-MUC16 x CD3 antibody are co-formulated.

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