EA046291B1 - METHODS FOR TREATING MALIGNANT NEOPLOGMS WITH BISPECIFIC ANTIBODIES AGAINST CD3XMUC16 AND ANTIBODIES AGAINST PD-1 - Google Patents

Description

Link to sequence list

The Sequence Listing is incorporated by reference into this application in machine-readable form as file 10469WO01-Sequence.txt, created on June 12, 2019 and measuring 33,567 bytes.

Technical field

This invention relates to methods of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds to programmed cell death receptor 1 (PD-1), in combination with a bispecific antibody that binds to mucin 16 (MUC16). ) and CD3.

State of the art

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 main domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with the sea urchin sperm protein, enterokinase, and agrin domains (SEA domain), and a C-terminal domain that contains a segment of the transmembrane region and a short cytoplasmic tail. . Proteolytic cleavage releases the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in malignancies including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma, massive form, cervical adenocarcinoma and gastric adenocarcinoma, as well as in diseases and conditions including inflammatory bowel disease, cirrhosis of the liver , 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) Treatments for ovarian cancer using antibodies to MUC16 have been investigated. Oregovomab and abgovomab are anti-MUC16 antibodies that have limited effectiveness. (Felder, supra, Das, S. and Batra, S.K. 2015, Cancer Res. 75:4660-4674.)

CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor (TCR) complex and is required for T cell activation. Functional CD3 is formed by the dimeric association of two of four different chains: epsilon, zeta, delta and gamma. CD3 dimeric structures include gamma/epsilon, delta/epsilon, and zeta/zeta. Anti-CD3 antibodies have been shown to form CD3 clusters on T cells, thereby causing T cell activation similar to TCR engagement by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes, including T cell activation. In addition, bispecific antibodies that are capable of binding CD3 and the target antigen have been proposed for use in therapeutic applications, including targeting the T-cell immune response to tissues and cells expressing the target antigen.

Programmed cell death receptor 1 (PD-1) signaling in the tumor microenvironment plays a key role in allowing tumor cells to evade immune surveillance by the host immune system. Blockade of the PD-1 signaling pathway has demonstrated clinical activity in patients with multiple tumor types, and therapeutic antibodies that block PD-1 (eg, 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).

Ovarian cancer represents the deadliest of gynecologic malignancies; Although the estimated number of new cases of ovarian cancer among American women is much lower than for some other malignancies, the death-to-incidence ratio of ovarian cancer is significantly higher (Siegal et al., CA Cancer J Clin 66:7-30, 2016). Ovarian cancer is often diagnosed at an advanced stage, which contributes to its mortality rate. Currently, the standard treatment for ovarian cancer is surgery followed by chemotherapy, namely a combination of platinum drugs and taxanes. While most patients respond to initial treatment, most experience disease recurrence, leading to a cycle of repeat surgeries and additional courses of chemotherapy. Although recurrent ovarian cancer may respond to further treatment, virtually all types will eventually become resistant to currently available treatments. Despite recent advances in therapy, such as the use of PARP inhibitors for patients carrying BRCA or other homologous recombination mutations (HRD), advanced ovarian cancer remains a disease with high unmet need.

Evidence suggests that ovarian cancer is amenable to some forms of immunotherapy (Kandalaft et al., J. Clin. Oncol., 29:925-933, 2011). For example, patients with ovarian cancer, koto tumors

- 1046291 patients were positive for intraepithelial CD8+ T-lymphocyte infiltration and had significantly better overall survival and progression-free survival than patients without intraepithelial infiltration of CD8' T-lymphocytes (Hamanishi et al., PNAS, 104:3360-65, 2007; and Zhang et al., N. Engl. J. Med., 348:203-213, 2003). Moreover, some patients exhibit a spontaneous immune response to tumors, as demonstrated by the detection of tumor-reactive T cells and antibodies in the blood, tumor, or ascites of patients with advanced disease (Schliengar et al., Clin Cancer Res, 9:1517-1527, 2003 ). Blockade of the PD-1/PD-L1 checkpoint pathway has demonstrated some benefit in ovarian cancer; PD-1 blockade as monotherapy resulted in an overall response rate (ORR) of approximately 10–15% in early phase clinical trials (Hamanishi et al., supra). However, blocking this path alone is clearly not enough.

Due to the high unmet need for effective treatments for ovarian cancer, it may be useful, as demonstrated herein, to combine treatment with an agent to enhance T cell function (eg, a PD-1 inhibitor, such as an anti-PD-1 antibody) along with an agent against the target antigen (bispecific antibody against MUC16/CD3).

Brief description of the invention

In accordance with certain embodiments, the present invention provides methods for treating, reducing the occurrence of at least one symptom or indication, or inhibiting the growth of a cancer in a subject. Methods according to this aspect of the invention include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to programmed cell death protein 1 (PD-1), in combination with a therapeutically effective amount of a bispecific antibody that specifically binds to MUC16 and CD3, to a subject who needs it.

In some embodiments, the present invention provides methods for treating, reducing the occurrence of at least one symptom or indication, or inhibiting the growth of a cancer in a subject. In some embodiments, the present invention provides methods for delaying tumor growth or preventing tumor recurrence. Methods in accordance with this and other aspects of the invention include sequential administration of one or more doses of a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to PD-1, in combination with one or more doses of a therapeutically effective amount of a bispecific antibody that specifically binds to MUC16 and CD3, in the subject in need. In one aspect of the present invention, there is provided a method of treating or inhibiting tumor growth, comprising administering to a subject in need thereof (a) a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody comprising a first antigen binding arm that specifically binds MUC16 and a second antigen binding arm that specifically binds CD3. In some cases, the anti-PD-1 antibody is administered before, simultaneously with, 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 an anti-PD-1 antibody are administered in combination with one or more doses of a bispecific antibody. In some cases, the anti-PD-1 antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight. In some cases, each dose of anti-PD-1 antibody ranges from 10 to 8,000 mcg. In some cases, the bispecific antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight. In some cases, each dose of bispecific antibody ranges from 10 to 8000 mcg. In some cases, each dose of anti-PD-1 antibody is administered 0.5 to 12 weeks after the immediately preceding dose. In some cases, each dose of the bispecific antibody is administered 0.5 to 12 weeks after the immediately preceding dose. In various embodiments, the antibodies are administered intravenously, subcutaneously, or intraperitoneally.

In some embodiments, the tumor includes ovarian cancer. In some embodiments, the subject is resistant to, has had an insufficient response to, or has relapsed after prior therapy.

In some cases, the method further includes administering to the subject a third therapeutic agent or therapy. In some embodiments, the third therapeutic agent or therapy is selected from the group consisting of radiation therapy, surgery, a chemotherapy agent, a cancer vaccine, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, a TLM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor , CD47 inhibitor, indoleamine 2,3 dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist, angiopoietin-2 (Ang2) inhibitor, transforming growth factor beta (TGF-β) inhibitor, epidermal growth factor receptor (EGFR) inhibitor , antibodies to tumor-specific antigen, bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor, cytotoxin, interleukin-6 receptor inhibitor (IL-6K). interleukin-4 receptor (IL-4R) inhibitor, IL-10, IL-2, IL-7, IL-21, IL-15 inhibitor, antibody-drug conjugate, anti-inflammatory

- 2 046291 medicinal product and biologically active food supplement.

In some embodiments, the anti-PD-1 antibody or antigen binding fragment thereof comprises heavy chain complementarity determining regions (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 complementarity determining regions (LCDR1, LCDR2 and LCDR3) light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34. In some cases, HCDR1 contains the amino acid sequence of SEQ ID NO: 35; HCDR2 contains the amino acid sequence SEQ ID NO: 36; HCDR3 contains the amino acid sequence SEQ ID NO: 37; LCDR1 contains the amino acid sequence SEQ ID NO: 38; LCDR2 contains the amino acid sequence SEQ ID NO: 39; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO: 33 and LCVR contains 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 the sequence of SEQ ID NO: 41, and a light chain containing the amino acid sequence of SEQ ID NO: 42.

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 the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light CDRs chains (A-LCDR1, A-LCDR2 and A-LCDR3) of the light chain variable region (A-LCVR) containing the amino acid sequence of SEQ ID NO 2. In some cases, A-HCDR1 contains the amino acid sequence of SEQ ID NO 8; A-HCDR2 contains the amino acid sequence SEQ ID NO 9; A-HCDR3 contains the amino acid sequence SEQ ID NO 10; A-LCDR1 contains the amino acid sequence SEQ ID NO 11; A-LCDR2 contains the amino acid sequence SEQ ID NO 12; and A-LCDR3 contains the amino acid sequence of SEQ ID NO: 13. In some cases, A-HCVR contains the amino acid sequence of SEQ ID NO: 1, and A-LCVR contains the amino acid sequence of SEQ ID NO: 2.

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 NO 3, 4, 5, 6 and 7, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2. In some cases, B -HCDR1, B-HCDR2 and B-HCDR3 contain, respectively, amino acid sequences 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; and B-LCDR1, B-LCDR2 and B-LCDR3 contain, respectively, the amino acid sequences of SEQ ID NOs 11-12-13. In some cases, B-HCVR contains an amino acid sequence selected from the group consisting of SEQ ID NOs 3, 4, 5, 6 and 7, and B-LCVR contains the amino acid sequence of SEQ ID NO 2.

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) containing the amino acid sequence of SEQ ID NO 3, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2.

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) containing the amino acid sequence of SEQ ID NO 4, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2.

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) containing the amino acid sequence of SEQ ID NO 5, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2.

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) containing the amino acid sequence of SEQ ID NO 6, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2.

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) containing the amino acid sequence of SEQ ID NO 7, and three light chain CDRs (B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO 2.

- 3 046291

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

In another aspect, the present invention provides a method of treating or inhibiting tumor growth, comprising administering to a subject in need thereof (a) a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3, wherein: (a) the anti-PD-1 antibody or antigen-binding fragment thereof contains complementarity-determining regions of severe chain (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence SEQ ID NO: 34; (b) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (ALCVR) containing the amino acid sequence SEQ ID NO: 2; and (c) the second antigen binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and BHCDR3) of a heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs ( B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence SEQ ID NO: 2.

In some embodiments of the method, the anti-PD-1 antibody and the bispecific antibody, respectively, comprise the following: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 35; HCDR2 containing the amino acid sequence SEQ ID NO: 36; HCDR3 containing the amino acid sequence SEQ ID NO: 37; LCDR1 containing the amino acid sequence SEQ ID NO: 38; LCDR2 containing the amino acid sequence SEQ ID NO: 39; and LCDR3 containing the amino acid sequence SEQ ID NO: 40; (b) AHCDR1 containing the amino acid sequence of SEQ ID NO: 8; A-HCDR2 containing the amino acid sequence SEQ ID NO: 9; A-HCDR3 containing the amino acid sequence SEQ ID NO: 10; A-LCDR1 containing the amino acid sequence SEQ ID NO: 11; A-LCDR2 containing the amino acid sequence SEQ ID NO: 12; and A-LCDR3 containing the amino acid sequence SEQ ID NO: 13; and (c) B-HCDR1 containing the amino acid sequence of SEQ ID NO: 14; B-HCDR2 containing the amino acid sequence SEQ ID NO: 15; B-HCDR3 containing the amino acid sequence SEQ ID NO: 16; B-LCDR1 containing the amino acid sequence SEQ ID NO: 11; B-LCDR2 containing the amino acid sequence SEQ ID NO: 12; and B-LCDR3 containing the amino acid sequence SEQ ID NO: 13.

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

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 containing 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.

In some embodiments of the method, the tumor is ovarian cancer.

In another aspect, the present invention provides a method of treating or inhibiting tumor growth, comprising administering to a subject in need thereof (a) a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1); and (b) a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3, wherein: (a) the anti-PD-1 antibody or antigen-binding fragment thereof contains complementarity-determining regions of severe chains (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR) containing the amino acid sequence SEQ ID NO: 33, and three complementary determining

- 4 046291 light chain region (LCDR1, LCDR2 and LCDR3) light chain variable region (LCVR) containing the amino acid sequence SEQ ID NO: 34; (b) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (ALCVR) containing the amino acid sequence SEQ ID NO: 2; and (c) the second antigen binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and BHCDR3) of a heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 7, and three light chain CDRs ( B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence SEQ ID NO: 2.

In some embodiments of the method, the anti-PD-1 antibody and the bispecific antibody, respectively, comprise the following: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 35; HCDR2 containing the amino acid sequence SEQ ID NO: 36; HCDR3 containing the amino acid sequence SEQ ID NO: 37; LCDR1 containing the amino acid sequence SEQ ID NO: 38; LCDR2 containing the amino acid sequence SEQ ID NO: 39; and LCDR3 containing the amino acid sequence SEQ ID NO: 40; (b) AHCDR1 containing the amino acid sequence of SEQ ID NO: 8; A-HCDR2 containing the amino acid sequence SEQ ID NO: 9; A-HCDR3 containing the amino acid sequence SEQ ID NO: 10; A-LCDR1 containing the amino acid sequence SEQ ID NO: 11; A-LCDR2 containing the amino acid sequence SEQ ID NO: 12; and A-LCDR3 containing the amino acid sequence SEQ ID NO: 13; and (c) B-HCDR1 containing the amino acid sequence of SEQ ID NO: 26; B-HCDR2 containing the amino acid sequence SEQ ID NO: 27; B-HCDR3 containing the amino acid sequence SEQ ID NO: 28; B-LCDR1 containing the amino acid sequence SEQ ID NO: 11; B-LCDR2 containing the amino acid sequence SEQ ID NO: 12; and B-LCDR3 containing the amino acid sequence SEQ ID NO: 13.

In some embodiments of the method, the anti-PD-1 antibody and the bispecific antibody, respectively, comprise the following: (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 33 and an LCVR containing the amino acid sequence of SEQ ID NO: 34; (b) AHCVR containing the amino acid sequence of SEQ ID NO: 1, and A-LCVR containing the amino acid sequence of SEQ ID NO: 2; and (c) a B-HCVR containing the amino acid sequence of SEQ ID NO: 7, and a B-LCVR containing the amino acid sequence of SEQ ID NO: 2.

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 containing 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.

In some embodiments of the method, the tumor is ovarian cancer.

In various embodiments of any of the methods discussed above or herein, circulating CA-125 at concentrations up to 10 kU/ml does not significantly interfere with the antitumor activity of the bispecific antibody. In various embodiments of any of the methods discussed above or herein, a subject is diagnosed with ovarian cancer and the subject has circulating levels of CA-125 of up to 10 kU/mL. In some embodiments of the methods discussed above or herein, the subject has elevated serum CA-125 levels prior to treatment. In some embodiments of the methods discussed above or herein, the subject has a pre-treatment serum CA-125 level greater than or equal to 2 times the upper limit of normal serum CA-125 levels. Some variations of the methods discussed above or herein include monitoring serum CA-125 levels, for example, to evaluate the effectiveness of treatment by comparing serum CA-125 levels at various points during or after treatment with baseline serum CA-125 levels in a given patient or with baseline serum CA-125 levels in a general patient population.

In some embodiments, the antibodies discussed herein are used in the manufacture of a medicament for use in any of the methods discussed above or herein. In some embodiments, the antibodies discussed herein are for use in medicine or for the treatment of cancer, as discussed above or herein. For example, the present description includes:

(A) Use of a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3 in the manufacture of a medicament for treating or inhibiting tumor growth in

- 5,046,291 to a subject in need thereof, in combination with an antibody or an antigen-binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1);

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

(C) The use of a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3 in the manufacture of a medicament for treating or inhibiting tumor growth in a subject in need thereof, in combination with the antibody or an antigen binding fragment that specifically binds programmed cell death protein 1 (PD-1), wherein: (i) the anti-PD-1 antibody or antigen binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region ( HCVR) containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (ALCVR) containing the amino acid sequence SEQ ID NO: 2; and (iii) the second antigen binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and BHCDR3) of a heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs ( B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence SEQ ID NO: 2;

(D) Use of an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1) in the manufacture of a medicament for treating or inhibiting tumor growth in a subject in need thereof, in combination with a bispecific antibody comprising a first antigen binding an arm that specifically binds MUC16, and a second antigen-binding arm that specifically binds CD3, wherein: (i) the anti-PD-1 antibody or antigen-binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region ( HCVR) containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (ALCVR) containing the amino acid sequence SEQ ID NO: 2; and (iii) the second antigen binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and BHCDR3) of a heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs ( B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence SEQ ID NO: 2;

(E) The use of a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3 in the manufacture of a medicament for treating or inhibiting tumor growth in a subject in need thereof, in combination with the antibody or an antigen binding fragment that specifically binds programmed cell death protein 1 (PD-1), wherein: (i) the anti-PD-1 antibody or antigen binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region ( HCVR) containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (ALCVR) containing the amino acid sequence SEQ ID NO: 2; and (iii) the second antigen binding arm of the bispecific antibody comprises three heavy chain CDRs (B-HCDR1, B-HCDR2 and BHCDR3) of a heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 7, and three light chain CDRs ( B-LCDR1, B-LCDR2 and B-LCDR3) light chain variable region (B-LCVR) containing the amino acid sequence SEQ ID NO: 2;

(F) The use of an antibody or antigen-binding fragment thereof that specifically binds

- 6 046291 et programmed cell death protein 1 (PD-1), in the production of a medicament for treating or inhibiting tumor growth in a subject in need thereof, in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16, and a second an antigen binding arm that specifically binds CD3, wherein: (i) the anti-PD-1 antibody or antigen binding fragment thereof comprises the heavy chain complementarity determining regions (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 complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (A-LCDR1, A-LCDR2 and A-LCDR3) light chain variable region (A-LCVR) containing the amino acid sequence SEQ ID 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 the heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 7, and three light CDRs chains (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (BLCVR) containing the amino acid sequence of SEQ ID NO: 2;

(G) A bispecific antibody comprising a first antigen binding arm that specifically binds MUC16 and a second antigen binding arm that specifically binds CD3, for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with an antibody or an antigen binding fragment thereof, which specifically binds programmed cell death protein 1 (PD-1);

(H) An antibody or antigen-binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1), for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16, and a second antigen-binding arm that specifically binds CD3;

(I) A bispecific antibody comprising a first antigen binding arm that specifically binds MUC16 and a second antigen binding arm that specifically binds CD3, for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with an antibody or an antigen binding fragment thereof, which specifically binds programmed cell death protein 1 (PD-1), wherein: (i) the anti-PD-1 antibody or antigen binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR), containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (AHCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (ALCDR1 , A-LCDR2 and A-LCDR3) light chain variable region (A-LCVR) containing the amino acid sequence SEQ ID 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) containing the amino acid sequence of SEQ ID NO: 3, and three light CDRs chains (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO: 2;

(J) An antibody or antigen-binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1), for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with a bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16, and a second antigen-binding arm that specifically binds CD3, wherein: (i) the anti-PD-1 antibody or antigen-binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR), containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (AHCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (ALCDR1 , A-LCDR2 and A-LCDR3) light chain variable region (A-LCVR) containing the amino acid sequence SEQ ID 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) containing the amino acid sequence of SEQ ID NO: 3, and three light CDRs chains (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) containing

- 7 046291 amino acid sequence SEQ ID NO: 2;

(K) A bispecific antibody comprising a first antigen-binding arm that specifically binds MUC16 and a second antigen-binding arm that specifically binds CD3, for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with an antibody or an antigen-binding fragment thereof, which specifically binds programmed cell death protein 1 (PD-1), wherein: (i) the anti-PD-1 antibody or antigen binding fragment thereof contains the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR), containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (AHCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (ALCDR1 , A-LCDR2 and A-LCDR3) light chain variable region (A-LCVR) containing the amino acid sequence SEQ ID 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 the heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 7, and three light CDRs chains (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO: 2; and (L) An antibody or antigen-binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1), for use in treating or inhibiting tumor growth in a subject in need thereof, in combination with a bispecific antibody comprising a first antigen-binding arm, that specifically binds MUC16, and a second antigen-binding arm that specifically binds CD3, wherein: (i) the anti-PD-1 antibody or antigen-binding fragment thereof contains heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 33, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 34; (ii) the first antigen binding arm of the bispecific antibody contains three heavy chain CDRs (A-HCDR1, A-HCDR2 and A-HCDR3) of the heavy chain variable region (AHCVR) containing the amino acid sequence of SEQ ID NO: 1, and three light chain CDRs (ALCDR1 , A-LCDR2 and A-LCDR3) light chain variable region (A-LCVR) containing the amino acid sequence SEQ ID 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 the heavy chain variable region (B-HCVR) containing the amino acid sequence of SEQ ID NO: 7, and three light CDRs chain (B-LCDR1, B-LCDR2 and B-LCDR3) of the light chain variable region (B-LCVR) containing the amino acid sequence of SEQ ID NO: 2. Other embodiments of the present invention will become apparent from a review of the following detailed description.

Brief description of graphic materials

In FIG. 1 shows the binding of various concentrations of anti-MUC 16 clone 3A5 and BSMUC16/CD3-001 to CA125 as measured by ELISA (described in Example 2 herein). BSMUC16/CD3-001 and its parent MUC16 antibody exhibit a markedly reduced binding signal at all concentrations tested compared to anti-MUC 16 clone 3A5, which binds to the MUC16 repeat region.

In FIG. Figure 2 shows average tumor growth curves for groups of mice (5 per group) treated with CD3-binding control+isotype control (A, BSMUC16/CD3-005+isotype control ( ⁿ )'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.

In fig. Figure 3 shows the effect of incubating T cells with BSMUC16/CD3-001 on the percentage of PD-1 positive T cells.

Detailed description of the invention

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims. Any embodiments or features of embodiments may be combined with each other, and such combinations are clearly within the scope of the present invention. Any specific value described above or herein may be combined with another related value described above or herein to indicate a range with values

- 8 046291 representing the upper and lower limits of the range, and such ranges are included within the scope of the present disclosure.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. As used herein, the term about, when used in relation to a specific numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although 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 will now be described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

Methods for treating or suppressing the growth of malignant neoplasms

The present invention includes methods of treating, reducing the occurrence or severity of at least one symptom or indication, or inhibiting the growth of a cancer in a subject. Methods according to this aspect of the invention include administering a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds PD-1, in combination with a therapeutically effective amount of a bispecific antibody against MUC16 and CD3, to a subject in need thereof. As used herein, the terms treat, treatment, etc. means relief of symptoms, elimination of causes of symptoms on a temporary or permanent basis, delay or suppression of tumor growth, reduction of tumor cell mass or tumor burden to promote tumor regression, cause tumor shrinkage, necrosis and/or disappearance, prevent tumor recurrence and/or prolong duration subject's survival.

As used herein, the expression subject in need means a human or mammal, other than a human, who exhibits one or more symptoms or signs of a cancer, and/or who has been diagnosed with a cancer, including ovarian cancer, and who requires treatment for him. In many embodiments, the term subject may be used interchangeably with the term patient. For example, a human subject may be diagnosed with a primary or metastatic tumor and/or one or more symptoms or indications, including, but not limited to, swollen lymph nodes, abdominal bloating, chest pain/pressure, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, itching. The expression includes subjects with primary or established ovarian tumors. In specific embodiments, the expression includes people who have and are in need of treatment for ovarian cancer or other tumor that expresses MUC16. In other specific embodiments, the expression includes subjects with MUC16+ tumors (eg, a tumor with MUC16 expression determined by flow cytometry). In some embodiments, the expression subject in need thereof includes patients with ovarian cancer that is resistant to, or refractory to, or not adequately controlled by prior therapy (eg, treatment with a conventional anticancer agent). For example, the expression includes subjects who have been treated with chemotherapy, such as a platinum-based chemotherapy agent (eg, cisplatin) or a taxol compound (eg, docetaxel). The expression also includes subjects with an ovarian tumor for whom standard anticancer therapy is inappropriate, for example due to toxic side effects. For example, the expression includes patients who have undergone one or more cycles of chemotherapy with toxic side effects. In some embodiments, subject in need includes patients with an ovarian tumor that has been treated but subsequently relapses or metastasizes. For example, patients with an ovarian tumor who may have been treated with one or more anticancer agents leading to tumor regression; however, subsequently resulting in recurrence of a cancer resistant to one or more anticancer agents (eg, chemotherapy-resistant cancer) is treated with the methods of the present invention.

The expression subject in need also includes subjects who are at risk of developing ovarian cancer, such as persons with a family history of ovarian cancer, persons with a history of ovarian cancer-associated infections, persons with mutations in the BRCA1/2 genes, or persons with a weakened immune system due to HIV infection or due to immunosuppressive medications.

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

- 9 046291

MUC16/CD3 in a patient with elevated PD-L1 and/or CA125 levels.

In some embodiments, the methods of the present invention are used in a subject with ovarian cancer. The terms tumor, cancer and malignancy are used interchangeably herein. As used herein, the term ovarian cancer refers to tumors of the ovary and fallopian tube and includes serous carcinoma, endometrioid carcinoma, clear cell carcinoma and mucinous carcinoma.

In some embodiments, the present invention includes methods for treating, delaying, or inhibiting tumor growth. In certain embodiments, the present invention includes methods for promoting tumor regression. In some embodiments, the present invention includes methods for reducing tumor cell mass or reducing tumor burden. In certain embodiments, the present invention includes methods for preventing tumor recurrence. Methods according to this aspect of the invention involve sequentially administering a therapeutically effective amount of an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody to a subject in need thereof, each antibody being administered to the subject in multiple doses, for example, as part of a specific therapeutic dosing regimen . For example, a therapeutic dosing regimen may include administering to a subject multiple doses of anti-PD-1 antibody at a frequency of about once per day, once every two days, once every three days, once every four days, once every five days, once 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 often. 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 an anti-MUC16/CD3 bispecific antibody, wherein one or more doses of the bispecific antibody are administered to a subject at a frequency of about once per day, one 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.

In some embodiments, each dose of anti-MUC16/CD3 antibody is administered in more than 1 fraction, such as 2-5 fractions (split dose administration) over a given dosing period. The anti-MUC16/CD3 bispecific antibody can be administered in divided doses to reduce or eliminate the cytokine surges induced in response to the antibody. Cytokine surges are among the clinical symptoms of cytokine release syndrome (cytokine storm) and infusion-related reactions. In some embodiments, the methods of the present invention comprise administering one or more doses of an anti-PD-1 antibody in combination with one or more doses of an anti-MUC16/CD3 bispecific antibody to a subject in need thereof, wherein: the dose of the bispecific antibody is administered in divided doses, or in more than 1 fraction, for example, as 2 fractions, 3 fractions, 4 fractions or 5 fractions during a given dosing period. In some embodiments, the dosage of bispecific antibody is divided into 2 or more fractions, with each fraction containing an amount of antibody equal to the other fractions. For example, a 1000 microgram dose of anti-MUC16/CD3 antibody can be administered once per week, with the dose administered in 2 fractions over the course of a week, each fraction being 500 micrograms. In some embodiments, the dosage of the bispecific antibody is administered divided into 2 or more fractions, wherein the fractions contain unequal amounts of antibody, for example, more or less than the first fraction. For example, a 1000 microgram dose of anti-MUC16/CD3 antibody can be administered once per week, with the dose administered in 2 fractions over the course of a week, with the first fraction being 700 micrograms and the second fraction being 300 micrograms. As another example, a 1000 microgram dose of anti-MUC16/CD3 antibody may be administered once every 2 weeks, with the dose administered in 3 fractions over a 2 week period, with the first fraction being 400 micrograms and the second fraction being 300 micrograms , and the third fraction is 300 mcg.

In some embodiments, the present invention includes methods for inhibiting, slowing, or stopping tumor metastasis or tumor infiltration into peripheral organs. Methods in accordance with this aspect include administering a therapeutically effective amount of an anti-PD-1 antibody to a subject in need thereof in combination with a bispecific anti-MUC16/CD3 antibody. In specific embodiments, the present invention provides methods for enhancing antitumor efficacy or enhanced tumor inhibition. Methods in accordance with this aspect of the invention include administering to a subject with ovarian cancer a therapeutically effective amount of an anti-PD-1 antibody before administering a therapeutically effective amount of an anti-MUC16/CD3 bispecific antibody, wherein the anti-PD-1 antibody may be administered about 1 day before, more than 1 day before , 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 methods provide increased tumor inhibition, e.g., about 20%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater

- 10 046291 than 80% compared to a subject in whom the bispecific antibody was administered before the anti-PD-1 antibody.

In certain embodiments, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody and a therapeutically effective amount of an anti-CD3xMUC16 bispecific antibody to a subject with ovarian cancer. In specific embodiments, the ovarian cancer is a serous cancer. In additional embodiments, the ovarian cancer is slow growing or aggressive. In certain embodiments, the subject does not respond to prior therapy or relapses from prior therapy. In certain embodiments, the methods of the present invention further comprise administering to the subject an additional therapeutic agent.

In certain embodiments, the methods of the present invention comprise administering a therapeutically effective amount of an anti-MUC16/CD3 bispecific antibody to a subject with a MUC16+ cancer. In specific embodiments, the malignancy is ovarian cancer. In additional embodiments, the ovarian cancer is slow growing or aggressive. In some embodiments, the malignancy is platinum-resistant ovarian cancer. In some embodiments, the cancer is taxol-resistant ovarian cancer. In some embodiments, the malignancy is a fallopian tube cancer. In some embodiments, the malignancy is a primary peritoneal cancer in which the patient may have elevated serum CA-125 levels. In specific embodiments, the malignancy is pancreatic cancer (eg, pancreatic adenocarcinoma). In some embodiments, the subject does not respond to prior therapy or relapses from prior therapy (eg, chemotherapy).

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

In some embodiments, the methods of the present invention are used to treat a patient with an MRD-positive disease. Minimal residual disease (MRD) refers to the small number of cancer cells that remain in a patient during or after treatment, and the patient may or may not show symptoms or signs of disease. These residual cancer cells, if not removed, often lead to recurrence of the disease. The present invention includes methods for inhibiting and/or eliminating residual cancer cells in a patient after MRD testing. MRD can be analyzed according to methods known in the art (eg, MRD flow cytometry). Methods according to this aspect of the invention include administering an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody to a subject in need thereof.

The methods of the present invention, in certain embodiments, comprise administering to a subject a therapeutically effective amount of each of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody in combination with a third therapeutic agent. The third therapeutic agent may be an agent selected from the group consisting of, for example, radiation therapy, chemotherapy, surgery, cancer vaccine, PD-L1 inhibitor (e.g., anti-PD-L1 antibody), LAG3 inhibitor (e.g., anti-LAG3 antibody) , CTLA-4 inhibitor (eg, anti-CTLA-4 antibody), TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, indoleamine 2,3-dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist, Ang2 inhibitor , transforming growth factor beta (TGF-β) inhibitor, epidermal growth factor receptor (EGFR) inhibitor, tumor-specific antigen antibody (eg, CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, M2 -Tumor PK, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9), vaccine (eg, bacillus Calmette-Guerin), granulocyte-macrophage colony-stimulating factor, cytotoxin, chemotherapeutic agent, Kn-6R inhibitor, inhibitor KH-4R, an IL-10 inhibitor, or a cytokine such as IL-2, IL-7, IL-21 and IL-15, an anti-inflammatory drug such as a corticosteroid and a non-steroidal anti-inflammatory drug, and a dietary supplement , for example, antioxidants. In some embodiments, the antibodies may be used in combination with therapy including a chemotherapy agent (eg, paclitaxel, carboplatin, doxorubicin, cyclophosphamide, cisplatin, gemcitabine, or docetaxel), radiation therapy, and surgery. Are you using

- May 11 046291 As used herein, the phrase in combination with means that the antibodies are administered to the subject simultaneously with, immediately before, or immediately after the administration of the third therapeutic agent. In some embodiments, the third therapeutic agent is administered as a co-formulation with the antibodies. In a related embodiment, the present invention includes methods comprising administering a therapeutically effective amount of an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody to a subject who is receiving a background anticancer therapy regimen. The primary anticancer therapy regimen may include a course of, for example, a chemotherapy agent or radiation therapy. An anti-PD-1 antibody in combination with a bispecific anti-MUC16/CD3 antibody can be added to complement a background cancer therapy regimen. In some embodiments, the antibodies are added as part of a background step therapy regimen in which the background cancer therapy is gradually withdrawn from the subject over time (e.g., in stages) while the antibodies are administered to the subject at a constant dose, or at an increasing or decreasing dose over time.

In some embodiments, the methods of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody in combination with a therapeutically effective amount of an anti-MUC16/CD3 bispecific antibody, wherein administration of the antibodies results in enhanced tumor growth suppression. In some embodiments, tumor growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to an untreated subject or subject , which is administered one of two antibodies as monotherapy. In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody results in increased tumor regression, shrinkage, and/or resolution. In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody results in a delay in tumor growth and development, e.g., tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days , more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years compared with an untreated subject or a subject administered one of the two antibodies as monotherapy. In some embodiments, administration of an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody prevents tumor recurrence and/or increases the duration of survival of the subject, e.g., increases the duration of survival by more than 15 days, more than 1 month, more than 3 months , more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months compared with an untreated subject or a subject administered one of the two antibodies as monotherapy. In certain embodiments, administration of antibodies in combination increases progression-free survival or overall survival. In some embodiments, administration of an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody increases the response and duration of response in a subject, e.g., by greater than 2%, greater than 3%, and greater than 4%, greater 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 those who did not receive treatment by a subject or subject who has received one of the two antibodies as monotherapy. In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody to a subject with ovarian cancer results in complete resolution of all evidence of tumor cells (complete response). In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody to a subject with ovarian cancer results in at least a 30% or greater reduction in the number of tumor cells or tumor size (partial response). In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody to a subject with ovarian cancer results in complete or partial clearance of tumor cells/lesions, including new measurable lesions. Tumor reduction can be measured by any of the methods known in the art, for example, using radiography, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology or molecular genetic analysis. In some embodiments, administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody produces a synergistic antitumor effect that is superior to the combined effects of the two agents when administered separately.

In some embodiments, the combination of antibodies administered is safe and well tolerated by the patient, with no increase in side effect (e.g., increased cytokine release (cytokine storm) or increased T cell activation) compared to a patient administered the bispecific antibody as monotherapy .

Antibodies against PD-1 and their antigen-binding fragments

In certain illustrative embodiments of the present invention, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof. As used herein, the term antibody includes molecules

- 12 046291 immunoglobulins containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (for example, IgM). In a typical antibody, each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and V _L consists of three CDRs and four FRs, located from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, FR antibodies against IL-4R (or an antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

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

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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a designated complementarity determining region (CDR) such as the CDR3 peptide) or a conformationally constrained peptide FR3-CDR3-FR4. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-antibodies, nanoantibodies (e.g. monovalent nanoantibodies, divalent nanobodies, etc.), immunopharmaceuticals small modular protein (SMIP)-based agents and shark IgNAR variable domains are also included in the expression antigen binding fragment used herein.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and typically may contain at least one CDR that is adjacent or in frame with one or more framework sequences. In antigen binding fragments having a VH domain linked to a _VL domain, the VH and _VL domains may be positioned relative to each other in any suitable orientation. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric VH or _VL domain.

In some embodiments, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen binding fragment of an antibody of the present invention include: (i) V _h -C _h 1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-C _h 2-CH3; (vi) VHCH ^2-C H ^{3; (vii) V} H ^- CL ^{; (viii) V} L ^- CH ^{1; (ix)} VL ^- CH ^{2; (x) V} L ^- CH ^3; (xi) ^V L ^-C H ^1- CH ^{2; (xii) V} L ^-C H ^1-C H ^2- CH ^3; (xiii) VL-CH2-CH3; 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 either be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of the antibody of the present invention may contain a homodimer or

- 13 046291 heterodimer (or other multimer) of any of the variable and constant domain configurations listed above, in non-covalent association with each other and/or with one or more monomeric VH OR VL domains (for example, via disulfide bond(s) )).

As used herein, the term antibody also includes multispecific (eg, bispecific) antibodies. A multispecific antibody or antigen binding fragment of an antibody will typically comprise at least two different variable domains, with each variable domain being capable of specifically binding to a different antigen or to 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 standard methods available in the art. For example, the present invention includes methods involving the use of bispecific antibodies in which one immunoglobulin arm is specific for PD-1 or a fragment thereof and the other immunoglobulin arm is specific for a second therapeutic target or conjugated to a therapeutic fragment. Exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv or diabody based bispecific formats, IgG-scFv fusion proteins, Double Variable Domain (DVD) Ig, Quadroma, overhangs -to-tooth, common light chain (e.g., conventional toe-to-tooth light chain, etc.), CrossMab, CrossFab, antibody-like protein containing chain exchange engineered domain, leucine zipper, Duobody, IgG1/IgG2 , IgG with Dual Action Fab (DAF) and bispecific Mab ₂ formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the above formats). Bispecific antibodies can also be constructed through peptide-nucleic acid conjugation, for example, whereby non-naturally occurring amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

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

Antibodies used in the methods of the present invention may be recombinant human antibodies. As used herein, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies, isolated from a recombinant, combinatorial library of human antibodies (described further below), antibodies isolated from an animal (eg, mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al., 1992, Nucl. Acids Res 20: 6287-6295), or antibodies produced, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VL and VH regions of the recombinant antibodies are the sequences , which, although derived from and related to human germline VL and VH sequences, may not naturally exist within the human germline antibody repertoire in vivo.

In certain embodiments, the antibodies used in the methods of the present invention specifically bind PD-1. The term specifically binds or the like. means that the antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that

- 14 046291 physically binds PD-1, used in the context of the present invention, includes antibodies that bind PD-1 or a portion thereof with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by surface plasmon resonance analysis. However, an isolated antibody that specifically binds human PD-1 may be cross-reactive with other antigens, such as PD-1 molecules from other (non-human) species.

In certain illustrative embodiments of the present invention, an anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising any of the amino acid sequences of the anti-PD antibodies -1, as set forth in US Patent Publication No. 20150203579. In some illustrative embodiments, an anti-PD-1 antibody or antigen binding fragment thereof that can be used in the context of the methods of the present invention comprises heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, an anti-PD-1 antibody or the antigen binding fragment contains three HCDR (HCDR1, HCDR2 and HCDR3) and three LCDR (LCDR1, LCDR2 and LCDR3), wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 35; HCDR2 contains the amino acid sequence SEQ ID NO: 36; HCDR3 contains the amino acid sequence SEQ ID NO: 37; LCDR1 contains the amino acid sequence SEQ ID NO: 38; LCDR2 contains the amino acid sequence SEQ ID NO: 39; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 40. In still other embodiments, the anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR comprising SEQ ID NO: 33 and an LCVR comprising SEQ ID NO: 34. In certain embodiments, the methods of the present invention include 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. Illustrative the antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a light chain containing the amino acid sequence of SEQ ID NO: 42 is a fully human anti-PD-1 antibody known as REGN2810 (also known as cemiplimab). In some illustrative embodiments, the methods of the present invention include the use of REGN2810 or a bioequivalent thereof. As used herein, the term bioequivalent refers to anti-PD-1 antibodies or PD-1 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives whose rate and/or extent of absorption do not demonstrate a significant difference from that of REGN2810 when administered intravenously. the same molar dose under similar experimental conditions, both in a single dose and in multiple doses. As used herein, the term refers to antigen binding proteins that bind to PD-1 that are not clinically significantly different from REGN2810 in their safety, purity and/or potency.

Other anti-PD-1 antibodies that may be used in the context of the methods of the present invention include, for example, antibodies referred to and known in the art as nivolumab (US Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (Pat. US No. 8609089), pidilizumab (US Patent No. 8686119) or any of the anti-PD-1 antibodies as set forth in US patents. No. 6808710, 7488802, 8168757, 8354509, 8779105 or 8900587.

Anti-PD-1 antibodies used in the context of the methods of the present invention may have pH-dependent binding characteristics. For example, an anti-PD-1 antibody for use in the methods of the present invention may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, the anti-PD-1 antibody of the invention may exhibit increased binding to its antigen at acidic pH compared to neutral pH. The expression acidic pH includes pH values less than about 6.2, for example about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6 , 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less . As used herein, the expression neutral pH means a pH of about 7.0 to about 7.4. The expression neutral pH includes pH values around 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In certain cases, decreased binding to PD-1 at acidic pH compared to neutral pH is expressed as the ratio of the _KD value of antibody binding to PD-1 at acidic pH to the _KD value for antibody binding to PD-1 at neutral pH (or vice versa ). For example, an antibody or antigen binding fragment thereof may be considered to exhibit reduced binding to PD-1 at acidic pH compared to neutral pH for purposes of the present invention if

- 15 046291 the antibody or antigen binding fragment thereof exhibits a ratio of K _D at acidic pH to K _D at neutral pH of about 3.0 or more. In some illustrative embodiments, the ratio of _KD at acidic pH to _KD at neutral pH for an antibody or antigen binding fragment of the present invention 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.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for decreased (or increased) binding to a particular antigen 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, by replacing one or more amino acids of the antigen binding domain (eg, in the CDR) with a histidine residue, an antibody with reduced antigen binding at an acidic pH relative to a neutral pH can be obtained. As used herein, the expression acidic pH means a pH of 6.0 or less.

Bispecific antibodies against MUC16/CD3

In certain illustrative embodiments of the present invention, the methods comprise administering a therapeutically effective amount of a bispecific antibody that specifically binds CD3 and MUC16. Such antibodies may be referred to herein as, for example, anti-MUC16/anti-CD3, or anti-MUC16xCD3 antibodies, or MUC16xCD3 bispecific antibodies, or other similar terminology.

As used herein, the expression bispecific antibody refers to an immunoglobulin protein comprising at least a first antigen binding domain and a second antigen binding domain. In the context of the present invention, the first antigen binding domain specifically binds a first antigen (eg, MUC16), and the second antigen binding domain specifically binds a second, different antigen (eg, CD3). Each antigen binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each of which contains three CDRs. In the context 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. Thus, the CDRs of the first antigen binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3; and the second antigen binding domain CDRs may be referred to herein as B-HCDR1, B-HCDR2 and B-HCDR3.

Each of the first antigen binding domain and the second antigen binding domain is associated with a separate multimerizing domain. As used herein, the term multimerization domain means any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or structure. In the context of the present invention, the multimerizing component is the Fc portion of an immunoglobulin (containing the C _H 2 -CH3 domain), for example, an IgG Fc domain selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group.

The bispecific antibodies of the present invention typically contain two multimerizing domains, for example, two Fc domains, each of which is individually part of a separate heavy chain of the antibody. The first and second multimerizing domains may be of the same IgG isotype, such as, for example, IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes, such as, for example, IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

Any format or technology of bispecific antibodies can be used to produce the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity may be operably linked (e.g., by chemical binding, 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 obtain a bispecific antigen binding molecule. Specific exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, for example, scFv or diabody based bispecific formats, IgG-scFv fusion proteins, Double Variable Domain (DVD) Ig, Quadrama, knobs-to-cavities, common light chain (e.g., conventional knobs-to-cavities light chain, etc.), CrossMab, CrossFab, antibody-like protein containing a strand exchange engineered domain, leucine zipper, Duobody, IgG1/ IgG2, IgG with Dual Action Fab (DAF), and bispecific Mab ₂ formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a review of the above formats).

In the context of the bispecific antibodies of the present invention, the Fc domains may contain one or more amino acid substitutions (eg, insertions, deletions or substitutions) compared to the natural wild-type version of the Fc domain. For example, the invention includes bispecific antigen binding molecules containing one or more modifications in the Fc domain that result in modi

- 16 046291 an infected Fc domain having a modified binding interaction (eg, increased or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule contains a modification at the CH2 or CH3 region, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (eg, 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 US Patent Publication No. 20150266966, which is incorporated herein in its entirety.

The present invention also includes bispecific antigen binding molecules comprising a first CH3 domain and a second CH3 Ig domain, wherein the first and second CH3 Ig domains differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibodies with protein A versus a bispecific antibody with no amino acid differences. In one embodiment, the first CH3 Ig domain binds to protein A, and the second CH3 Ig domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (IMGT exon numbering, H435R according to EU numbering). The second CH3 may additionally contain the Y96F modification (IMGT; EU Y436F). See, for example, US Pat. No. 8,586,713. Other modifications that may be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in case of IgG1 antibodies; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) for IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) for IgG4 antibodies.

In some embodiments, the Fc domain may be chimeric by combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and part or all of a CH3 sequence derived from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a lower hinge sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that can be included in any of the antigen-binding molecules provided herein contains from the N- to C-terminus: [CH1 IgG4]-[upper hinge IgG4]-[lower hinge IgG2]-[ _CH 2 IgG4]-[C _H 3 IgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein contains from the N- to C-terminus: [CH1 IgG1]-[upper hinge IgG1]-[lower hinge IgG2]-[ _CH 2 IgG4]-[C _H 3 IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules of the present invention are described in US Patent Publication No. 20140243504, which is incorporated herein in its entirety. Chimeric Fc domains sharing these common structural arrangements and variants thereof may have altered Fc receptor binding, which in turn affects Fc effector function.

In some exemplary embodiments of the present invention, the anti-MUC16/CD3 bispecific antibody or antigen binding fragment thereof comprises heavy chain variable regions (A-HCVR and B-HCVR), light chain variable regions (A-LCVR and BLCVR), and/or complementarity determining regions (CDR) comprising any of the amino acid sequences of the anti-MUC16/CD3 bispecific antibodies as set forth in U.S. Patent Publication No. 20180112001. In certain illustrative embodiments, an anti-MUC16/CD3 bispecific antibody or antigen binding fragment thereof that can be used in the context of the methods of the present invention , contains: (a) a first antigen-binding arm containing the heavy chain complementarity determining regions (A-HCDR1, A-HCDR2 and AHCDR3) of the heavy chain variable region (A-HCVR) containing the amino acid sequence of SEQ ID NO: 1 and the light complementarity determining regions chains (A-LCDR1, A-LCDR2 and ALCDR3) of the light chain variable region (A-LCVR) containing the amino acid sequence of SEQ ID NO: 2; and (b) a second antigen binding arm containing the heavy chain CDRs (BHCDR1, B-HCDR2 and B-HCDR3) of HCVR (B-HCVR) containing 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 a light chain CDR (B-LCDR1, B-LCDR2, and B-LCDR3) of LCVR (B-LCVR) comprising the amino acid sequence of SEQ ID NO 2. In some embodiments, A-HCDR1 comprises the amino acid sequence SEQ ID NO 8; A-HCDR2 contains the amino acid sequence SEQ ID NO 9; AHCDR3 contains the amino acid sequence SEQ ID NO 10; A-LCDR1 contains the amino acid sequence SEQ ID NO 11; A-LCDR2 contains the amino acid sequence SEQ ID NO 12; A-LCDR3 contains the amino acid sequence SEQ ID NO 13; B-HCDR1 contains 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 contains 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 contains amino acids

- 17 046291 new sequence SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25 or SEQ ID NO: 28; and B-LCDR1 contains the amino acid sequence SEQ ID NO: 11; B-LCDR2 contains the amino acid sequence SEQ ID NO 12; B-LCDR3 comprises the amino acid sequence of SEQ ID NO 13. In other embodiments, the anti-MUC16/CD3 bispecific antibody or antigen binding fragment thereof comprises: (a) a first antigen binding arm comprising HCVR (A-HCVR) comprising SeQ ID NO 1, and LCVR (A-LCVR) containing SEQ ID NO: 2; and (b) a second antigen binding arm containing HCVR (B-HCVR) containing SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, and LCVR ( B-LCVR) comprising SEQ ID NO: 2. In some illustrative embodiments, the anti-CD3xMUC16 bispecific antibody comprises a MUC16 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO 30 , and a CD3 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO 31, and a light chain comprising the amino acid sequence of SEQ ID NO 30. In some illustrative embodiments, the anti-CD3xMUC16 bispecific antibody comprises a MUC16 binding arm containing a heavy chain, containing the amino acid sequence of SEQ ID NO: 29, and a light chain containing the amino acid sequence of SEQ ID NO: 30, and a CD3 binding arm containing a heavy chain containing the amino acid sequence of SEQ ID NO: 32, and a light chain containing the amino acid sequence of SEQ ID NO: 30 .

In some embodiments, the antitumor activity of the anti-CD3xMUC16 bispecific antibodies of the present invention is not significantly hampered by the presence of high levels (eg, up to 10,000 U/ml) of circulating CA125. Serum CA125 levels are elevated in most patients with ovarian cancer (published median levels are approximately 656 U/mL). As shown in Example 2 below, high levels of CA125 in serum or in ascites will not significantly affect the antitumor profile of the bispecific antibodies of the present invention.

Other anti-MUC16/CD3 bispecific antibodies that may be used in the context of the methods of the present invention include, for example, any of the antibodies set forth in US Patent Publication No. 20180112001.

Combination Therapy Options

The methods of the present invention, in certain embodiments, comprise administering to a subject an anti-MUC16/CD3 bispecific antibody in combination with an anti-PD-1 antibody. In some embodiments, the methods of the present invention include administering antibodies with additional or synergistic activity to treat a cancer, preferably ovarian cancer. As used herein, in combination with means that the anti-MUC16/CD3 bispecific antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. The term in combination with also includes sequential or simultaneous administration of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody. For example, when administered before the anti-MUC16/CD3 bispecific 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 before anti-MUC16 bispecific antibody administration /CD3. When administered after the anti-MUC16/CD3 bispecific antibody, the anti-PD-1 antibody can be administered after about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than 72 hours after administration of the anti-MUC16/CD3 bispecific antibody. Coadministration with an anti-MUC16/CD3 bispecific antibody means that the anti-PD-1 antibody is administered to a subject in a separate dosage form less than 5 minutes (before, after, or simultaneously) of administration of the anti-MUC16/-CD3 bispecific antibody, or administered to the subject as a single combination dosage form containing both an anti-PD-1 antibody and a bispecific anti-MUC16/CD3 antibody.

In some embodiments, the methods of the present invention include administering a third therapeutic agent, wherein the third therapeutic agent is an anticancer drug. As used herein, the term anticancer drug means any agent useful for the treatment of cancer, including, but not limited to, cytotoxins and an agent such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P'-(DDD)), biological drugs (eg antibodies and interferons) and radioactive agents. As used herein, the term cytotoxin or cytotoxic agent also refers to a chemotherapeutic agent and means any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolomide, cytochalasin B, gramicidin D, bromide

- 18 046291 ethidium, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracyndione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, proprano lol and puromycin and their analogues or homologues.

In some embodiments, the methods of the present invention include administering a third therapeutic agent selected from the group consisting of radiation therapy, surgery, a cancer vaccine, a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody), a LAG-3 inhibitor, a CTLA inhibitor -4 (eg, ipilimumab), TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, another T cell coinhibitor antagonist or ligand (eg, anti-CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA antibodies), an indoleamine 2,3 dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., a VEGF decoy such as aflibercept or another VEGF inhibitory fusion protein as described in US Pat. No. 7,087,411, or an anti-VEGF antibody, or its antigen-binding moiety (eg, bevacizumab or ranibizumab), or small molecule VEGF receptor kinase inhibitor (eg, sunitinib, sorafenib, or pazopanib)], Ang2 inhibitor (eg, nesvacumab), transforming growth factor beta (TGF-β) inhibitor, epidermal factor receptor inhibitor growth factor (EGFR) (eg, erlotinib, cetuximab), costimulatory receptor agonist (eg, glucocorticoid-induced TNFR-related protein agonist), tumor-specific antigen antibodies (eg, CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, M2-PK tumor, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9), vaccine (eg, Bacillus Calmette-Guerin, cancer vaccine), adjuvant to increase antigen presentation (eg , granulocyte-macrophage colony-stimulating factor), cytotoxin, chemotherapeutic agent (eg, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel and vincristine), radiation therapy, inhibitor of IL- 6I (eg, sarilumab), IL-4K inhibitor (eg, dupilumab), IL10 inhibitor, cytokine such as IL-2, IL-7, IL-21 and IL-15, antibody-drug conjugate (ADC) (eg , anti-CD19-DM4 ADC and anti-DS6-DM4 ADC), chimeric antigen receptor T cells (eg, CD19-targeting T cells), an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs) and a dietary supplement, such as antioxidants.

In some embodiments, the methods of the present invention include administering an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody in combination with radiation therapy to produce long-term sustained antitumor responses and/or prolong survival of patients with cancer.

In some embodiments, the methods of the present invention include administering radiation therapy before, concomitantly, or after administration of anti-PD-1 antibody and anti-MUC16/CD3 bispecific antibody to patients with cancer. For example, radiation therapy may be applied in one or more doses to tumor lesions after administration of one or more doses of antibodies. In some embodiments, radiation therapy may be applied locally to a tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant ionizing radiation) and/or to kill tumor cells (destructive ionizing radiation) following systemic administration of an anti-PD-1 antibody and/or a bispecific anti-MUC16 antibody /CD3. In some embodiments, the antibodies can be used in combination with radiation therapy and a chemotherapeutic agent (eg, carboplatin and/or paclitaxel) or a VEGF antagonist (eg, aflibercept).

Pharmaceutical compositions and administration

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

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, micro

- 19 046291 capsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes of administration. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous integuments (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, in the case of subcutaneous delivery, the pen can easily be used in delivering the pharmaceutical composition of the present invention. This syringe pen can be reusable or disposable. A reusable delivery pen typically uses a replaceable cartridge containing a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The delivery pen can then be reused. The disposable delivery pen does not have a replaceable cartridge. Preferably, the disposable pen comes filled with a pharmaceutical composition held in a reservoir within the device. Immediately after the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment of the invention, a pump may be used. In another embodiment of the invention, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment of the invention, the controlled release system can be placed in close proximity to the target of the composition, requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in Langer, 1990, Science 249: 1527-1533.

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

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

Administration regimens

The present invention includes methods comprising administering an anti-PD-1 antibody to a subject at a dosing frequency of about four times per week, twice per week, once per week, once every two weeks, once every three weeks, once every four weeks , once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently until a therapeutic response is achieved. In certain embodiments, the present invention includes methods comprising administering an anti-MUC16/CD3 bispecific antibody to a subject at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently until a therapeutic response is achieved. In certain embodiments, the methods include administering an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific 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 nine weeks, once every twelve weeks or less frequently until a therapeutic response is achieved.

- 20 046291

In certain embodiments of the present invention, multiple doses of an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody can be administered to a subject over a period of time. The methods of this aspect of the invention involve sequentially administering to a subject one or more doses of an anti-PD-1 antibody in combination with one or more doses of an anti-MUC16/CD3 bispecific antibody. As used herein, the term sequential administration means that each dose of antibody is administered to a subject at different points in time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of an anti-PD-1 antibody, followed by one or more secondary doses of an anti-PD-1 antibody, and optionally followed by one or more tertiary doses of an anti-PD-1 antibody. In certain embodiments, the methods further comprise sequentially administering to the patient a single initial dose of the anti-MUC16/CD3 bispecific antibody, followed by one or more secondary doses of the bispecific antibody, and optionally followed by one or more tertiary doses of the bispecific antibody.

In accordance with some embodiments of the present invention, multiple doses of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody may be administered to a subject over a period of time. Methods according to this aspect of the invention involve sequentially administering multiple doses of an anti-PD-1 antibody and an anti-MUC16/CD3 bispecific antibody to a subject. As used herein, sequential administration means that each dose of an anti-PD-1 antibody in combination with a bispecific antibody is administered to a subject at different points in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). .

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may contain the same amount of antibody (anti-PD-1 antibody or bispecific antibody). However, in some embodiments, the amounts contained in the initial, secondary, and/or tertiary doses are different from each other (eg, adjusted up or down) over the course of treatment. In some embodiments, one or more (eg, 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered less frequently (eg, maintenance doses). For example, an anti-PD-1 antibody may be administered to a patient with ovarian cancer at a loading dose of about 1 to 3 mg/kg, followed by one or more maintenance doses of about 0.1 to about 20 mg/kg of the patient's body weight.

In one illustrative embodiment of the present invention, each secondary and/or tertiary dose is administered from 1/2 to 14 (eg, 1/2, 1, 11/2, 2, 21/2, 3, 31/2, 4, 41/ 2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14, 141/2, or more) weeks after the immediately preceding dose. As used herein, the phrase immediately preceding dose means, in a multiple dosing sequence, the dose of anti-PD-1 antibody (and/or anti-MUC16/CD3 bispecific antibody) that is administered to a patient before the next dose in the sequence, with no intervening doses.

Methods according to this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of an anti-PD-1 antibody (and/or an anti-MUC16/CD3 bispecific antibody). For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses.

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

In some embodiments, one or more doses of anti-PD-1 antibody and/or bispecific anti-MUC16/CD3 antibody are administered at the beginning of the treatment regimen as induction doses more frequently (twice weekly, once weekly, or once every 2 weeks) with subsequent doses (consoles

- 21 046291 administration doses or maintenance doses), which are administered less frequently (for example, once every 412 weeks).

The present invention includes methods comprising sequentially administering an anti-PD-1 antibody in combination with an anti-MUC16/CD3 bispecific antibody to a patient for the treatment of ovarian cancer (eg, serous cancer). In some embodiments, the methods comprise administering one or more doses of an anti-PD-1 antibody followed by one or more doses of an anti-MUC16/CD3 bispecific antibody. In certain embodiments, these methods include administering a single dose of an anti-PD-1 antibody followed by one or more doses of a bispecific anti-MUC16/CD3 antibody. In some embodiments, one or more doses of about 0.1 mg/kg to about 20 mg/kg of anti-PD-1 antibody may be administered followed by one or more doses of about 0.1 mg/kg to about 20 mg/kg a bispecific antibody for inhibiting tumor growth and/or preventing tumor recurrence in a subject with ovarian cancer. In some embodiments, an anti-PD-1 antibody is administered in one or more doses followed by one or more doses of a bispecific antibody, resulting in increased antitumor efficacy (e.g., greater inhibition of tumor growth, increased prevention of tumor recurrence, as compared to an untreated subject or a subject who was administered one of the antibodies as monotherapy). Alternative embodiments of the invention relate to the simultaneous administration of an anti-PD-1 antibody and a bispecific antibody that is administered at a separate dose at the same or different frequency than 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 unit dosage form with the anti-PD-1 antibody.

Dosage

The amount of anti-PD-1 antibody and/or anti-MUC16/CD3 bispecific antibody administered to a subject in accordance with 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 anti-MUC16/CD3 bispecific antibody) that results in one or more of the following: (a) a reduction in the severity or duration of a symptom of a malignancy (eg, ovarian cancer) ; (b) inhibiting tumor growth or increasing tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and development; (d) inhibiting, slowing or stopping tumor metastasis; (f) preventing relapse of tumor growth; (f) increasing the survival rate of a subject with a malignancy (eg, ovarian cancer); and/or (g) reducing the use or need for conventional anticancer therapy (eg, reducing or eliminating the use of chemotherapeutic or cytotoxic agents) compared to an untreated subject or a subject administered one of the two antibodies as monotherapy.

For an anti-PD-1 antibody, a therapeutically effective amount may 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, 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. 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 anti-PD-1 antibody. In some embodiments, 250 mg of anti-PD-1 antibody is administered.

For an anti-MUC16/CD3 bispecific antibody, the therapeutically effective amount may be from about 10 μg (μg) to about 8000 μg, for example, about 10 μg, about 20 μg, about 50 μg, about 70 μg, about 100 μg, about 120 μg , 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 900 mcg, about 1000 mcg, about 1050 mcg, about 1100 mcg, about 1500 mcg, about 1700 mcg, about 2000 mcg, about 2050 mcg, about 2100 mcg, about 2200 mcg, about 2500 mcg, about 2700 mcg, about 2800 mcg , about 2900 μg, about 3000 μg, about 4000 μg, about 5000 μg, about 6000 μg, about 7000 μg or about 8000 μg of anti-MUC16/CD3 bispecific antibody.

The amount of either anti-PD-1 antibody or anti-MUC16/CD3 bispecific antibody contained in individual doses may be expressed in milligrams of antibody per kilogram of subject body weight (ie, mg/kg). In some embodiments, either the anti-PD-1 antibody or the anti-MUC16/CD3 bispecific antibody used in the methods of the present invention can be administered to a subject at a dose of from about 0.0001 to about 100 mg/kg of the subject's body weight. For example, an anti-PD-1 antibody can be administered at a dose of from about 0.1 mg/kg to about 20 mg/kg of the patient's body weight. The anti-MUC16/CD3 bispecific antibody can be administered at a dose of from about 0.1 mg/kg to about 20

- 22 046291 mg/kg of patient’s body weight.

A summary of the sequences and corresponding SEQ ID NOs mentioned in this document is shown in table. 1 below.

Table 1

Brief description of sequences

SEQ ID NO: Description 1 Anti-MiC16 heavy chain variable region 2 Anti-MiC16 and anti-POP light chain variable region 3 Anti-POP-G heavy chain variable region 4 Anti-POP-O5 heavy chain variable region 5 Anti-POP-O9 heavy chain variable region 6 Anti-POP-O heavy chain variable region 10 7 Anti-POP-O20 heavy chain variable region 8 HCDR1 anti-MiC16 9 HCDR2 anti-MiC16 10 HCDR3 anti-MiC16 eleven LCDR1 anti-MiC16 and anti-POP 12 LCDR2 anti-MiC16 and anti-POP 13 LCDR3 anti-MiC16 and anti-POP 14 HCDR1 anti-POP-G 15 HCDR2 anti-POP-G 16 HCDR3 anti-POP-G 17 HCDR1 anti-POP-O5 18 HCDR2 anti-POP-O5 19 HCDR3 anti-POP-O5 20 HCDR1 anti-POP-O9 21 HCDR2 anti-POP-O9 22 HCDR3 anti-POP-O9 23 HCDR1 anti-POP-GIO 24 HCDR2 anti-POP-GIO 25 HCDR3 anti-POP-GIO 26 HCDR1 anti-POP-O20 27 HCDR2 anti-POP-O20 28 HCDR3 anti-POP-O20 29 Anti-MiS16 heavy chain thirty Anti-MCS16 and anti-POP light chain 31 Heavy chain anti-POP-G 32 Anti-POP-O20 heavy chain 33 Anti-PD-l heavy chain variable region 34 Anti-PD-l light chain variable region 35 HCDR1 anti-PD-l 36 HCDR2 anti-PD-l 37 HCDR3 anti-PD-l 38 LCDR1 anti-PD-l 39 LCDR2 anti-PD-l 40 LCDR3 anti-PD-l 41 Anti-PD-l heavy chain 42 Anti-PD-l light chain

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Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to develop and apply the methods and compositions of the invention, and are not intended to limit the scope of what the inventors believe to be their invention. Efforts have been made to ensure accuracy in terms of numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Preparation of bispecific antibodies that bind ovarian cell specific (MUC16) and CD3

The present invention relates to bispecific antigen binding molecules that bind CD3 and MUC16; such bispecific antigen binding molecules are also referred to herein as anti-MUC16/CD3 or anti-MUC16xCD3 bispecific molecules. The anti-MUC16 portion of the anti-MUC16/CD3 bispecific molecule is useful for targeting tumor cells that express MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is useful for T cell activation. Simultaneous binding of MUC16 to the tumor cell and CD3 to the T cell promotes targeted killing (cell lysis) of the target tumor cell by the activated T cell.

Bispecific antibodies containing an anti-MUC16-specific binding domain and an anti-CD3-specific binding domain were constructed using standard methodologies in which the anti-MUC16 antigen binding domain and the anti-CD3 antigen binding domain each contain different, separate HCVRs paired with a common LCVR. In the exemplary bispecific antibodies, molecules were constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a common light chain from an anti-MUC16 antibody. In other cases, bispecific antibodies can be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody or an antibody light chain that is known to bind indiscriminately or efficiently to multiple heavy chain arms .

Examples of bispecific antibodies have been produced with the IgG1 Fc domain (BSMUC16/CD3-001, -002, -003 and -004) or a modified (chimeric) IgG4 Fc domain (BSMUC16/CD3-005) as specified in US Patent Application Publication No. US20140243504A1 , published August 28, 2014

A brief description of the constituent parts of the antigen-binding domains of various engineered bispecific antibodies against MUC16xCD3 is presented in Table. 2.

table 2

Brief description of the components of bispecific antibodies against MUC16xCD3

Bispecific Antibody Identifier Anti-MiS16 Ahth-CD3 Common light chain variable region antigen binding domain Antigen binding domain Heavy chain variable region Heavy chain variable region BSMUC16/CD3- 001 (SEQIDNO:1) CD3-VH-G (SEQ ID NO:3) (SEQ ID NO:2) BSMUC16/CD3- 002 (SEQ ID NO: 1) CD3-VH-G5 (SEQ ID NO:4) (SEQ ID NO:2) BSMUC16/CD3- 003 (SEQ ID NO: 1) CD3-VH-G9 (SEQ ID NO:5) (SEQ ID NO:2) BSMUC16/CD3- 004 (SEQ ID NO: 1) CD3-VH-G10 (SEQ ID NO:6) (SEQ ID NO:2) BSMUC16/CD3- 005 (SEQ ID NO: 1) CD3-VH-G20 (SEQ ID NO:7) (SEQ ID NO:2)

Example 2. CA-125 does not affect the activity of antibodies against MUC16xCD3 in vitro

The effect of soluble CA-125 (the exfoliated form of MUC16) on the activity of BSMUC16/CD3-001 was assessed using FACS binding assay and cytotoxicity assay in the presence of high levels of CA-125 purified from ascitic fluid of ovarian cancer patients.

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CA-125 levels are elevated in the serum of most patients with ovarian cancer, and circulating levels may interfere with any MUC16-targeted therapy through antigen-dependent clearance. The CA-125 levels used in the assay (10,000 U/mL) are significantly higher than the published median levels of 656.6 U/mL in patients with ovarian cancer. Ability of BSMUC16/CD3-001 to kill OVCAR-3 cells expressing MUC16 in the presence of soluble CA-125 enriched from human ascitic fluid (Creative Biomart, New York, USA) or with a membrane-proximal construct expressing five C-terminal SEAs -domains and the juxtamembrane region of MUC16 (MUC16A). determined at a 4:1 effector/target ratio with a fixed concentration of BSMUC16/CD3-001 or CD3-binding control antibody (100 μM) and serial dilution of either MUC16-1H or MUC16A for 72 hours at 37°C. To track specific killing of MUC16-bearing target cells, OVCAR-3 cells were labeled with 1 μM Violet Cell Tracker. After labeling, cells were incubated overnight at 37°C. Separately, human PBMCs were placed in medium supplemented with RPMI at a concentration of 1x10 ⁶ cells/ml and incubated overnight at 37°C to enrich lymphocytes by depleting adherent cells. The next day, target cells were co-incubated with naïve PBMCs with adherent cell depletion (4:1 effector cell/target cell ratio) and serial dilution of either BSMUC16/CD3-001 or CD3-binding control for 72 hours at 37°C. Cells were removed from cell culture plates using trypsin and analyzed by FACS. For FACS analysis, cells were stained with Live/Dead Far Red Cell Tracker (Invitrogen). To assess killing specificity, cells were gated on CellTracker Violet-labeled populations. The percentage of target cells alive was specified to calculate adjusted survival as follows: adjusted survival = (R1/R2)*100, where R1 = % target cells alive in the presence of antibody and R2 = % target cells alive in the absence of the test antibody. T cell activation was assessed by incubating cells with directly conjugated antibodies to CD2, CD69, and CD25 and by recording the percentage of activated (CD69+) T cells or (CD25+) T cells from total T cells (CD2+).

The binding of BSMUC16/CD3-001 and an antibody known to bind CA-125 (clone 3A5) to CA125 derived from human ascites fluid was measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, soluble CA-125 (Creative Biomart, New York, USA) at a concentration of 4000 U/mL in PBS was passively adsorbed onto 96-well microtiter plates overnight at 4°C. The plates were then washed with PBST and blocked with 0.5% BSA in PBS for 1 hour. Biotinylated BSMUC16/CD3-001, parent anti-MUC16, anti-MUC16 3A5, and non-binding controls (isotype control BSMUC16/CD3-001 and isotype control 3A5 anti-MUC16) were added to the plate at concentrations of 10, 1, 0.3, or 0.1 nM in 0.5% BSA in PBS for 1 hour followed by rinsing with PBST. Streptavidin conjugated to horseradish peroxidase (SA-HRP) (ThermoFisher Scientific, Waltham, MA, USA) at a 1:10,000 dilution of 1.0 mg/mL stock solution was added to the wells and incubated for 1 hour to detect biotinylated antibodies bound to the tablet. The plate was washed and developed with 3-3',5-5'tetramethylbenzidine substrate (BD Biosciences, Franklin Lake, NJ, USA) according to the manufacturer's instructions. Absorbance at 450 nm was recorded for each well on a VICTOR Multilabel Plate Reader (Perkin Elmer; Melville, NY). Data were analyzed using GraphPad Prism software.

Excess CA-125 had minimal effect on BSMUC16/CD3-0001 binding to OVCAR-3 cells, indicating minimal binding to CA-125 (Figure 1). In contrast, CA-125 strongly inhibited the ability of the reference antibody, which likely binds to the repeat region of MUC16 (the in-house version of antibody clone 3A) (Fig. 1). Subsequently, a soluble MUC16 construct containing the membrane-proximal region up to the 5th SEA domain of MUC16 (MUC16A) dramatically inhibited BSMUC16/CD3-001 binding, demonstrating that BSMUC16/CD3-001 binds the membrane-proximal region, as discussed in more detail. in WO 2018/067331, which is incorporated herein by reference. Consistent with 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 MUC16A (data not shown). Thus, BSMUC16/CD3-001 can bind to MUC16 and induce redirected T cell killing even in the presence of high concentrations of CA-125.

Example 3. PD-1 blockade enhances the antitumor activity of bispecific antibodies against MUC16xCD3 in xenogeneic and syngeneic tumor models

The in vivo efficacy of a bispecific antibody against MUC16/CD3 in combination with PD-1 blockade was assessed in xenogeneic and syngeneic tumor models.

A. Xenogeneic model - OVCAR-3/Luc

For the xenogeneic model, immunodeficient NSG mice were injected intraperitoneally (ip) with OVCAR-3/Luc cells, previously passaged in vivo (day 0), thirteen days after

- 25 046291 development of human ICPCs. Mice were administered ip 12.5 μg/mouse BSMUC16/CD3-001 or administered 12.5 μg CD3-binding control alone or in combination with 100 μg REGN2810 on days 5 and 8. Tumor burden was assessed using BLI at days 4, 8, 12, 15, 20, and 25 after tumor implantation. As determined by BLI measurements at day 25, treatment with 12.5 μg BSMUC16/CD3-001 resulted in significant antitumor efficacy as determined by BEL measurements, and combination with REGN2810 (anti-PD-1) further enhanced antitumor efficacy. All groups had similar tumor burden as assessed by BLI before dosing. There were no significant differences in tumor burden between groups.

BSMUC16/CD3-001 significantly reduced tumor burden at 12.5 μg, and the addition of antiPD-1 enhanced antitumor efficacy compared with BSMUC16/CD3-001 alone. NSG mice inoculated with human T cells were implanted with human OVCAR-3/Luc cells. On days 5 and 8, mice were treated with 12.5 μg of BSMUC16/CD3-001 administered intravenously or treated with CD3-binding control or non-binding control (12.5 μg i.v.). The data presented in table. 3 below represent tumor burden assessed by BLI at day 25 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-001 +/- REGN2810 was compared with CD3-binding control (* p < 0.05 for BSMUC16/CD3-001, ** p < 0.01 for BSMUC16/CD3-001 and REGN2810), and treatment BSMUC16/CD3-001 monotherapy was compared with combination with REGN2810 (# p < 0.05).

Table 3

Bioluminescence on the 25th day after tumor implantation

CD3-binding control hlgG4P-PVA

7.71е+06± 1.07е+06

BSMUC16/CD3-001 (12

7.44е+03 ± 3.11е+03

9.29е+06± 1.82е+06

BSMUC16/CD3-001 (12) + REGN2810 (100

Average emission intensity

1f/s/cm ² /sr1 25 days after implantation (median ± SEM)

12) + REGN2810 (100

B. Syngeneic model - ID8-VEGF/huMUC16

CD3 binding control h!gG4P-PVA

To test efficacy in an immunocompetent model, the mouse CD3 gene was replaced with human CD3, and part of the mouse MUC16 gene was replaced with human sequence. The substitutions resulted in a mouse whose T cells express human CD3 and which express a chimeric MUC16 molecule containing a portion of human MUC16 that is bound by the bispecific antibodies BSMUC16/CD3-001 and BSMUC16/CD3-005.

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

BSMUC16/CD3-001 significantly prolonged median survival time in the ID8VEGF ascites model, and additional PD-1 blockade (REGN2810) allowed several mice to survive. Mice expressing human CD3 instead of mouse CD3 and a chimeric MUC16 molecule were implanted with a mouse ovarian tumor line expressing a portion of human MUC16. Mice were treated with BSMUC16/CD3001 (5 mg/kg i.v.) or treated with CD3-binding control (5 mg/kg i.v.) with isotype control or anti-PD-1 on day 3 postimplantation. Mice received treatment on days 3, 7, 10, 14, and 17 after tumor implantation. Data presented represent median survival. Mice were sacrificed when they gained more than 20% weight due to ascites-induced abdominal bloating. Statistical significance was determined using the Cox-Mantel test. Treatment with BSMUC16/CD3-001 and BSMUC16/CD3-001+anti-PD-1 resulted in an increase in median survival time, and the combination of BSMUC16/CD3-001+anti-PD-1 resulted in 50% survival, demonstrating a synergistic effect between the bispecific anti-MUC16xCD3 antibody and anti-PD-1 antibody. The results are shown in table. 4 below.

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Table 4

Median survival in the ID8-VEGF/huMUC16 model

Antibody (mg/kg) Median survival (days) POP-binding control (5) + isotype control (5) 36 BSMUC16/CD3-001 (5) + isotype control (5) 46 POP-binding control (5) + PD-1 (5) 32 BSMUC16/CD3-001 (5) + PD-1 (5) 69.5

Similar results were observed when BSMUC16/CD3-001 was administered at a dose of 1 mg/kg in combination with anti-PD-1 antibody.

C. Syngeneic model - MC38/huMUC16

As discussed above, the mice used in this experiment were modified so that the mouse CD3 gene was replaced with human CD3, and part of the mouse MUC16 gene was replaced with human sequence. The substitutions resulted in a mouse whose T cells express human CD3 and which express a chimeric MUC16 molecule containing a portion of human MUC16 that is bound by the bispecific antibodies BSMUC16/CD3-001 and BSMUC16/CD3005.

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

The combination of BSMUC16/CD3-005 and anti-PD-1 blockade resulted in better antitumor efficacy than BSMUC16/CD3-005 alone in a subcutaneous MC model38. Mice expressing human CD3 instead of mouse CD3 and a chimeric MUC16 molecule were implanted with the MC38 mouse tumor line expressing part of human MUC16. Mice were treated with BSMUC16/CD3-005 or treated with CD3-binding control (1 mg/kg i.v.) with isotype control or anti-PD-1 antibody (5 mg/kg i.v.) on day 7 postimplantation. Mice were treated on days 7, 11, and 14 after tumor implantation. The results are shown in Fig. 2. Statistical significance was determined using two-way analysis of variance with Tukey's correction for multiple comparisons. BSMUC16/CD3-005 with anti-PD-1 significantly and synergistically inhibited tumor growth compared with CD3-binding control.

Example 4: Immuno-PET Imaging in Modified Mice Demonstrates Localization of Anti-MUC16xCD3 Bispecific Antibody in T Cell-Rich Organs

In vivo localization of BSMUC16/CD3-001 and BSMUC16/CD3-005 and MUC16 protein expression were assessed in wild-type and genetically humanized mice using PET imaging. The biodistribution of ⁸⁹ Zr-labeled anti-MUC16 antibody (a divalent anti-MUC16 antibody produced using the same anti-MUC16 heavy and light chain as the bispecific antibodies, referred to herein as parent antibodies) was similar in both wild-type and humanized mice , indicating low expression of the humanized MUC16 protein or its accessibility to the antibody. In contrast, when mice were administered therapeutically relevant doses of ⁸⁹ Zr-labeled bispecific antibody BSMUC16/CD3-001, distribution in the spleen and lymph nodes was evident due to the recognition of CD3-positive T cells in these lymphoid organs (data not shown). Ex vivo biodistribution analysis in selected tissues confirmed localization to the lymph nodes and spleen (data not shown). The uptake of ⁸⁹ Z-labeled bispecific antibody BSMUC16/CD3-005 into lymphoid tissues was significantly reduced compared to BSMUC16/CD3-001 due to its lower affinity for CD3. To evaluate whether BSMUC16/CD3001 and BSMUC16/CD3-005 could accumulate in MUC16-expressing tumors, mice bearing ID8-VEGF-huMUC16Δ tumors were treated with ⁸⁹ Zr-labeled BSMUC16/CD3-001 and ⁸⁹ Zr-labeled BSMUC16/CD3005. Tumor uptake did not differ significantly between bispecific antibodies, despite higher lymphoid uptake of BSMUC16/CD3-001 (data not shown).

Preparation of immunoconjugate and small animal PET: BSMUC16/CD3-001 and control antibody were conjugated to DFO with glutamine residues at position 295 via transamidation using microbial transglutaminase after deglycosylation of the antibodies with PNGase F. Then conjugate

- 27 046291 DFO-jugated antibodies were chelated with zirconium-89 ( ⁸⁹ Zr). Mice received the antibody at a final dose of 0.5 mg/kg via tail vein injection. PET imaging was then performed to assess in vivo localization of the radioimmunoconjugate on day 6 post-dose prior to ex vivo biodistribution studies. For mouse tumor experiments, mice were implanted subcutaneously with 10x10 ⁶ ID8-VEGF-huMUC16Δ tumor cells. Tumor-bearing mice were dosed with radiolabeled ⁸⁹ Zr antibody 20 days after implantation, when tumors averaged 150 mm ³ .

A pre-calibrated Sofie Biosciences G8 PET/CT instrument (Sofie Biosciences (Culver City, CA) and Perkin Elmer) was used to acquire PET and CT images. The energy window ranged from 150 to 650 keV with a recovered resolution of 1.4 mm at the center of the field of view. On day 6 post-dose, mice were induced with isoflurane anesthesia and maintained in a continuous flow of isoflurane during 10 min of PET data collection in statistical mode. CT images were obtained after PET data collection. PET images were subsequently reconstructed using predefined settings. Decay-corrected PET and CT data were processed using VivoQuant software (inviCRO Imaging Services) into co-registered false-colored PET-CT maximum intensity projections on a color scale calibrated to indicate the signal range from 0 to 30% of the administered dose per volume expressed as % BP/g. For ex vivo biodistribution analysis, mice were sacrificed after imaging on day 6 post-dose. Blood was collected by cardiac puncture into scintillation vials. Normal tissues (inguinal and axillary lymph nodes, thymus, spleen, heart, lungs, stomach, small intestine, liver, kidneys, bones and ovaries) were then excised and placed in scintillation vials. Tumors were similarly collected into scintillation vials. All vials were preweighed and then reweighed to determine blood and tissue weights. The γ-radiation activity for all samples was then counted on an automatic gamma counter (Wizard 2470, Perkin Elmer) and the results were recorded as counts per minute (cpm). The % ID/g for each sample was determined using sample counts relative to the number of standard doses obtained from the original input material. Subsequently, individual %VD/g values were obtained by dividing the %VD value by the corresponding weight of the corresponding blood, tissue, or tumor sample.

⁸⁹ Zr-labeled BSMUC16/CD3-001 and ⁸⁹ Zr-labeled BSMUC16/CD3-005 demonstrated specific localization in MUC16+ tumors and CD3+ lymphoid tissues, with distribution in lymphoid tissues correlating with relative affinity for CD3. Both bispecific antibodies against MUC16xCD3 demonstrated equivalent tumor localization in the presence of CD3+ tissues.

Example 5 Toxicology studies in cynomolgus monkeys show no apparent toxicity for anti-MUC16xCD3 bispecific antibodies

BSMUC16/CD3-001 cross-reacts with monkey MUC16 and CD3. To determine safety and tolerability and characterize the pharmacokinetics of the bispecific antibodies, a multi-dose toxicity study was conducted in cynomolgus monkeys. Six monkeys/sex/group received weekly BSMUC16/CD3-001 for a total of five doses of 0.01, 0.1, or 1 mg/kg. At the end of the dosing period, 3 animals/sex/group were sacrificed and tissue examined for changes by microscopy, while the remaining 3 animals/sex/group underwent 12 weeks of recovery without treatment to assess the reversibility or persistence of any effects associated with BSMUC16 /CD3-001. BSMUC16/CD3-001 was well tolerated and all animals survived to scheduled necropsy. Toxicokinetic analysis demonstrated dose-dependent exposure effects and linear kinetics across dose groups without any gender differences (data not shown). Continuous exposure to BSMUC16/CD3-001 was observed throughout the dosing phase, and exposure to BSMUC16/CD3-001 was maintained until the end of the recovery phase in all (n=6) and 50% of animals in the 0.1 and 1.0 mg/kg groups. respectively. BSMUC16/CD3-001 was not detected in the serum of any animal in the 0.01 mg/kg group after 8 weeks of recovery. The half-life of BSMUC16/CD3-001 was approximately 10 days.

There were no clinical observations associated with BSMUC16/CD3-001, nor were there any changes in urinalysis parameters, peripheral blood immunophenotyping, food intake, or body weight during the dosing or recovery period. It is important to note that administration of BSMUC16/CD3-001 did not result in any changes in pharmacological safety assessments regarding respiratory, nervous or cardiovascular CD3 function, including ECG parameters. There were no BSMUC16/CD3-001-related changes in organ weight, and no macroscopic changes were detected in either terminal necropsy or recovery necropsy. A dose-dependent, reversible increase in circulating inflammatory markers (C-reactive protein (CRP) and IL-6) was observed within 1 day after an initial dose of 1.0 or 0.1 mg/kg, but this increase was not evident after subsequent doses (data not available). shown). IN

- 28046291 Consistent with minimal increases in serum cytokines, T cell redistribution was not detected following administration of BSMUC16/CD3-001 (data not shown), in contrast to what has been reported for several CD3 bispecific molecules against hematologic tumors.

The cynomolgus macaque study was conducted in accordance with IACUC guidelines. Cynomolgus monkeys (6 animals/sex/group) were administered control (diluted placebo) or BSMUC16/CD3-001 (0.01, 0.1, or 1 mg/kg) once weekly via a 30-minute intravenous infusion. The control was 10 mM histidine with 10% sucrose and 0.05% polysorbate 20, pH 6, diluted in 0.9% sodium chloride injection, USP (sterile saline). Blood or tissue samples were collected at various time points for clinical pathology and histopathology. The concentration of BSMUC16/CD3-001 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 using MSD (Meso Scale Diagnostics, Rockville, MD). The number of T cells was determined using flow cytometry. Briefly, blood was collected into potassium EDTA tubes, lysed, stained for CD3, CD4, and CD8 (BD Biosciences), and relative values were determined for each phenotype using FACS Canto II. These values were then multiplied by the absolute lymphocyte count (using a hematology assay) to calculate the absolute cell count for each phenotype.

Immunohistochemical staining for MUC16 was observed in the expected tissues: pancreas (mesothelium, ductal epithelium), heart and ovary (data not shown), as well as salivary gland (goblet cells), liver (mesothelium, bile duct), lungs (mesothelium, bronchiolar/ bronchial epithelium), small intestine (mesothelium), testes (mesothelium, rete testis/effurent duct), and tonsils (epithelium, mucous glands) (not shown). BSMUC16/CD3-001-associated microscopic changes assessed by histological hematoxylin and eosin (H&E) staining included inflammation (leukocyte infiltration) and increased mesothelial cell size and mesothelial cell richness, resulting in non-adverse serosa and/or submesothelial thickening. connective tissue of many organs of the chest cavity and peritoneum. These changes were typically focal or multifocal in nature and were minimal or minor in severity and were considered to be a target of BSMUC16/CD3-001, resulting from the involvement of MUC16 expressed on serous epithelial (mesothelial) cells and activation of T cells. Importantly, serous changes were reversed or tended to return to baseline at the end of the recovery period (data not shown).

Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein following administration of BSMUC16/CD3-001 without apparent toxicity.

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

Because cytokine release syndrome (CRS) is a common serious side effect of anti-CD3 bispecific antibody and CAR-T cell therapy, a study was conducted to monitor serum cytokines in appropriate models after treatment with BSMUC16/CD3-001. Genetically humanized tumor-free MUC16/CD3 mice had no obvious serum cytokine response to BSMUC16/CD3-001 administration.

To assess T cell activation in vivo by BSMUC16/CD3-001, cytokine levels were measured in the serum of tumor-bearing mice. Serum samples were collected 4 hours after the first dose of antibody in the 0.5 mg/kg BSMUC16/CD3-001, CD3-binding control, and non-binding control groups. Treatment with activated BSMUC16/CD3-001 T cells, as determined by induction of IFN-γ, TNF-α, IL-2, IL-6, IL-8, and IL-10, compared with non-binding control and CD3-binding control (data not shown). The BSMUC16/CD3-001-induced cytokine response required the presence of both T cells and OVCAR-3/Luc cells, since mice carrying only OVCAR3/Luc cells had no detectable levels of human IFN-γ in serum, and tumor-free mice cells providing MUC16 for cross-linking did not demonstrate an increase in serum IFN-γ in response to BSMUC16/CD3-001 (data not shown).

Measurement of serum cytokine levels: T cell activation in response to BSMUC16/CD3-001 treatment was assessed by measuring serum concentrations of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL- 2), IL-4, IL-6, IL-8, IL-10, IL-12p70, IL13 and IL-LB four hours after the first dose of 0.5 mg/kg. Cytokine levels were analyzed using the V-plex Human Prolnflamasted-10 Plex kit according to the manufacturer's instructions (Meso Scale Diagnostics, Rockville, MA). Cytokines were measured in two separate studies with 4-6 mice per group.

Example 7. Expression of MUC16 in humanized mice and the effect of bispecific antibodies against MUC16xCD3 on M11C16-positive tissues

To investigate the antitumor efficacy of BSMUC16/CD3-001 in mice with completely

- 29 046291 with an intact immune system, mice were genetically modified to express human CD3 on T cells and the MUC16 region covering the antigen binding region, both at endogenous mouse loci (knockin mice). To validate these mice, MUC16 expression was examined by RT-PCR and IHC. RNA expression was detected in the trachea and also at low levels in the lungs, heart, ovaries, pancreas, and bladder (data not shown), similar to published data on MUC16 expression in mice. To assess MUC16 protein expression, IHC was performed on selected tissues using an anti-human MUC16 antibody that recognizes the membrane-proximal region of MUC16. MUC16 protein expression was confirmed in the surface epithelium of the ovaries and stomach of these mice. MUC16 was also observed in the tracheal lining/epithelium as well as in glands in the submucosal layer as described in humans (data not shown).

Mouse Tissue Histology: Tissues from humanized or WT mice were collected and stained with anti-MUC16 antibody binding to the membrane-proximal domain of MUC16 by IHC using Ventana Discovery XT (Ventana; Tucson, AZ). 5-μm paraffin sections were cut onto Superfrost PLUS slides and baked for one hour at 60°C. Immunohistochemical staining was performed on a Discovery XT automated staining system for IHC using the Ventana DAB Map detection kit. Dewaxing was carried out using EZ Prep solution at 75°C for 8 minutes. Gentle antigen retrieval was performed using Tris-EDTA pH 9 (CC1) buffer from Ventana (95°C for 8 minutes, then 100°C for 24 minutes). This was followed by several stages of blocking. Tissue sections were incubated with anti-MUC16 antibody (2 μg/ml) for 8 hours at room temperature. An isotype control antibody recognizing an irrelevant nonbinding antibody was used as a negative control. Primary antibodies and negative controls were applied manually. Biotinylated goat anti-human IgG (Jackson ImmunoResearch) was used as a secondary antibody (1 μg/ml), and samples were incubated for an hour at room temperature. The chromogenic signal was obtained using the Ventana DAB MAP kit. Microslides were manually counterstained with hematoxylin (2 minutes), dehydrated and covered with coverslips. Images were acquired on an Aperio AT 2 slide scanner (Leica Biosystems; Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs; Corrales, NM). Hematoxylin and eosin staining was performed by Histoserv, Inc (Germantown, MD, USA).

T cells in these mice are polyclonal, as assessed by Ve T cell receptor (TCR) frequency, express human CD3, and are present in numbers similar to those in wild-type mice (data not shown). To determine whether BSMUC16/CD3-001 induces any T cell activation or effects on normal tissues in these animals, tumor-free mice were administered a high dose of BSMUC16/CD3-001 (10 mg/kg) and the amount of T -cells in the blood, serum cytokines and histopathology were examined. Although T cells can be activated by anti-human CD3 antibody (OKT3), as measured by T cell accumulation in the blood and elevated serum cytokine levels (data not shown), BSMUC16/CD3001 did not induce such effects, suggesting limited availability of the MUC16 target ( data not shown). To determine whether BSMUC16/CD3-001 induces any microscopic changes in tissues expressing MUC16, MUC16 and CD3 humanized mice received two doses of 10 mg/kg BSMUC16/CD3-001 on day 0 and day 3 . At day 5, several MUC16-expressing tissues were examined (trachea, stomach, and ovary), and no cellular infiltration or necrosis was observed in these tissues following administration of BSMUC16/CD3-001 (data not shown).

Histopathological examination revealed no inflammation or infiltration in MUC16-expressing tissues in mice after administration of BSMUC16/CD3-001 at the time studied.

The results of this study, as well as the cynomolgus monkey study described in Example 5, demonstrate the safety profile of BSMUC16/CD3-001. BSMUC16/CD3-001 induced only minimal serum cytokines, and although focal induction of inflammation and serosal thickening was observed in MUC16-expressing activity, indicating targeted activity, these effects disappeared by the end of the recovery period and were consistent with inflammation and increased cellularity, suggesting about restoration. The observed serous changes did not correlate with any clinical observations, clinical pathology (other than the inflammatory response) or microscopic changes in the underlying parenchyma. In summary, studies in genetically humanized mice and cynomolgus monkeys demonstrated that BSMUC16/CD3001 was well tolerated.

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

To monitor specific killing of MUC16-bearing target cells by flow cytometry, the ovarian cell line OVCAR-3 was labeled with 1 μM Violet Cell Tracker. After labeling, cells were incubated overnight at 37°C. Separately, human PBMCs were placed in medium from up to

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Claims (26)

adding RPMI at a concentration of 1x 10 ⁶ cells/ml and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells and some monocytes. The next day, target cells were co-incubated with naïve PBMCs with adherent cell depletion (effector cells/target cells 4:1) and serial dilution of either BSMUC16/CD3001 or CD3-binding control for 72 hours at 37°C. Cells were removed from cell culture plates using trypsin and analyzed by FACS. For FACS analysis, cells were stained with Live/Dead Far Red Cell Tracker (Invitrogen). To assess killing specificity, cells were gated on CellTracker Violet-labeled populations. PD-1 expression was assessed by incubating cells with directly conjugated antibodies to CD2, CD4, CD8 and PD-1, recording the percentage of PD-1/CD4-positive T cells or PD-1/CD8-positive T cells from the total number T cells (CD2+). Incubation with BSMUC16/CD3-001 increased the percentage of PD-1+ T cells by more than 10-fold (CD4+ T cells) or more than 3-fold (CD8+ T cells) compared to controls. The results are shown in Fig. 3. The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. CLAIM 1. A method of treating or inhibiting the growth of a MUC16-expressing tumor in a subject, comprising administering an antibody or antigen-binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1); and administering a bispecific antibody comprising a first antigen binding arm that specifically binds MUC16 and a second antigen binding arm that specifically binds CD3, wherein (a) the anti-PD-1 antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions, HCDR1, HCDR2, HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NO: 35, 36, 37, 38, 39 and 40, respectively; (b) the first antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, A-HCDR1, A-HCDR2 and A-HCDR3, and three light chain complementarity determining regions, A-LCDR1, A-LCDR2 and A-LCDR3, where A -HCDR1, A-HCDR2, AHCDR3, A-LCDR1, A-LCDR2 and A-LCDR3 contain the amino acid sequences SEQ ID NO: 8, 9, 10, 11, 12 and 13, respectively; and (c) the second antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, B-HCDR1, B-HCDR2 and B-HCDR3, and three light chain complementarity determining regions, B-LCDR1, B-LCDR2 and B-LCDR3, wherein B-HCDR1, B-HCDR2, BHCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 contain the amino acid sequences SEQ ID NO: 14, 15, 16, 11, 12 and 13, respectively, or SEQ ID NO: 26, 27 , 28, 11, 12 and 13, respectively. 2. The method according to claim 1, characterized in that (a) the anti-PD-1 antibody is administered before, simultaneously or after the bispecific antibody; or (b) one or more doses of an anti-PD-1 antibody is administered in combination with one or more doses of a bispecific antibody. 3. The method according to claim 1 or 2, characterized in that (a) the anti-PD-1 antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight; (b) the bispecific antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight; (c) each dose of anti-PD-1 antibody is administered 0.5 to 12 weeks after the immediately preceding dose; (d) each dose of the bispecific antibody is administered 0.5 to 12 weeks after the immediately preceding dose; and/or (e) the antibodies are administered intravenously, subcutaneously or intraperitoneally. 4. The method according to any one of claims 1 to 3, characterized in that (a) the tumor is ovarian cancer, or the tumor is pancreatic cancer; and/or (b) the subject is resistant to, has had an insufficient response to, or has relapsed after prior therapy. 5. The method according to any one of claims 1 to 4, wherein the antibody or antigen binding fragment thereof and the bispecific antibody are administered in combination with a third therapeutic agent or therapy. 6. The method according to claim 5, characterized in that the third therapeutic agent or therapy is selected from the group consisting of radiation therapy, surgery, a chemotherapeutic agent, - 31 046291 cancer vaccine, PD-L1 inhibitor, LAG-3 inhibitor, CTLA-4 inhibitor, TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, indoleamine 2,3-dioxygenase (IDO) inhibitor, vascular endothelial growth factor antagonist (VEGF), angiopoietin-2 (Ang2) inhibitor, transforming growth factor beta (TGF-β) inhibitor, epidermal growth factor receptor (EGFR) inhibitor, tumor-specific antigen antibody, bacillus Calmette-Guérin vaccine, granulocyte-macrophage colony-stimulating factor, cytotoxin , an inhibitor of the interleukin-6 receptor (IL-6L). an interleukin-4 receptor (IL-4R) inhibitor, an IL-10, IL-2, IL-7, IL-21, IL-15 inhibitor, an antibody-drug conjugate, an anti-inflammatory drug, and a dietary supplement. 7. The method according to any one of claims 1 to 6, characterized in that the first antigen binding arm of the bispecific antibody comprises an HCVR containing the amino acid sequence of SEQ ID NO: 1, and an LCVR containing the amino acid sequence of SEQ ID NO: 2. 8. The method according to any one of claims 1 to 7, wherein B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and BLCDR3 of the second antigen-binding arm of the bispecific antibody contain the amino acid sequences SEQ ID NO: 14, 15, 16, 11, 12 and 13, respectively. 9. The method according to claim 8, characterized in that the second antigen-binding arm of the bispecific antibody contains a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: : 2. 10. The method according to any one of claims 1 to 7, characterized in that B-HCDR1, B-HCDR2, B-HCDR3, BLCDR1, B-LCDR2 and B-LCDR3 of the second antigen-binding arm of the bispecific antibody contain the amino acid sequences of SEQ ID NO: 26 , 27, 28, 11, 12 and 13, respectively. 11. The method of claim 10, wherein the second antigen-binding arm of the bispecific antibody comprises an HCVR containing the amino acid sequence of SEQ ID NO: 7, and an LCVR containing the amino acid sequence of SEQ ID NO: 2. 12. The method according to any one of claims 1 to 11, wherein the anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 33, and an LCVR containing the amino acid sequence of SEQ ID NO: 34. 13. The method according to any one of claims 1-9 or 12, where the bispecific antibody contains a first heavy chain containing the amino acid sequence of SEQ ID NO: 29, paired with a light chain containing the amino acid sequence of SEQ ID NO: 30, and a second heavy chain, comprising the amino acid sequence of SEQ ID NO: 31 paired with a light chain containing the amino acid sequence of SEQ ID NO: 30. 14. The method according to any one of claims 1-7 or 10-12, where the bispecific antibody contains a first heavy chain containing the amino acid sequence of SEQ ID NO: 29, paired with a light chain containing the amino acid sequence of SEQ ID NO: 30, and a second heavy chain a chain containing the amino acid sequence of SEQ ID NO: 32 paired with a light chain containing the amino acid sequence of SEQ ID NO: 30. 15. The method according to any one of claims 1 to 14, wherein the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain containing the amino acid sequence of SEQ ID NO: 42. 16. Use of an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 (PD-1) in the manufacture of a medicament for treating or inhibiting the growth of a MUC16 expressing tumor in a subject in combination with a bispecific antibody comprising a first antigen binding arm that specifically binds MUC16, and a second antigen binding arm that specifically binds CD3, wherein (a) the anti-PD-1 antibody or antigen binding fragment thereof contains three heavy chain complementarity determining regions, HCDR1, HCDR2, HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NO: 35, 36, 37, 38, 39 and 40, respectively; (b) the first antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, A-HCDR1, A-HCDR2 and A-HCDR3, and three light chain complementarity determining regions, A-LCDR1, A-LCDR2 and A-LCDR3, where A -HCDR1, A-HCDR2, AHCDR3, A-LCDR1, A-LCDR2 and A-LCDR3 contain the amino acid sequences SEQ ID NO: 8, 9, 10, 11, 12 and 13, respectively; and (c) the second antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, B-HCDR1, B-HCDR2 and B-HCDR3, and three light chain complementarity determining regions, B-LCDR1, B-LCDR2 and B-LCDR3, wherein B-HCDR1, B-HCDR2, BHCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 contain the amino acid sequences SEQ ID NO: 14, 15, 16, 11, 12 and 13, respectively, or SEQ ID NO: 26, 27 , 28, 11, 12 and 13, respectively. 17. Use of a bispecific antibody containing a first antigen-binding arm, which - 32 046291 which specifically binds MUC16, and a second antigen binding arm that specifically binds CD3, in the production of a medicament for treating or inhibiting the growth of a MUC16 expressing tumor in a subject in combination with an antibody or antigen binding fragment thereof that specifically binds programmed cell death protein 1 ( PD-1), and: (a) an anti-PD-1 antibody or antigen binding fragment thereof contains three heavy chain complementarity determining regions, HCDR1, HCDR2 and HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences SEQ ID NO: 35, 36, 37, 38, 39 and 40, respectively; (b) the first antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, A-HCDR1, A-HCDR2 and A-HCDR3, and three light chain complementarity determining regions, A-LCDR1, A-LCDR2 and A-LCDR3, where A -HCDR1, A-HCDR2, AHCDR3, A-LCDR1, A-LCDR2 and A-LCDR3 contain the amino acid sequences SEQ ID NO: 8, 9, 10, 11, 12 and 13, respectively; and (c) the second antigen binding arm of the bispecific antibody comprises three heavy chain complementarity determining regions, B-HCDR1, B-HCDR2 and B-HCDR3, and three light chain complementarity determining regions, B-LCDR1, B-LCDR2 and B-LCDR3, wherein B-HCDR1, B-HCDR2, BHCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 contain the amino acid sequences SEQ ID NO: 14, 15, 16, 11, 12 and 13, respectively, or SEQ ID NO: 26, 27 , 28, 11, 12 and 13, respectively. 18. Use according to claim 16 or 17, characterized in that (a) the anti-PD-1 antibody is administered before, simultaneously or after the bispecific antibody; or (b) one or more doses of an anti-PD-1 antibody is administered in combination with one or more doses of a bispecific antibody. 19. Use according to any one of claims 16 to 18, characterized in that (a) the anti-PD-1 antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight; (b) the bispecific antibody is administered at a dose of 0.1 mg/kg to 20 mg/kg of the subject's body weight; (c) each dose of anti-PD-1 antibody is administered 0.5 to 12 weeks after the immediately preceding dose; (d) each dose of the bispecific antibody is administered 0.5 to 12 weeks after the immediately preceding dose; and/or (f) the antibodies are administered intravenously, subcutaneously or intraperitoneally. 20. Use according to any one of claims 16 to 19, characterized in that (a) the tumor is ovarian cancer, or the tumor is pancreatic cancer; and/or (b) the subject is resistant to, has had an insufficient response to, or has relapsed after prior therapy. 21. Use according to any one of claims 16 to 20, wherein the antibody or antigen binding fragment thereof and the bispecific antibody are further combined with a third therapeutic agent or therapy. 22. The use of claim 21, wherein the third therapeutic agent or therapy is selected from the group consisting of radiation therapy, surgery, a chemotherapy agent, a cancer vaccine, a PD-L1 inhibitor, a LAG-3 inhibitor, a CTLA-4 inhibitor, TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, indoleamine 2,3 dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist, angiopoietin-2 (Ang2) inhibitor, transforming growth factor beta (TGF-β) inhibitor, epidermal growth factor receptor (EGFR) inhibitor, antibody to tumor-specific antigen, bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor, cytotoxin, interleukin-6 receptor inhibitor (IL-6C). an interleukin-4 receptor (IL-4R) inhibitor, an IL-10, IL-2, IL-7, IL-21, IL-15 inhibitor, an antibody-drug conjugate, an anti-inflammatory drug, and a dietary supplement. 23. Use according to any one of claims 16 to 22, characterized in that the first antigen binding arm of the bispecific antibody comprises an HCVR containing the amino acid sequence of SEQ ID NO: 1, and an LCVR containing the amino acid sequence of SEQ ID NO: 2. 24. Use according to any one of claims 16 to 23, wherein B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 of the second antigen-binding arm of the bispecific antibody contain the amino acid sequences of SEQ ID NO: 14, 15, 16, 11, 12 and 13, respectively. 25. The use of claim 24, wherein the second antigen-binding arm of the bispecific antibody comprises a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 3 : 2. 26. Use according to any one of claims 16 to 23, characterized in that B-HCDR1, B-HCDR2, B-HCDR3, BLCDR1, B-LCDR2 and B-LCDR3 of the second antigen-binding arm of the bispecific antibody contains -