RU2822091C2 - Use of bispecific antigen-binding molecules that bind muc16 and cd3, in combination with costimulation 4-1bb - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions involving the use of a bispecific antigen-binding molecule to CD3/MUC16 and 4-1BB agonist for treating MUC16-positive cancer or inhibiting the growth of MUC16-positive tumour and a pharmaceutical combination for treating MUC16-positive cancer or inhibiting the growth of MUC16-positive tumour. In one embodiment, the bispecific antigen-binding molecule to CD3/MUC16 contains the heavy chain variable region amino acid sequence SEQ ID NO: 1 and light chain variable region SEQ ID NO: 3.

EFFECT: invention extends the range of products for treating MUC16-positive cancer.

24 cl, 6 dwg, 8 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to bispecific antigen binding molecules that bind mucin 16 (MUC16) and CD3, in combination with 4-1BB costimulation, as well as methods of using them.

LINK TO LIST OF SEQUENCES

[0002] An official copy of the sequence listing is submitted along with the description electronically via EFS-Web as a sequence listing in ASCII format with file name 10604WO01_SEQ_LIST_ST25, creation date June 19, 2020, and approximately 16,384 bytes in size. The sequence listing contained in this ASCII document is part of the specification and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125 or CA-125, is a highly glycosylated integral membrane glycoprotein with a single transmembrane domain that is expressed at high levels in ovarian cancer . MUC16 consists of three main domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with sea urchin sperm protein repeats, an enterokinase domain, and agrin (SEA) domains, and a carboxy-terminal domain, which contains a segment of the transmembrane region and a short cytoplasmic tail. . Proteolytic cleavage results in the shedding of most of the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma of the May conforming type, cervical adenocarcinoma and gastric adenocarcinoma, and in diseases and conditions including inflammatory bowel disease, liver cirrhosis, heart failure, peritoneal infection and abdominal surgery. (Haridas, D. et al., 2014, FASEB J., 28:4183-4199). 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 developed. Oregovomab and abgovomab are anti-MUC16 antibodies that have been used with limited success. (Felder, supra, Das, S. and Batra, S.K. 2015, Cancer Res. 75:4660-4674.)

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor (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 promote the formation of 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 involving T cell activation. In addition, bispecific antibodies capable of binding CD3 and the target antigen have been proposed for therapeutic applications, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0005] In T cell activation, costimulation through the TNF receptor superfamily is a key factor for survival, acquisition of effector functions, and differentiation of memory cells. 4-1BB (TnfrsfP), also known as CD137, is a member of the TNF receptor superfamily. Receptor expression is induced upon lymphocyte activation following TCR-mediated priming, but its levels can be increased by CD28 costimulation. Exposure of CD8 ⁺ T cells to ligands or agonistic monoclonal antibodies (mAbs) co-stimulates 4-1BB, promoting clonal expansion, T cell survival and development, induced proliferation of peripheral monocytes, NF-kappa B activation, increased T cell apoptosis induced by advanced via TCR/CD3 activation, and the formation of memory cells.

SHORT DESCRIPTION

[0006] Provided herein are methods of treating cancer in a subject. In some aspects, the methods include administering to the subject a pharmaceutical composition comprising an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen binding molecule and a pharmaceutically acceptable carrier or diluent, and further administering to the subject a 4-1BB agonist. In some aspects, the methods include administering to a subject a pharmaceutical composition comprising an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen binding molecule, a 4-1BB agonist, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and gastric adenocarcinoma. In some cases, the cancer is ovarian cancer. In some cases, the cancer is breast cancer.

[0007] Additionally provided herein are methods of treating cancer or inhibiting tumor growth. In some aspects, the methods include administering to a subject in need thereof a therapeutically effective amount of each of: (a) an anti-MUC16 antibody or antigen-binding fragment thereof or a bispecific antigen-binding molecule against CD3/MUC16 and (b) a 4-1BB agonist.

[0008] Also provided herein are therapeutic methods for targeting/killing tumor cells expressing MUC16. In some aspects, the therapeutic methods include administering a therapeutically effective amount of an anti-MUC16 antibody or an anti-CD3/MUC16 bispecific antigen-binding molecule and a therapeutically effective amount of a 4-1BB agonist to a subject in need thereof. In some aspects, the anti-MUC16 antibody or anti-CD3/MUC16 bispecific antigen-binding molecule and the 4-1BB agonist are separately formulated. In some aspects, an anti-MUC16 antibody or anti-CD3/MUC16 bispecific antigen-binding molecule and a 4-1BB agonist are formulated in a single pharmaceutical composition.

[0009] Also provided herein is the use of an anti-MUC16 antibody or a 4-1BB agonist anti-CD3/MUC16 bispecific antigen-binding molecule in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by MUC16-expressing cells.

[0010] Administration of an anti-MUC16 antibody or antigen binding fragment thereof or a bispecific antigen binding molecule to MUC16/CD3 in the presence of a 4-1BB agonist may reduce tumor volume compared to the tumor volume in a subject administered an anti-MUC16 antibody or bispecific antigen binding molecule to CD3 /MUC16 in the absence of the 4-1BB agonist.

[0011] Administration of an anti-MUC16 antibody or antigen binding fragment thereof or a bispecific antigen binding molecule to MUC16/CD3 in the presence of a 4-1BB agonist may improve tumor-free survival in a subject compared to tumor-free survival in a subject administered an anti-MUC16 antibody or bispecific antigen binding molecule to CD3/MUC16 in the absence of the 4-1BB agonist. In some aspects, the increase in tumor-free survival occurs without weight loss in the subject.

[0012] Administration of an anti-MUC16 antibody or antigen binding fragment thereof or a bispecific antigen binding molecule to MUC16/CD3 in the presence of a 4-1BB agonist may initiate a memory cell response in a subject treated with an anti-MUC16 antibody or bispecific antigen binding molecule to CD3/ MUC16 in the presence of the 4-1BB agonist, with subsequent exposure to tumor cells.

[0013] The 4-1BB agonist may be a small molecule or biological 4-1BB agonist, and in some aspects is an antibody. Exemplary 4-1BB agonists include commercially available antibodies, such as mouse 4-1BB agonists, and therapeutic antibodies, such as urelumab and utomilumab.

[0014] In accordance with the methods provided herein, antibodies to MUC16 or antigen-binding fragments thereof, as well as bispecific antibodies and antigen-binding fragments thereof that bind human MUC16 and human CD3, are useful. Bispecific antibodies are useful, among other things, for targeting T cells expressing CD3 and for promoting T cell activation, for example in conditions where T cell-mediated killing of MUC16 expressing cells is useful or necessary. For example, bispecific antibodies are capable of targeting CD3-mediated T cell activation to specific MUC16-expressing cells, such as ovarian tumor cells.

[0015] Antibodies to MUC16 or antigen binding fragments thereof that bind MUC16 are useful in combination with a 4-1BB agonist for the treatment of diseases and disorders associated with or caused by MUC16-expressing tumors, and in particular tumors that are very large and/or more difficult to treat. Exemplary MUC16 antibodies and antigen binding fragments thereof are described in detail in U.S. 2018/0112001. In some aspects, the anti-MUC16 antibody comprises HCVR of SEQ ID NO: 18 and LCVR of SEQ ID NO: 26, referred to in U.S. 2018/0112001. In some aspects, the kMUC16 antibody is the H1H8767P antibody referred to in U.S. 2018/0112001.

[0016] In addition, exemplary bispecific antigen binding molecules that bind both MUC16 and CD3 are described in US Publication No. 2018/0112001, which is incorporated herein by reference.

[0017] Bispecific antigen binding molecules (eg antibodies) that bind MUC16 and CD3 are also referred to herein as “MUC16/CD3 bispecific molecules”, “CD3/MUC16 bispecific molecules”, “MUC16xCD3 bsAb” or simply “MUC16xCD3” " The MUC16 binding portion of the MUC16/CD3 bispecific molecule is useful for targeting cells (eg, tumor cells) that express MUC16 (eg, ovarian tumors), and the CD3 binding portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of MUC16 on the tumor cell and CD3 on the T cell facilitates targeted killing (cell lysis) of the targeted tumor cell by the activated T cell. Therefore, MUC16/CD3 bispecific molecules are useful, inter alia, for the treatment of diseases and disorders associated with or caused by MUC16-expressing tumors (eg, ovarian tumors). MUC16/CD3 bispecific molecules are also useful in combination with a 4-1BB agonist to treat diseases and disorders associated with or caused by MUC16-expressing tumors and to initiate a memory cell response and/or epitope spreading. In some aspects, MUC16/CD3 bispecific antigen-binding molecules in combination with a 4-1BB agonist are useful to stimulate an antitumor response independent of the presence of or response to the MUC16 antigen, particularly to suppress secondary tumor formation.

[0018] Bispecific antigen binding molecules comprise a first antigen binding domain that specifically binds human CD3 and a second antigen binding domain that specifically binds MUC16.

[0019] Exemplary bispecific antibodies useful in accordance with the methods provided herein are CD3/MUC16 bispecific molecules, wherein the first antigen binding domain that specifically binds CD3 comprises any of the heavy chain variable region (HCVR) amino acid sequences, any of the light chain variable region (LCVR) amino acid sequences, any of the HCVR/LCVR amino acid sequence pairs, any of the heavy chain amino acid sequences CDR1-CDR2-CDR3, or any of the light chain amino acid sequences CDR1-CDR2-CDR3 set forth in US Publication No. 2018/0112001.

[0020] In accordance with the methods provided herein, useful are bispecific antigen binding molecules for CD3/MUC16, wherein the first antigen binding domain that specifically binds CD3 comprises any of the HCVR amino acid sequences and/or any of the LCVR amino acid sequences or substantially highly similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, as set forth in Tables 16, 18, 19, 22, and 24 of US Publication No. 2018 /0112001. In some aspects, the first antigen binding domain that specifically binds CD3 comprises the amino acid sequence of HCVR-1 of SEQ ID NO: 2. In some aspects the first antigen binding domain that specifically binds CD3 comprises the full length heavy chain amino acid sequence of SEQ ID NO: 5.

[0021] In accordance with the methods provided herein, bispecific molecules for MUC16/MUC16 are applicable, wherein the second antigen binding domain, which specifically binds CD3, contains any of the HCVR amino acid sequences and/or any of the LCVR amino acid sequences or to a significant extent sequence similarity thereto having at least 90%, at least 95%, at least 98%) or at least 99% sequence identity, as set forth in Table 1 of US Publication No. 2018/0112001. In some aspects, the second antigen binding domain that specifically binds MUC16 comprises the amino acid sequence of HCVR-2 of SEQ ID NO: 1. In some aspects the second antigen binding domain that specifically binds MUC16 comprises the full length heavy chain amino acid sequence of SEQ ID NO: 4.

[0022] In accordance with the methods provided herein, bispecific molecules for CD3/MUC16 containing any of the sequences, or a substantially similar sequence thereto, characterized by at least 90%, at least 95%, are useful. at least 98%, or at least 99% sequence identity, as set forth in Table 4 of US Publication No. 2018/0112001.

[0023] In accordance with the methods provided herein, useful are CD3/MUC16 bispecific molecules, wherein the first antigen binding domain, which specifically binds CD3, contains the amino acid sequence of HCVR-1 of SEQ ID NO: 2, and wherein the second antigen binding domain , which specifically binds MUC16, contains the amino acid sequence of HCVR-2 of SEQ ID NO: 1. In some aspects, the bispecific CD3/MUC16 molecule contains the amino acid sequence of the general LCVR of SEQ ID NO: 3.

[0024] In accordance with the methods provided herein, useful are bispecific antigen-binding molecules for CD3/MUC16, wherein the first antigen-binding domain, which specifically binds CD3, contains the full-length heavy chain amino acid sequence of SEQ ID NO: 5, and wherein the second antigen-binding domain the domain that specifically binds MUC16 contains the full-length heavy chain amino acid sequence of SEQ ID NO: 4. In some aspects, the CD3/MUC16 bispecific molecule contains the full-length light chain amino acid sequence of SEQ ID NO: 6.

[0025] In one aspect, provided herein is a pharmaceutical composition comprising an anti-MUC16 antigen-binding molecule or a bispecific anti-MUC16/CD3 antigen-binding molecule and a pharmaceutically acceptable carrier or diluent. In some aspects, the pharmaceutical composition further comprises a 4-1BB agonist.

[0026] Anti-MUC16 antibodies and antigen-binding fragments thereof, as well as anti-CD3/MUC16 bispecific antigen-binding molecules having a modified glycosylation profile, are useful in the methods of the present invention. In some applications, modification to remove undesired glycosylation sites or an antibody without the fucose moiety present on the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) functionality (see Shield et al. (2002) JBC 277:26733 ). In other applications, galactosylation modification can be performed to modify complement dependent cytotoxicity (CDC).

[0027] In one aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-MUC16 antibody or antigen binding fragment thereof or a CD3/MUC16 bispecific antigen-binding molecule disclosed herein, a 4-1BB agonist, and a pharmaceutically acceptable carrier. In a related aspect of the present invention, a composition is described that is a combination of an anti-MUC16 antibody or antigen binding fragment thereof or a bispecific antigen-binding molecule against CD3/MUC16, a 4-1BB agonist, and a third therapeutic agent. In one embodiment, the third therapeutic agent is any agent that provides benefit when combined with an anti-MUC16 antibody or antigen binding fragment thereof or a bispecific antigen-binding molecule against CD3/MUC16. Exemplary agents that can be combined with a CD3/MUC16 bispecific antigen-binding molecule to advantage are discussed in detail elsewhere in this document.

[0028] In another aspect, provided herein are radiolabeled anti-MUC16 antibody conjugates or anti-CD3/MUC16 bispecific antigen-binding molecule conjugates for use in immune PET imaging. The conjugate contains an antibody to MUC16 or a bispecific antigen-binding molecule to CD3/MUC16, a chelating moiety and a positron emitter.

[0029] This document provides methods for the synthesis of these conjugates and synthetic intermediates useful therefor.

[0030] Provided herein are methods for imaging tissue that expresses MUC16, the methods comprising introducing into the tissue a radiolabeled anti-MUC16 antibody conjugate or a CD3/MUC16 bispecific antigen-binding molecule conjugate described herein and visualizing observation of MUC16 expression by positron emission tomography (PET) imaging.

[0031] Provided herein are methods for imaging tissue containing MUC16-expressing cells, the methods comprising introducing into the tissue a radiolabeled anti-MUC16 antibody conjugate or a CD3/MUC16 bispecific antigen-binding molecule conjugate described herein, and visual observation of MUC16 expression by PET imaging.

[0032] Provided herein are methods for detecting MUC16 in tissue, the methods comprising introducing into the tissue a radiolabeled anti-MUC16 antibody conjugate or a CD3/MUC16 bispecific antigen-binding molecule conjugate described herein and visually observing expression MUC16 by PET imaging. In one embodiment, the tissue is present in a human subject. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer, an inflammatory disease, or an infection.

[0033] Provided herein are methods for detecting MUC16 in tissue, the methods comprising contacting the tissue with an anti-MUC16 antibody conjugate or an anti-CD3/MUC16 bispecific antigen-binding molecule conjugated to a fluorescent molecule described herein and visually observing expression of MUC16 by fluorescence imaging.

[0034] Provided herein are methods for identifying a subject suitable for anticancer therapy, the methods comprising selecting a subject with a solid tumor, administering a radiolabeled anti-MUC16 antibody conjugate or a bispecific antigen-binding molecule anti-CD3/MUC16 conjugate described in herein, and visual observation of the administered radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor allows the subject to be identified as suitable for antitumor therapy.

[0035] Provided herein are methods of treating a tumor, the methods including selecting a subject with a tumor, determining whether the tumor is MUC16 positive, and administering an anti-tumor therapy to a subject in need thereof. In certain embodiments, the antitumor therapy comprises an inhibitor of the PD-1/PD-L1 signaling axis (eg, an anti-PD-1 antibody or an anti-PD-L1 antibody), an example of an immune checkpoint inhibitor therapy. In certain embodiments, the subject is administered a radiolabeled anti-MUC16 antibody conjugate or a CD3/MUC16 bispecific antigen binding molecule conjugate described herein, and the localization of the radiolabeled antibody conjugate is visualized by positron emission imaging tomography (PET) to determine whether the tumor is MUC16 positive. In certain embodiments, the subject is further administered a radiolabeled anti-PD-1 antibody conjugate and localization of the radiolabeled antibody conjugate is visualized by positron emission tomography (PET) imaging to determine whether the tumor is PD-1 -positive.

[0036] Provided herein are methods for monitoring the effectiveness of an anticancer therapy in a subject, the methods comprising selecting a subject with a solid tumor where the subject is being treated with an anticancer therapy, administering to the subject a radiolabeled anti-MUC16 antibody conjugate or an anti-MUC16 conjugate. based on the CD3/MUC16 bispecific antigen-binding molecule described herein, visualizing the localization of the administered radiolabeled conjugate in the tumor via PET imaging and determining tumor growth, where a decrease in conjugate uptake or radiolabel signal relative to baseline indicates the effectiveness of the antitumor therapy.

[0037] In certain embodiments, the anticancer therapy includes a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, as well as those disclosed in US Patent Publication No. 2015-0203580), a CTLA-4 inhibitor (eg ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, another T cell coinhibitor or ligand antagonist (eg antibody to LAG3, CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), indoleamine 2,3-dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist [e.g. “VEGF-Trap”, such as aflibercept or other VEGF inhibitory fusion protein as set forth in US 7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (eg bevacizumab or ranibizumab), or a small molecule VEGF receptor kinase inhibitor (eg sunitinib, sorafenib or pazopanib)], an Ang2 inhibitor (eg nesvacumab), transforming growth factor beta (TGFβ) inhibitor, epidermal growth factor receptor (EGFR) inhibitor (eg erlotinib, cetuximab), CD20 inhibitor (eg anti-CD20 antibody such as rituximab), tumor-specific antigen antibody (eg e.g. CA9, CA125, melanoma associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor protein M2-PK, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), mucin-1, MART-1 and CA19-9], vaccine (eg, Bacillus Calmette-Guerin, cancer vaccine), adjuvant to enhance antigen presentation (eg, granulocyte-macrophage colony-stimulating factor), bispecific antibody (eg, bispecific anti-CD3xCD20 antibody or anti-PSMAxCD3 bispecific antibody), cytotoxin, poly-ADP-ribose polymerase (PARP), chemotherapy agent (eg, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin , paclitaxel and vincristine), cyclophosphamide, radiation therapy agent, IL-6R inhibitor (eg sarilumab), IL-4R inhibitor (eg dupilumab), IL-10 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).

[0038] Other embodiments will be apparent from a review of the following detailed description.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0039] Figure 1 shows the results of the OVCAR-3 study model 1 (average radiation intensity [f/s/cm ² /sr] on day 4). All groups had similar tumor burdens assessed by BLI before dosing. Data shown are tumor burden assessed by BLI at day 4 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. There was no significant difference in tumor burden between groups after dosing began.

[0040] Figure 2 shows the results of the OVCAR-3 study model 1 (average radiation intensity [f/s/ ^cm2 /sr] on day 25). BSMUC16/CD3-001 significantly reduced tumor burden at 12.5 μg, and the addition of anti-4-1BB antibody enhanced antitumor efficacy compared to BSMUC16/CD3-001 alone. NSG mice engrafted with human T cells were implanted with human OVCAR-3/Luc cells. Mice were treated on days 5 and 8 after tumor implantation with BSMUC16/CD3-001 (12.5 μg IV) or control CD3-binding antibody (12.5 μg IV) alone or in combination with the 4-1BB agonist (100 μg IV ). Data shown are tumor burden assessed by BLI at day 25 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. The groups were compared with a control CD3-binding antibody (*p=0.0159 for BSMUC16/CD3-001, **p<0.0079 for BSMUC16/CD3-001 and combination with anti-4-1BB). The combination of BSMUC16/CD3-001 and anti-4-1BB was also compared with BSMUC16/CD3-001 alone (#p=0.0159).

[0041] Figure 3 shows results in the ID8-VEGF/hu MUC16 mouse model after treatment with BSMUC16/CD3-001 + anti-4-1BB combination. Treatment with BSMUC16/CD3-001 significantly improved median survival in the ID8-VEGF/huMUC16 ascites model, and the addition of 4-1BB co-stimulation 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 cell line expressing part of human MUC16. Mice were treated with either BSMUC16/CD3-001 (1 mg/kg IV) or control CD3-binding antibody (1 mg/kg IV) with isotype control on days 3, 6, and 10 postimplantation or with anti-4-1BB antibody (clone LOB12.3, 2.5 mg/kg IV) on day 3 postimplantation, followed by two additional doses of BSMUC16/CD3-001 (1 mg/kg IV) on days 6 and 10. Mice were sacrificed if they showed weight gain, accounting for more than 30%, due to ascites-induced bloating of the abdominal cavity. Statistical significance was determined using the Gehan-Breslow-Wilcoxon method. For statistical analysis, groups were compared to a control CD3-binding antibody (**p=0.0026 for BSMUC16/CD3-001, ****p<0.0001 for BSMUC16/CD3-001 with anti-4-1BB). Additionally, to determine whether the addition of anti-4-1BB resulted in any benefit compared to BSMUC16/CD3-001 alone, anti-4-1BB was compared with BSMUC16/CD3-001 alone (#p=0.0168 ). BSMUC16/CD3-001 increased median survival compared to the control CD3-binding antibody group, as well as the % of mice surviving (from 0 to 27%). The median survival for the BSMUC16/CD3-001 + anti-4-1BB combination group could not be determined because more than 50% of the mice survived. Overall survival in the combination-treated group was 55%.

[0042] Figure 4 depicts the change in weight over time. Treatment with the BSMUC16/CD3-001 + anti-4-1BB combination, as shown in Figure 3, did not initiate any significant weight loss during the administration period, which was used as an indicator of toxicity.

[0043] Figure 5 presents data from a second study in the ID8-VEGF/huMUC16 mouse model treated at 3 different dosing schedules with the BSMUC16/CD3-001 + anti-4-1BB combination: A) combination of either BSMUC16/CD3-001 ( 1 mg/kg IV) or control CD3-binding antibody (1 mg/kg IV) with either isotopic control (2.5 mg/kg IV) or anti-4-1BB antibody (2.5 mg/kg IV) in days 3, 7 and 10 after implantation; B) one dose of the combination BSMUC16/CD3-001 (1 mg/kg IV) plus anti-4-1BB (2.5 mg/kg IV) on day 3 after implantation followed by doses of BSMUC16/CD3-001 (1 mg/kg IV) kg intravenously) on days 7 and 10 after implantation; C) one dose of the combination BSMUC16/CD3-001 (1 mg/kg IV) or control CD3-binding antibody (1 mg/kg IV) plus anti-4-1BB antibody (2.5 mg/kg IV) on day 3 after implantation without additional processing. Data shown are median survival. Mice were sacrificed if they exhibited a weight gain of more than 30% due to ascites-induced abdominal distension. Statistical significance was determined using the Gehan-Breslow-Wilcoxon method. For statistical analysis, groups were compared with a control CD3-binding antibody (**p=0.002 for BSMUC16/CD3-001, ****p<0.0001 for all three groups consisting of BSMUC16/CD3-001 and combination with anti-CD3-binding antibody). 4-1ВВ). In addition, to determine whether the combination with anti-4-1BB resulted in any benefit compared with BSMUC16/CD3-001 alone, all groups were compared to this group (#p=0.011 for Gp4 (3 doses of bispecific antibodies to CD3 + 3 doses of antibody to 4-1BB), #p=0.027 for Gp5 (3 doses of bispecific antibody to CD3 + only 1 dose of antibody to 4-1BB), #p=0.011 for Gp7 (one dose of bispecific antibody to CD3 + antibody to 4-1BB)).

[0044] Figure 6 depicts the change in weight over time. Treatment with the BSMUC16/CD3-001 + anti-4-1BB combination at three different dosing schedules, as shown in Figure 5, did not initiate any significant weight loss during the dosing period, which was used as an indicator of toxicity.

[0045] 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 for purposes of describing 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.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein, the term "approximately" when used in relation to a specific referenced numerical value means that the value may differ from the referenced 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.).

[0047] Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, only the preferred methods and materials are described herein. All patents, patent applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0048] As shown in the Examples section, bispecific antibodies to MUC16xCD3 were effective in the treatment of ovarian tumors in several mouse models. However, the present inventors sought to enhance and prolong MUC16xCD3-induced T cell activity by providing a co-stimulatory signal using a 4-1BB agonist. The 4-1BB signaling pathway can enhance the intensity and duration of T cell responses by promoting T cell survival, reversing T cell anergy, and subsequently leading to the generation of memory T cells to promote effective antitumor activity and epitope spreading.

[0049] As shown herein, combining the anti-MUC16xCD3 bispecific antibody with 4-1BB co-stimulation resulted in significant antitumor efficacy in ovarian tumors. Antitumor effects were observed in the absence of weight loss. This combination has also been shown to induce the formation of tumor-specific memory T cells and initiate epitope spreading. This combination has also been shown to stimulate an antitumor response independent of the presence of or response to the MUC16 antigen, particularly to suppress secondary tumor formation.

[0050] The ability of anti-MUC16 antibodies and bispecific anti-MUC16xCD3 antibodies in combination with 4-1BB costimulation increases the intensity and duration of the T cell response, resulting in significant antitumor efficacy, as demonstrated herein. Combining anti-MUC16 antibodies or bispecific anti-MUC16xCD3 antibodies with 4-1BB costimulation is useful in tumor treatment methods to achieve better overall survival.

Routes of therapeutic use of antigen-binding molecules

[0051] The present invention provides methods comprising administering to a subject in need thereof an anti-MUC16 antibody or an antigen binding fragment thereof or a bispecific antigen binding molecule that specifically binds CD3 and MUC16 with a 4-1BB agonist. A therapeutic composition useful according to the methods described herein may comprise a kMUC16 antibody or a bispecific antigen-binding molecule to MUC16xCD3 and a pharmaceutically acceptable carrier or diluent. As used herein, the expression “subject in need thereof” means a human or non-human animal that exhibits one or more symptoms or signs of cancer (for example, a subject who has a tumor or is suffering from any of the types of cancer mentioned herein below) or that would otherwise benefit from inhibition or reduction of MUC16 activity or depletion of MUC16+ cells (eg ovarian cancer cells).

[0052] The antibodies and bispecific antigen-binding molecules disclosed herein (and therapeutic compositions containing them) are useful, among other things, in combination with a 4-1BB agonist for the treatment of any disease or disorder that would benefit from stimulation, activation and /or targeting the immune response. In particular, anti-MUC16 antibodies and anti-CD3/MUC16 bispecific antigen-binding molecules in combination with a 4-1BB agonist can be used to treat, prevent and/or alleviate any disease or disorder associated with or mediated by the expression of MUC16 or the activity or proliferation of MUC16+ cells . The mechanism of action by which the therapeutic methods disclosed herein are achieved involves killing cells expressing MUC16 in the presence of effector cells, for example, through CDC, apoptosis, ADCC, phagocytosis, or through a combination of two or more of these mechanisms. Cells expressing MUC16 that can be suppressed or killed using antibodies or bispecific antigen binding molecules include, for example, ovarian tumor cells. Additional therapeutic effects are achieved through 4-1BB costimulation, including contributions to clonal expansion, T cell survival and development, induced proliferation of peripheral monocytes, NF-kappa B activation, increased T cell apoptosis induced by TCR/CD3 triggered activation, formation of memory cells.

[0053] Antigen-binding molecules, including anti-MUC16 antibodies and bispecific anti-MUC16/CD3 antibodies in combination with a 4-1BB agonist, can be used to treat, for example, primary and/or metastatic tumors such as ovarian cancer, breast cancer, pancreatic cancer glands, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, intrahepatic mass-forming cholangiocarcinoma, cervical adenocarcinoma and gastric adenocarcinoma, as well as diseases and conditions including inflammatory bowel disease, cirrhosis, heart failure, peritoneal infection and abdominal surgery . In certain embodiments, antibodies or bispecific antigen-binding molecules are used to treat one or more of the following types of cancer: ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, adenocarcinoma cervix and adenocarcinoma of the gastric tract. In certain embodiments of the present invention, anti-MUC16 antibodies or anti-MUC16/CD3 bispecific antibodies in combination with a 4-1BB agonist are useful for treating a patient with ovarian cancer or breast cancer. In other related embodiments disclosed herein, methods are provided comprising administering a CD3/MUC16 bispecific antigen binding molecule in combination with a 4-1BB agonist to a patient with ovarian cancer or breast cancer.

[0054] The present invention also includes methods for initiating a response with the formation of memory cells and/or epitope spreading. The present invention also includes methods for promoting an antitumor response independent of the presence of or response to the MUC16 antigen, for example to suppress the formation of a secondary tumor.

[0055] The present invention also includes methods for treating residual cancer in a subject. As used herein, the term “residual cancer” means the presence or persistence of one or more cancer cells in a subject after treatment with anticancer therapy.

[0056] In certain aspects, the present invention provides methods for treating a disease or disorder associated with MUC16 expression (eg, ovarian cancer), comprising administering one or more bispecific antigen binding molecules described elsewhere in combination with a 4-1BB agonist to a subject after the subject was determined to have ovarian cancer. For example, the present invention provides methods for treating ovarian cancer comprising administering a CD3/MUC16 bispecific antigen binding molecule to a patient after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks , 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received hormonal therapy (eg, androgen deprivation therapy).

Definitions

[0057] As used herein, the expression “CD3” refers to an antigen that is expressed on T cells as part of a multimolecular T cell receptor (TCR) and that consists of a homodimer or heterodimer formed by the association of two of the four receptor chains: CD3- epsilon, CD3 delta, CD3 zeta and CD3 gamma. All references to proteins, polypeptides and protein fragments herein refer to the human version of the corresponding protein, polypeptide or protein fragment unless explicitly stated to be from a non-human species. Thus, the expression "CD3" means human CD3 unless specified to be from a non-human species, such as "mouse CD3", "monkey CD3", etc.

[0058] As used herein, “an antibody that binds CD3” or “an anti-CD3 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and their antigen-binding fragments that specifically recognize a dimeric complex of two CD3 subunits (eg, CD3 gamma/epsilon, delta/epsilon and zeta/zeta dimers). Antibodies and antigen-binding fragments as used herein can bind soluble CD3 and/or CD3 expressed on the surface of a cell. Soluble CD3 includes natural CD3 proteins as well as recombinant variants of CD3 proteins, such as, for example, monomeric and dimeric CD3 constructs that lack the transmembrane domain or are not otherwise associated with the cell membrane.

[0059] As used herein, “cell surface expressed CD3” means one or more CD3 proteins that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of the CD3 protein is located on the extracellular side of the cell membrane and accessible to the antigen-binding portion of the antibody. “Cell surface expressed CD3” includes CD3 proteins contained within a functional T cell receptor in the cell membrane. The expression “CD3 expressed on the cell surface” includes CD3 protein expressed as part of a homodimer or heterodimer on the cell surface (eg, CD3 gamma/epsilon, delta/epsilon and zeta/zeta dimers). The expression “CD3 expressed on the cell surface” also includes a CD3 chain (eg, CD3 epsilon, CD3 delta, or CD3 gamma) that is expressed alone, without other types of CD3 chains, on the cell surface. "CD3 expressed on the cell surface" may comprise or consist of CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface expressed CD3" may comprise or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0060] As used herein, the expression “MUC16” refers to mucin 16, also known as cancer antigen 125 (CA125). MUC16 is a large membrane-associated mucin containing a single transmembrane domain. This cell surface glycoprotein is highly expressed in ovarian cancer and plays a role in stimulating the growth of cancer cells. As used herein, the expressions “antibody that binds MUC16” or “antibody to MUC16” include antibodies and antigen binding fragments thereof that specifically recognize MUC16.

[0061] As used herein, "4-1BB", also known as CD137, refers to an activation-induced co-stimulatory molecule. 4-1BB is an important regulator of immune responses and is a member of the TNF receptor superfamily. The expression "4-1BB agonist" means any ligand that binds 4-1BB and activates the receptor. Exemplary 4-1BB agonists include urelumab (BMS-663513) and utomilumab (PF-05082566), as well as commercially available murine 4-1BB antibodies. In addition, the term “4-1BB agonist” refers to any molecule that partially or completely stimulates, induces, enhances and/or activates the biological activity of 4-1BB. Suitable agonist molecules particularly include agonist antibodies or antibody fragments, including bispecific antibodies, for example, a bispecific antibody containing one arm that binds 4-1BB on an immune cell and another arm that binds, for example, an antigen on tumor target. The term also includes fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. In some embodiments, activation in the presence of an agonist occurs in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 100% greater than the signal measured using the negative control under comparable conditions. The effectiveness of an agonist can also be determined using functional assays, such as the ability of the agonist to activate or stimulate the function of the polypeptide. For example, a functional assay may involve contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The effectiveness of an agonist is usually determined by its EC ₅₀ value (the concentration required to activate 50% of the response involving the agonist). The lower the EC ₅₀ value, the higher the potency of the agonist and the lower the concentration required to activate a maximal biological response. The 4-1BB agonist may also include a molecule containing a 4-1BB ligand or a fragment of a 4-1BB ligand, for example a bispecific molecule containing one arm that contains 4-1BBL or a fragment thereof and another arm that binds, for example, to an antigen on tumors. These fragments may include the Fc region.

[0062] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0063] As used herein, the term “antibody” means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen (eg, MUC16 or CD3). The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (for example, IgM). 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 _CH 1 , _CH 2 and _CH 3 . 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 (C _L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments disclosed herein, FR anti-MUC16 antibodies or anti-CD3 antibodies (or antigen binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on a side-by-side comparison analysis of two or more CDRs.

[0064] As used herein, the term “antibody” also includes antigen-binding portions of complete antibody molecules. As used herein, the terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like. 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 genetic engineering technologies involving manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration or to introduce codons, insert cysteine residues, modify, add or remove amino acids, and etc.

[0065] 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 dedicated complementarity determining region (CDR) such as the CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide conformational freedom. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.) , small modular protein (SMIP) immunopharmaceuticals and shark IgNAR variable domains are also covered by the expression “antigen binding fragment” as used herein.

[0066] An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in reading frame to one or more framework sequences. In antigen binding fragments containing a V _H domain associated with a V _L domain, the V _H and V _L domains may be arranged in any suitable order relative to each other. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric V _H or V _L domain.

[0067] In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen binding fragment of an antibody as used herein include (i) V _H -C _H 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H -C _H 1-C _H 2; (v) V _H -C _H 1-C _H 2-C _H 3; (vi) V _H -C _H 2-C _H 3; (vii) V _H -C _L ; (viii) V _L -C _H 1; (ix) V _L -C _H 2; (x) V _L -C _H 3; (xi) V _L -C _H 1-C _H 2; (xii) V _L -C _H 1-C _H 2-C _H 3; (xiii) V _L -C _H 2-C _H 3 and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly connected to each other or can be connected through all or part of a hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen binding fragment of an antibody as used herein may comprise a homodimer or heterodimer (or other multimer) in any of the variable and constant domain configurations set forth above, in non-covalent association with each other and/or with one or more monomeric domains V _H or V _L (eg via disulfide bond(s)).

[0068] As with full antibody molecules, antigen binding fragments can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use within the antigen binding fragment of an antibody as used herein using conventional techniques available in the art.

[0069] Antibodies useful herein may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity” (CDC) refers to the lysis of antigen-expressing cells by an antibody disclosed herein in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils and macrophages) recognize bound antibody on the cell -target and due to this, lysis of the target cell is carried out. CDC and ADCC can be measured using assays that are well known and available in the art (see, for example, US Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 9.5:652 656). The constant region of an antibody is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether the antibody is required to mediate cytotoxicity.

[0070] In certain embodiments, the anti-MUC16 antibodies or anti-MUC16/CD3 bispecific antibodies used herein are human antibodies. The term “human antibody” as used herein is intended to include antibodies containing variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR and, in particular, CDR3. However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0071] In some embodiments, antibodies useful in accordance with the methods disclosed herein 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 methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (further described below ), antibodies isolated from a recombinant combinatorial library of human antibodies (further described 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, generated or released by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, to somatic mutagenesis in vivo) and thus the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences which, although derived from and related to human germline V _H and V _L sequences, may not naturally exist in the human germline antibody repertoire in vivo.

[0072] Human antibodies can exist in two forms, which are associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain disulfide bond at the heavy chains. In the second form, the dimers are not linked by interchain disulfide bonds, and a molecule of approximately 75-80 kDa in size is formed, consisting of covalently linked light and heavy chains (half-antibody). These forms were extremely difficult to separate even after affinity purification.

[0073] The frequency of occurrence of the second form in different isotypes of intact IgG is due, without limitation, to structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the human IgG4 hinge region can significantly reduce the incidence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. The present invention covers antibodies containing one or more mutations in the hinge, C _H 2 or C _H 3 region, which may be necessary, for example, during production, to provide improved yield of the desired form of the antibody.

[0074] Antibodies used herein may be isolated antibodies. As used herein, the term “isolated antibody” means an antibody that has been identified and separated from and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component from a body, or from a tissue or cell in which the antibody exists or is naturally produced is an “isolated antibody” for purposes of the present invention. The isolated antibody also includes the antibody in situ within the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In accordance with certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

[0075] Anti-MUC16 antibodies and anti-MUC 16/CD3 antibodies used in the methods disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework region and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to produce the corresponding residue(s) ) from the germline sequence from which the antibody is derived, or deriving the corresponding residue(s) from another human germline sequence, or deriving a conservative amino acid substitution from the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). One of ordinary skill in the art, using the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all residues from the framework region and/or CDRs in the V _H and/or V _L domains are reverse mutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are reverse mutated to the original germline sequence, e.g., only residues within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4 are mutated, or only residues within CDR1 are mutated. CDR2 or CDR3. In other embodiments, one or more residues from the framework region and/or CDR are mutated to produce the corresponding residue(s) from a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally obtained). In addition, antibodies useful herein may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, where certain individual residues are mutated to a corresponding residue from a specific germline sequence, where whereby certain other residues that differ from the original germline sequence are retained or mutated to produce a corresponding residue from another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate) , reduced immunogenicity, etc. The present invention covers antibodies and antigen binding fragments obtained using this general method.

[0076] Anti-MUC16 antibodies and anti-MUC16/CD3 antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein containing one or more conservative substitutions are useful in the methods provided herein. For example, the present invention includes anti-MUC16 antibodies and anti-MUC16/CD3 antibodies containing HCVR, LCVR and/or CDR amino acid sequences, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions compared to any of the heavy chain or light chain amino acid sequences set forth in Table 1 herein.

[0077] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. One antigen may contain more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is formed by amino acids from different segments of a linear polypeptide chain, located nearby in space. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may contain saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

[0078] The term "substantially identical" or "substantially identical" with respect to a nucleic acid or fragment thereof indicates that, when optimally aligned, taking into account corresponding nucleotide insertions or deletions with another nucleic acid sequence (or complementary strand thereof), the of the nucleotide sequence is at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% nucleotide bases, as measured by a well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0079] When applied to polypeptides, the term “essentially similar” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs, using default gap opening penalty values, are characterized by at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not essentially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for effecting such correction are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic side chains with hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acidic side chains: aspartate and glutamate and (7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood function matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood function matrix.

[0080] For polypeptides, sequence similarity, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity scores assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, the GCG software package includes programs such as Gap and Bestfit that can be used with default parameters to determine the degree of sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein . See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and calculations of percent sequence identity in regions of greatest overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the BLAST computer program, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-110 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

Sequence options

[0081] Antibodies and bispecific antibodies used herein contain one or more amino acid substitutions, insertions and/or deletions in the framework region and/or CDR regions of the heavy chain variable domains compared to the corresponding germline sequences from which the antibodies were derived .

[0082] Also applicable herein are antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to produce the corresponding(- their) residue(s) from the germline sequence from which the antibody is derived, or by deriving the corresponding residue(s) from another human germline sequence, or by deriving a conservative amino acid substitution from the corresponding residue(s) germline mutations (such sequence changes are referred to collectively herein as “germline mutations”).

[0083] In addition, antibodies useful herein may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, where certain individual residues are mutated to produce a corresponding residue from a particular germline sequence lineage, wherein certain other residues that differ from the original germline sequence are retained or mutated to produce a corresponding residue from another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. . Antibodies and antigen-binding fragments obtained using this general method in accordance with the instructions of the present invention are covered by the present invention.

[0084] Antibodies and bispecific antibodies comprising variants of any of the heavy chain or light chain amino acid sequences provided herein containing one or more conservative substitutions are useful in accordance with the present invention. The antibodies and bispecific antigen binding molecules used herein contain one or more amino acid substitutions, insertions and/or deletions in the framework region and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual sequences were derived. antigen-binding domains, while maintaining or improving the necessary antigen-binding characteristics. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not essentially change the functional properties of the protein. Examples of groups of amino acids that contain side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic side chains with hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acidic side chains: aspartate and glutamate and (7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood function matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood function matrix.

[0085] The present invention also includes antigen binding molecules comprising an antigen binding domain with an HCVR, LCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein while maintaining or improving the required affinity to the antigen. The term "substantially identical" or "substantially identical" with respect to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as using the GAP or BESTFIT programs using default gap penalty values, have less at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for effecting such correction are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference.

[0086] For polypeptides, sequence similarity, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity scores assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, the GCG software package includes programs such as Gap and Bestfit that can be used with default parameters to determine the degree of sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein . See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and calculations of percent sequence identity in regions of greatest overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the BLAST computer program, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-110 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-102.

[0087] Once produced, antigen binding domains that contain one or more germline mutations are tested for reduced binding affinity using one or more in vitro assays. Although antibodies that recognize a particular antigen are typically screened for high (ie, strong) affinity binding to the antigen, antibodies as used herein exhibit weak or no detectable binding. Bispecific antigen binding molecules containing one or more antigen binding domains prepared in this general manner are also encompassed by the present invention and have been found to be useful as avidity-based tumor therapies.

[0088] Unexpected benefits, such as improved pharmacokinetic properties and low patient toxicity, can be realized using the methods described herein.

Antibody binding properties

[0089] As used herein, the term “binding” in the context of binding of an antibody, immunoglobulin, antibody binding fragment, or Fc-containing protein to any, for example, predetermined antigen, such as a cell surface protein or fragment thereof, generally refers to interaction or an association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0090] For example, binding affinity typically corresponds to a K _D value of about 10 ^-7 M or less, such as about 10 ^-8 M or less, such as about 10 ^-9 M or less, with determination, for example, using surface plasmon resonance (SPR) technology in the BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody binding fragment or Fc-containing protein as an analyte (or antiligand). Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays have also been traditionally used, and FACS data correlates well with other methods such as competitive binding of radiolabeled ligands and SPR (Benedict, CA , J Immunol Methods 1997, 201(2): 223-31; Geuijen, CA, et al. J Immunol Methods 2005, 302(1-2):68-77.

[0091] Accordingly, the antibody or antigen binding protein disclosed herein binds to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to a K _D value that is at least ten times lower than its binding affinity to nonspecific antigen (for example, BSA, casein). In accordance with the present invention, an antibody affinity corresponding to a K _D value that is equal to or less than ten times lower than that for a nonspecific antigen may be considered undetectable binding, but such an antibody may be coupled with a second antigen binding arm to produce the bispecific antibody disclosed in this document.

[0092] The term “K _D ” (M) refers to the equilibrium dissociation constant of a particular antibody-antigen interaction or the equilibrium dissociation constant of binding of an antibody or antibody binding fragment to an antigen. There is an inverse relationship between K _D and binding affinity, therefore, the lower the K _D value, the higher it is, i.e. stronger, affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to interact and therefore a lower K _D value, and conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to interact and, therefore, a higher value of K _D . In some cases, the higher binding affinity (or _KD ) of a particular molecule (e.g., antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of a molecule (e.g., antibody) to another interaction partner molecule (e.g. by antigen Y) can be expressed as a binding ratio determined by dividing the larger _KD (lower or weaker affinity) by the smaller _KD (higher or stronger affinity), e.g. expressed as 5-fold binding affinity or 10 times more, depending on the circumstances.

[0093] The term “k _d ” (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction or the dissociation rate constant of the binding of an antibody or antibody binding fragment. The specified value is also referred to as the k _off value.

[0094] The term " _ka " (M-1 x sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction or the association rate constant of the binding of an antibody or antibody binding fragment.

[0095] The term “K _A ” (M-1 or 1/M) refers to the equilibrium association constant of a particular antibody-antigen interaction or the equilibrium association rate constant of binding of an antibody or antibody binding fragment. The equilibrium association constant is obtained by dividing k _a by k _d .

[0096] The term "EC50" or "EC50" refers to the half-maximal effective concentration, which includes the concentration of antibody that produces half the response between baseline and maximum after a specified exposure time. EC ₅₀ actually represents the concentration of an antibody at which 50% of its maximum effect is observed. In some embodiments, the EC ₅₀ value is equal to the concentration of an antibody disclosed herein that produces half-maximal binding to cells expressing CD3 or tumor-associated antigen, as determined, for example, by a FACS binding assay. Thus, reduced or weaker binding is observed at increased EC ₅₀ or half-maximal effective concentration.

[0097] In one embodiment, reduced binding can be defined as an increase in the concentration of EC ₅₀ antibody that provides binding to half the maximum number of target cells.

[0098] In another embodiment, the EC ₅₀ value represents the concentration of antibody that causes half-maximal depletion of target cells due to cytotoxic T cell activity. Thus, increased cytotoxic activity (eg, T cell-mediated killing of tumor cells) is observed at a reduced EC ₅₀ or half-maximal effective concentration.

Bispecific antigen-binding molecules

[0099] Antibodies used herein may be monospecific, bispecific, or polyspecific. Polyspecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60 69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-MUC16/CD3 bispecific antibodies used herein may be coupled to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., through chemical bonding, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[00100] When used herein, the expression “anti-CD3 antibody” or “anti-MUC16 antibody” is intended to include both monospecific anti-CD3 antibodies or anti-MUC16 antibodies and bispecific antibodies comprising a CD3 binding arm and a MUC16 binding arm. Thus, the present invention includes monospecific antibodies that bind MUC16, such as the anti-MUC16 antibodies described in U.S. 2018/0112001. Exemplary antibodies to MUC16 include antibody H1H8767P and antibodies containing CDRs within antibody H1H8767P disclosed in the U.S. 2018/0112001. In addition, the present invention includes bispecific antibodies in which one immunoglobulin arm binds human CD3 and the other immunoglobulin arm is specific for human MUC16. Exemplary bispecific antibody sequences useful in accordance with the methods presented herein are shown in Table 1.

[00101] In certain embodiments, the CD3 binding arm binds to human CD3 and induces activation of human T cells. In certain embodiments, the CD3 binding arm weakly binds to human CD3 and induces activation of human T cells. In other embodiments, the CD3 binding arm weakly binds to human CD3 and induces the killing of cells expressing tumor-associated antigen within a bispecific or multispecific antibody. In other embodiments, the CD3 binding arm binds or weakly associates with human and cynomolgus CD3, and the binding interaction is not detected by in vitro assays known in the art.

[00102] In certain illustrative embodiments, the present invention includes bispecific antigen binding molecules that specifically bind CD3 and MUC16. Such molecules may be referred to herein as, for example, “CD3/MUC16 bispecific molecules” or “CD3xMUC16 bispecific molecules” or “CD3xMUC16” or other similar terms (eg, anti-MUC16 antibody/anti-CD3 antibody).

[00103] As used herein, the term “MUC16” refers to the human MUC16 protein unless indicated to be from a non-human species (eg, “mouse MUC16,” “monkey MUC16,” etc.).

[00104] The above-mentioned bispecific antigen binding molecules that specifically bind CD3 and MUC16 may comprise an anti-CD3 antigen binding molecule that binds to CD3 with weak binding affinity, for example characterized by a K _D of greater than about 40 nM as measured by a binding affinity assay in vitro.

[00105] As used herein, the expression "antigen binding molecule" means a protein, polypeptide, or molecular complex containing or consisting of at least one complementarity determining region (CDR), which alone or in combination with one or more additional CDRs and/or frameworks regions (FR), specifically binds to a specific antigen. In certain embodiments, the antigen binding molecule is an antibody or antibody fragment as those terms are defined elsewhere herein.

[00106] As used herein, the term “bispecific antigen binding molecule” means a protein, polypeptide, or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a bispecific antigen binding molecule contains at least one CDR that, alone or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the present invention, a first antigen binding domain specifically binds a first antigen (eg CD3) and a second antigen binding domain specifically binds a second, different antigen (eg MUC16).

[00107] In certain illustrative embodiments, the bispecific antigen binding molecule is a bispecific antibody. Each antigen binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). Within a bispecific antigen binding molecule comprising a first and a second antigen binding domain (eg, a bispecific antibody), the CDRs of the first antigen binding domain may be designated by the prefix "A1" and the CDRs of the second antigen binding domain may be designated by the prefix "A2". Thus, the CDRs of the first antigen binding domain may be referred to herein as A1-HCDR1, A1-HCDR2 and A1-HCDR3; a The CDRs of the second antigen binding domain may be referred to herein as A2-HCDR1, A2-HCDR2 and A2-HCDR3.

[00108] The first antigen binding domain and the second antigen binding domain can be directly or indirectly linked to each other to form a bispecific antigen binding molecule as used herein. Alternatively, each of the first antigen binding domain and the second antigen binding domain may be coupled to a separate multimerization domain. The association of one multimerization domain with another multimerization domain facilitates the association between the two antigen-binding domains, resulting in the formation of a bispecific antigen-binding molecule. As used herein, “multimerization domain” means any macromolecule, protein, polypeptide, peptide, or amino acid that is characterized by the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain may be a polypeptide containing an immunoglobulin C _H 3 domain. A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (containing the C _H 2 -C _H 3 domain), for example, the Fc domain of an IgG selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group.

[00109] Bispecific antigen binding molecules useful herein typically contain two multimerization domains, eg, two Fc domains, each of which is individually part of a distinct antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as, for example, IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as, for example, IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

[00110] In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence ranging from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides containing or consisting of a leucine zipper, helix-loop motif, or supercoil motif.

[00111] Any bispecific antibody format or technology can be used to produce the bispecific antigen binding molecules useful herein. For example, an antibody or fragment thereof having a first antigen-binding specificity may be operably linked (e.g., by chemical bonding, 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, resulting in 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, scFv- or diabody-based bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, quadromy, knobs-in-dugs, common light chain (e.g. common light chain with knob-in-groove structure, etc.), CrossMab, CrossFab, (SEED)-antibody, leucine zipper, duobody, IgG1/IgG2, Fab(DAF) Dual-acting IgG and bispecific Mab2 formats (for a review of the above formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[00112] In the context of bispecific antigen binding molecules as used herein, multimerization domains, e.g., Fc domains, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the naturally occurring wild version of the Fc domain type. For example, the present invention includes bispecific antigen binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain characterized by a modified binding interaction (eg, enhanced or weakened) between the Fc and FcRn. In one embodiment, the bispecific antigen binding molecule contains a modification at the C _H 2 or C _H 3 region, where the modification increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428 and/or 433 (for example, L/R/S/P/Q or K) and/or 434 (for example, H/F or Y); or modification at position 250 and/or 428, or modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 2591 (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L) and modification 307 and/or 308 (for example, 308F or 308P).

[00113] Also included in the present invention are bispecific antigen binding molecules comprising a C _H 3 domain of a first Ig and a C _H 3 domain of a second Ig, wherein the C _H 3 domains of the first and second Ig differ from each other by at least one amino acid, and where the difference is at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody in which there is no amino acid difference. In one embodiment, the C _H 3 domain of the first Ig binds Protein A, and the C _H 3 domain of the second Ig contains a mutation that reduces or eliminates Protein A binding, such as modification H95R (according to IMGT exon numbering; H435R according to EU numbering). The second C _H 3 may additionally contain modification Y96F (according to IMGT; Y436F according to EU). See, for example, US Patent No. 8586713. Additional modifications that may occur within the second C _H 3 include: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies, as well as Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) in case of IgG4 antibodies.

[00114] In certain embodiments, the Fc domain may be a chimeric domain combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a C _H 2 sequence derived from the C _H 2 region of human IgG1, human IgG2, or human IgG4, and part or all of a C _H 3 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 a “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 set forth herein comprises, from N- to C-terminus: [C _H 1 IgG4] - [upper hinge IgG4] - [lower hinge IgG2] - [CH2 IgG4] - [CH3 IgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [C _H 1 IgG1] - [upper hinge IgG1] - [lower hinge IgG2] - [CH2 IgG4] - [CH3 IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules used herein are described in US Patent Application Publication 2014/0243504, published August 28, 2014, which is incorporated herein in its entirety. completeness. Chimeric Fc domains containing these general structural patterns and variants thereof may exhibit altered binding to the Fc receptor, which in turn affects Fc effector function.

pH-dependent binding

[00115] The present invention includes anti-MUC16 antibodies and anti-CD3/MUC16 bispecific antigen-binding molecules with pH-dependent binding characteristics. For example, the MUC16 arm of a bispecific antigen binding molecule as used herein may exhibit reduced binding to MUC16 at acidic pH compared to binding at neutral pH. Alternatively, CD3/MUC16 bispecific antigen-binding molecules useful herein may exhibit enhanced binding to MUC16 at acidic pH compared to binding at neutral pH. The expression "acidic pH" includes pH values less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65 , 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5 ,0 or less. As used herein, the term “neutral pH” means a pH ranging from about 7.0 to about 7.4. The expression "neutral pH" includes pH values of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[00116] In some cases, "reduced binding ... at acidic pH compared to binding at neutral pH" is expressed as the ratio of the _KD value of binding of an antibody to its antigen at acidic pH to the _KD value of binding of an antibody to its antigen at neutral pH (or vice versa). For example, an antibody or antigen binding fragment thereof may be considered to have "reduced binding to MUC16 at acidic pH compared to neutral pH" for purposes of the present invention if the antibody or antigen binding fragment thereof has an acidic/neutral pH K _D ratio of approximately 3. 0 or more. In certain illustrative embodiments, the K _D ratio under acidic/neutral conditions for an antibody or antigen binding fragment may be approximately 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.

[00117] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a panel of antibodies for reduced (or enhanced) 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, within the CDR) with a histidine residue, an antibody with reduced antigen-binding ability at acidic pH compared to neutral pH can be generated.

Antibodies containing Fc variants

[00118] In certain embodiments useful herein, anti-MUC16 antibodies and anti-CD3/MUC16 bispecific antigen-binding molecules are provided comprising an Fc domain containing one or more mutations that enhance or weaken binding of the antibody to the FcRn receptor, e.g. acidic pH compared to neutral pH. For example, the present invention includes antibodies containing a mutation in the C _H 2 - or C _H 3 region of the Fc domain, where the mutation(s) provide(s) an increase in the affinity of the Fc domain for FcRn in an acidic environment (for example, in endosome, where the pH ranges from about 5.5 to about 6.0). The result of such mutations may be an increase in the half-life of the antibody in the blood serum when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K) and/or 434 (for example, H/F or Y); or modification at position 250 and/or 428, or modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 2591 (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L) and modification 307 and/or 308 (for example, 308F or 308P).

[00119] For example, the present invention includes anti-MUC16 antibodies and anti-CD3/MUC16 bispecific antigen-binding molecules comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L ); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); and 433K and 434F (for example, H433K and N434F). It is within the scope of the present invention to include all possible combinations of the aforementioned mutations in the Fc domain and other mutations within the variable domains of the antibodies disclosed herein.

Biological characteristics of bispecific antigen-binding molecules

[00120] Useful in the present invention are monospecific and bispecific antibodies and antigen binding fragments thereof that bind CD3-expressing human T cells and/or MUC16-expressing human T cells with high affinity (eg, K _D values at the subnanomolar level). Such antibodies and their properties are disclosed in US Publication No. 2018/0112001, which is incorporated herein by reference. Such bispecific antibodies are particularly useful in combination with a 4-1BB agonist in the treatment of tumors.

[00121] Applicable herein are anti-MUC16 antibodies and anti-CD3/MUC16 bispecific antigen-binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) suppression of tumor growth in immunocompromised mice bearing human ovarian cancer xenografts ; and (b); suppression of tumor growth of established tumors in immunocompromised mice bearing human ovarian cancer xenografts (see, for example, US publication No. 2018/0112001, example 8).

[00122] Useful herein are antibodies and antigen-binding fragments thereof that bind human CD3 with moderate to low affinity, depending on the therapeutic context and the specific targeting properties that are desired. For example, in the context of a bispecific antigen-binding molecule, where one arm binds CD3 and the other arm binds a target antigen (eg MUC16), it may be necessary for the target antigen-binding arm to bind the target antigen with high affinity, while the CD3 arm bound CD3 with moderate to low affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while preventing overall/off-target CD3 binding and subsequent unwanted side effects associated therewith.

[00123] Bispecific antigen binding molecules (eg, bispecific antibodies) as used herein are capable of simultaneously binding to human CD3 and human MUC16. The binding arm that interacts with cells expressing CD3 may exhibit weak or undetectable binding as measured in a suitable in vitro binding assay. The extent to which a bispecific antigen binding molecule binds cells that express CD3 and/or MUC 16 can be assessed by fluorescence-activated cell sorting (FACS), as illustrated in U.S. 2018/0112001, example 5.

[00124] For example, bispecific antibodies that specifically bind human T cell lines that express CD3 but do not express MUC16, such as Jurkat and/or primate T cells (for example, cynomolgus monkey peripheral blood mononuclear cells [PBMCs]) are useful herein. ). Also useful herein are bispecific antibodies that bind to MUC16-expressing cells and cell lines with an EC ₅₀ value of 7 nM (7 x 10 ^-9 ) or less, as determined using the FACS binding assay set forth in US Publication No. 2018 /0112001, example 5, or essentially similar analysis.

[00125] In some aspects, bispecific antibodies bind human CD3 with weak (ie, low) affinity or even no detectable affinity. In accordance with certain embodiments, the present invention includes antibodies and antigen-binding antibody fragments that bind human CD3 (eg, at 37°C) with a K _D of greater than about 11 nM, as measured by surface plasmon resonance.

[00126] In some aspects, bispecific antibodies bind monkey (eg, cynomolgus) CD3 with weak (ie, low) affinity or even no detectable affinity.

[00127] In some aspects, bispecific antibodies bind human CD3 and induce T cell activation. For example, certain anti-CD3 antibodies induce activation of human T cells at an EC ₅₀ value of less than about 113 pM, as measured by an in vitro T cell activation assay.

[00128] Bispecific antibodies useful herein can bind human CD3 and induce T cell-mediated killing of tumor cells expressing the antigen. For example, the present invention includes bispecific antibodies that induce T cell-mediated tumor cell killing at an EC ₅₀ of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay.

[00129] Bispecific antibodies useful herein can bind CD3 with a dissociative half-life (t½) of less than about 10 minutes, as measured by surface plasmon resonance at 25°C or 37°C.

[00130] The CD3/MUC16 bispecific antigen binding molecules used herein may further exhibit one or more characteristics selected from the group consisting of: (a) inducing PBMC proliferation in vitro; (b) activation of T cells through induction of IFN-gamma release and up-regulation of CD25 in human whole blood, and (c) induction of T cell-mediated cytotoxicity against tumor cells expressing MUC16.

[00131] The present invention includes bispecific CD3/MUC16 antigen binding molecules that are capable of depleting tumor antigen expressing cells in a subject (see, for example, US Publication No. 2018/0112001, Example 8). For example, in certain embodiments, CD3/MUC16 bispecific antigen binding molecules are provided, wherein a single administration of the bispecific antigen binding molecule to a subject (eg, at a dose of about 5.0 mg/kg, about 0.1 mg/kg, about 0.08 mg/kg) kg, about 0.06 mg/kg, about 0.04 mg/kg, about 0.02 mg/kg, about 0.01 mg/kg or less) causes a decrease in the number of MUC16-expressing cells in a subject (eg, tumor growth in subject is suppressed or suppressed) below detectable levels. Unless otherwise stated, bioluminescent emission intensity refers to [f/s/ ^cm2 /sr].

[00132] In certain embodiments, a single administration of a CD3/MUC16 bispecific antigen binding molecule at a dose of about 0.4 mg/kg causes tumor growth in a subject to decrease below detectable levels on about day 7, about day 6, about day 5, about day 4, about day 3, about day 2, or about day 1 after administration of the bispecific antigen binding molecule to the subject. In certain embodiments, a single administration of a bispecific antigen-binding molecule to CD3/MUC16 disclosed herein at a dose of at least about 0.01 mg/kg, such as at least about 85 μg/kg or at least about 100 μg /kg, results in the number of MUC16-expressing tumor cells remaining below detectable levels until at least about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days after administration or more. As used herein, “below detectable levels” means that tumor cells cannot be directly or indirectly detected as growing subcutaneously in a subject using standard caliper measurement techniques, such as those set forth in US Publication No. 2018/0112001, Example 8.

[00133] Also useful in the methods provided herein are CD3/MUC16 bispecific antigen-binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) suppression of tumor growth in immunocompromised mice bearing cancer xenografts human ovary; (b) suppressing tumor growth in immunocompetent mice bearing human ovarian cancer xenografts; (c) suppressing tumor growth of established tumors in immunocompromised mice bearing human ovarian cancer xenografts; and (d) reducing tumor growth of established tumors in immunocompetent mice bearing human ovarian cancer xenografts (see, for example, US Publication No. 2018/0112001, Example 8). Exemplary CD3/MUC16 bispecific antigen-binding molecules may further exhibit one or more characteristics selected from the group consisting of: (a) inducing a transient dose-dependent increase in circulating cytokine levels, (b) inducing a transient increase in circulating T cell levels, and (c) no depletion of effector T cells (eg CD4+ T cells, CD8+ T cells and regulatory T cells, i.e. Tregs).

[00134] Also useful in the methods provided herein are anti-MUC16 antibody-drug conjugates, including kMUC16xCD3 bispecific antigen-binding molecule-drug conjugates, which suppress tumor growth in MUC16-positive in vivo ovarian cancer xenograft models (see, for example, , U.S. 2018/0112001, example 10, in a bioluminescent imaging assay or a substantially similar assay).

Epitope mapping and related technologies

[00135] The epitope on CD3 and/or MUC16 to which antigen binding molecules used herein bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD3 or MUC16 protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD3 or MUC16. Antibodies useful in accordance with the methods disclosed herein may react with amino acids contained within one CD3 chain (e.g., CD3 epsilon, CD3 delta, or CD3 gamma) or may react with amino acids in two or more different CD3 chains. As used herein, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. One antigen may contain more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is formed by amino acids from different segments of a linear polypeptide chain, located nearby in space. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In certain circumstances, the epitope may contain saccharide moieties, phosphoryl groups, or sulfonyl groups in the antigen.

[00136] Various techniques known to those of ordinary skill in the art can be used to determine “whether the antigen binding domain of an antibody interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, traditional cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation assays, peptide blot assays (Reineke, 2004, Methods Mol Biol 248: 443–463) and peptide digestion assay. In addition, methods such as epitope excision, epitope isolation, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which the antigen binding domain of an antibody interacts is hydrogen-deuterium exchange detected by mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium, followed by coupling of the antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the antibody-protected residues (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease digestion and analysis by mass spectrometry, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 75:256A-265A. X-ray crystallography of the antigen/antibody complex can also be used for epitope mapping.

[00137] Exemplary bispecific antigen binding molecules useful herein may comprise a first antigen binding domain that specifically binds human and/or cynomolgus CD3 with low or detectable binding affinity, and a second antigen binding domain that specifically binds human MUC16, wherein the first the antigen binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen binding domains described herein, and/or wherein the second antigen binding domain binds to the same epitope on MUC16 as any of the specific exemplary MUC16- specific antigen binding domains described herein.

[00138] Likewise, bispecific antigen binding molecules useful herein may comprise a first antigen binding domain that specifically binds human CD3 and a second antigen binding domain that specifically binds human MUC16, wherein the first antigen binding domain competes for binding to CD3 with either the specific exemplary CO3-specific antigen binding domains described herein in Table 1, and/or wherein the second antigen binding domain competes for binding to MUC16 with any of the specific exemplary MUC16-specific antigen binding domains described herein in Table 1.

[00139] Using conventional methods known in the art, it can be readily determined whether a particular antigen binding molecule (e.g., antibody) or antigen binding domain thereof binds to or competes with the same epitope as the reference antigen binding molecule of the present invention . For example, to determine whether a test antibody binds to the same epitope on MUC16 (or CD3) as a reference bispecific antigen binding molecule of the present invention, first allowing the reference bispecific molecule to bind to the MUC16 protein (or CD3 protein). The ability of the test antibody to bind to the MUC16 (or CD3) molecule is then assessed. If the test antibody is able to bind to MUC16 (or CD3) after saturating binding to the reference bispecific antigen binding molecule, it can be concluded that the test antibody binds to an epitope of MUC16 (or CD3) different from the epitope to which the reference bispecific antigen binding molecule binds. On the other hand, if the test antibody is unable to bind to a MUC16 (or CD3) molecule after saturating binding to a reference bispecific antigen binding molecule, then the test antibody may bind to the same MUC16 (or CD3) epitope as the epitope bound by the reference bispecific antigen binding molecule . Additional conventional experiments (eg, peptide mutation assays and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope to which the reference bispecific antigen binding molecule binds, or whether the lack of binding observed is due to binding is steric blocking (or other phenomenon). Experiments of this type can be performed using ELISA, PJA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In accordance with certain embodiments of the present invention, two antigen binding proteins bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen binding proteins are considered to bind to the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antigen binding protein reduce or eliminate the binding of the other. Two antigen binding proteins are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that weaken or eliminate the binding of one antigen binding protein weaken or eliminate the binding of the other.

[00140] To determine whether an antibody or antigen binding domain competes for binding to a reference antigen binding molecule, the binding technique described above is performed in two directions. The first direction allows the reference antigen binding molecule to bind to the MUC16 protein (or CD3 protein) under saturation conditions and then assess the binding of the test antibody to the MUC16 (or CD3) molecule. The second pathway allows the test antibody to bind to a MUC16 (or CD3) molecule under saturation conditions, followed by an assessment of the binding of a reference antigen binding molecule to the MUC16 (or CD3) molecule. If in both directions only the first (saturating) antigen binding molecule is capable of binding to the MUC16 (or CD3) molecule, then it is concluded that the test antibody and the reference antigen binding molecule are competing for binding to MUC16 (or CD3). One of ordinary skill in the art will appreciate that an antibody that competes for binding to a reference antigen binding molecule need not bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Preparation of antigen-binding domains and construction of bispecific molecules

[00141] Antigen-binding domains specific for particular antigens can be produced using any antibody technology known in the art. Once prepared, two different antigen binding domains specific for two different antigens (eg CD3 and MUC16) can be suitably positioned relative to each other to produce a bispecific antigen binding molecule using conventional methods. (A discussion of exemplary bispecific antibody formats that can be used to construct bispecific antigen binding molecules of the present invention is provided elsewhere in this document.) In certain embodiments, one or more individual components (eg, heavy and light chains) of the polyspecific antigen binding molecules are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more heavy and/or light chains of bispecific antigen binding molecules useful herein can be produced using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE® or any other human antibody technology), chimeric high-affinity antibodies to a specific antigen (for example CD3 or MUC16) containing a human variable region and a murine constant region are initially isolated . Antibodies are analyzed and selected for required characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the required human constant region to produce fully human heavy and/or light chains that can be incorporated into the bispecific antigen binding molecules used herein.

[00142] Genetically engineered animals can be used to produce human bispecific antigen binding molecules. For example, a genetically modified mouse may be used that lacks the ability to rearrange and express an endogenous mouse immunoglobulin light chain variable region sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse constant gene kappa chain regions at the endogenous mouse kappa chain locus. Such genetically modified mice can be used to produce fully human bispecific antigen binding molecules containing two different heavy chains that are associated with an identical light chain that contains a variable domain derived from one of two different human light chain variable region gene segments. (For a detailed discussion of such genetically engineered mice and their application to the production of bispecific antigen-binding molecules, see, for example, US Pat. No. 10143186).

Bioequivalents

[00143] The methods disclosed herein involve the use of antigen binding molecules containing amino acid sequences that differ from the sequences of the exemplary molecules disclosed herein, but which retain the ability to bind CD3 and/or MUC16. Such variant molecules may contain one or more additions, deletions or substitutions of amino acids when compared to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described bispecific antigen binding molecules.

[00144] Applicable herein are antigen binding molecules that are bioequivalents of any of the exemplary antigen binding molecules set forth in Table 1. Two antigen binding proteins or antibodies are considered bioequivalents if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives in which the rate and/or the extent of absorption does not show significant difference when administered at the same molar dose under similar experimental conditions, either in the case of a single dose or in the case of multiple doses. Some antigen binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption but not in their rate of absorption, and may still be considered bioequivalents because such differences in absorption rates are intended and reflected in the package insert, are not necessary to achieve effective concentrations of the drug in the body, for example, during long-term use, and are considered insignificant from a medical point of view in the case of the particular drug product being studied.

[00145] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in terms of their safety, purity, and potency.

[00146] In one embodiment, two antigen binding proteins are bioequivalent if the patient can be switched one or more times from the reference product to the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or a decrease in the effectiveness of compared with that of continuing therapy without such a transfer.

[00147] In one embodiment, two antigen binding proteins are bioequivalent if they both act according to a common mechanism or mechanisms of action under a condition or conditions of use, to the extent such mechanisms are known.

[00148] Bioequivalence can be demonstrated using in vivo and in vitro methods. Measurements of the degree of bioequivalence include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other biological fluids over time; (b) an in vitro test that correlated with and predicted in vivo human bioavailability data with reasonable accuracy; (c) an in vivo test in humans and other mammals in which the relevant early pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

[00149] Bioequivalent variants of the exemplary bispecific antigen binding molecules set forth herein can be constructed by, for example, making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of intramolecular disulfide bridges that are unnecessary or incorrect during renaturation. In other situations, antigen binding bioequivalent proteins may include variants of the illustrative bispecific antigen binding molecules set forth herein that contain amino acid changes that modify the glycosylation characteristics of the molecules, such as mutations that eliminate or remove glycosylation.

Species selectivity and interspecific cross-reactivity

[00150] In accordance with certain embodiments, the bispecific antigen binding molecules used herein bind to human CD3, but not to CD3 of other species. Also useful herein are antigen-binding molecules that bind to human MUC16, but not to MUC16 of other species. The methods disclosed herein also involve the use of bispecific antigen binding molecules that bind to human CD3 and to CD3 of one or more non-human species, and/or bispecific antigen binding molecules that bind to human MUC16 and to MUC16 of one or more non-human species. from a person.

[00151] In certain illustrative embodiments, the antigen binding molecules used herein bind to human CD3 and/or human MUC16 and may or may not bind, as appropriate, to one or more of mouse CD3 and/or MUC16, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, monkey, rhesus monkey or chimpanzee. For example, a specific illustrative embodiment of the present invention provides bispecific antigen binding molecules comprising a first antigen binding domain that binds human CD3 and cynomolgus CD3, and a second antigen binding domain that specifically binds human MUC16.

Radiolabeled MUC16/CD3 antigen-binding molecule immunoconjugates for immune PET imaging

[00152] Provided herein are radiolabeled antigen binding proteins that bind MUC16 and/or CD3. In some embodiments, the radiolabeled antigen binding proteins comprise an antigen binding protein covalently linked to a positron emitter. In some embodiments, the radiolabeled antigen binding proteins comprise the antigen binding protein covalently linked to one or more chelating moieties, which are chemical moieties capable of chelating a positron emitter.

[00153] Suitable radiolabeled antigen binding proteins, such as radiolabeled antibodies, include those that do not or substantially impair T cell function when exposed to the radiolabeled antigen binding protein. In some embodiments, a radiolabeled antigen binding protein that binds an antigen binding molecule to MUC16/CD3 is a weak blocker of CD3 T cell function, i.e. T cell function is little or no affected by exposure to the radiolabeled antibody. The use of a radiolabeled CD3 binding protein that has minimal effect on CD3-mediated T cell function in accordance with the methods presented herein ensures that a subject treated with such a molecule will not suffer adverse effects due to its inability to T cells eliminate infection.

[00154] In some embodiments, anti-MUC16 antibodies or KMUC16/CD3 antigen-binding molecules are provided, such as bispecific antibodies, wherein said antigen-binding proteins are covalently linked to one or more moieties characterized by the following structure:

where L is a chelating moiety, M is a positron emitter, and z is independently in each case 0 or 1, and where at least one of z is 1.

[00155] In some embodiments, the radiolabeled antigen binding protein is a compound of formula (I):

A is an anti-MUC16 antibody or an anti-MUC16/CD3 antigen-binding molecule, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1 In some embodiments, k is 2.

[00156] In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (II):

where A is an anti-MUC16 antibody or an anti-MUC16/CD3 antigen-binding molecule, L is a chelating moiety, M is a positron emitter, and k is an integer from 1 to 30.

[00157] In some embodiments, provided herein are compositions containing a conjugate having the following structure:

where A is an anti-MUC16 antibody or an anti-MUC16/CD3 antigen-binding molecule, L is a chelating moiety, and k is an integer from 1 to 30, wherein the conjugate chelates a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging.

[00158] Chelating moieties and positron emitters are shown below.

Positron emitters and chelating moieties

[00159] Suitable positron emitters include, but are not limited to, those that form stable complexes with the chelating moiety and have physical half-lives suitable for immune PET imaging purposes. Exemplary positron emitters include, but are not limited to, ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ⁴⁴ Sc, and ⁸⁶ Y. Suitable positron emitters also include those that directly bind to an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule, including but not limited to ⁷⁶ Br and ¹²⁴ I, and those introduced through a prosthetic group, for example ¹⁸ F.

[00160] The chelating moieties described herein are chemical moieties that are covalently linked to an anti-MUC16 antibody or a KMUC16/CD3 antigen-binding molecule and contain a portion capable of chelating a positron emitter, i.e. capable of reacting with a positron emitter to form a coordinated chelate complex. Suitable moieties include those that provide efficient loading of a particular metal and form metal-chelator complexes that are sufficiently stable in vivo for diagnostic applications, such as immune PET imaging. Exemplary chelating moieties include those that minimize positron emitter dissociation and accumulation in bone mineral, plasma proteins, and/or bone marrow deposition to an extent suitable for diagnostic applications.

[00161] Examples of chelating moieties include, but are not limited to, those that form stable complexes with the positron emitters ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ⁴⁴ Sc and/or ⁸⁶ Y. Exemplary chelating moieties include, but are not limited to, those described in Nature Protocols, 5( 4): 739, 2010; Bioconjugate Chem., 26(12): 2579 (2015); Chem Commun (Camb), 51(12): 2301 (2015); Mol. Pharmaceutics, 12: 2142 (2015); Mol. Imaging Biol., 18: 344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007/s00259-016-3499-x; Bioconjugate Chem., 26(12): 2579 (2015); WO 2015/140212 A1 and US 5639879, which are incorporated by reference in their entirety.

[00162] Exemplary chelating moieties also include, but are not limited to, those containing desferroxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7 ,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), (1R,4R,7R,10R)-α'α''α'''-tetramethyl-1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H ₄ octapa, H ₆ phospa, H ₂ dedpa, H ₅ decap, H ₂ azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane- N,N,N''-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[ 6.6.2]hexadecane-4,11-diketic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (cyclen), 1,4,8,11-tetraazacyclotetradecane (cyclam), octadentate chelators, octadentate bifunctional chelators agents, for example, DFO*, hexadentate chelators, phosphonate chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinin C and chelators that are derivatives of fusarinin C, triacetylfusarin C (TAFC), ferroxamine E (FOXE), ferroxamine B (FOXB), ferrichrome A (FCHA), etc.

[00163] In some embodiments, the chelating moieties are covalently linked to an anti-MUC16 antibody or anti-MUC16/CD3 bispecific antigen-binding molecule via a linker moiety that covalently attaches the chelating moiety of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed by the reaction between a reactive moiety of a bispecific antigen binding molecule, such as a cysteine or lysine of an antibody, and a reactive moiety that is attached to a chelator, including, for example, a p-isothiocyanatobenyl group and the reactive moieties presented in methods of conjugation below. In addition, such linker moieties optionally contain chemical groups used to control the polarity, solubility, steric interactions, stringency and/or length between the chelating moiety and the anti-MUC16 antibody or anti-MUC16/CD3 bispecific antigen-binding molecule.

Preparation of radiolabeled conjugates based on a bispecific antigen-binding molecule to MUC16/CD3

[00164] Radiolabeled anti-MUC16 antibody conjugates and bispecific antigen-binding molecule conjugates against MUC16/CD3 can be prepared by: (1) reacting the antigen-binding molecule with a molecule containing a positron emitter chelator and a moiety that reacts with the desired site of conjugation of the bispecific binding protein, and (2) loading of the desired positron emitter.

[00165] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which, for example, may be native or engineered, and may, for example, be present on the heavy or light chain of the antibody. Cysteine conjugation sites include, but are not limited to, antibodies generated by mutation, insertion, or reduction of disulfide bonds. Methods for producing antibodies with an engineered cysteine residue include, but are not limited to, the methods disclosed in WO 2011/056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on an antibody, achieve the desired stoichiometry, and/or achieve the desired chelator-to-antibody ratio. Such conjugation methods are known to those of ordinary skill in the art and include, but are not limited to, cysteine residue engineering and enzymatic and chemoenzymatic methods, including, without limitation, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, as well as methods that described in J. Clin. Immunol., 36: 100 (2016), incorporated herein by reference in its entirety. Suitable moieties that react with the desired conjugation site will typically provide efficient and facile binding of the MUC16/CD3 bispecific antigen binding molecule, such as a bispecific antibody, and a positron emitter chelator. The moieties that react with lysine and cysteine sites include electrophilic groups that are known to those of ordinary skill in the art. In certain aspects, if the desired conjugation site is a lysine residue, the reactive moiety is an isothiocyanate, such as a p-isothiocyanatobenyl group or a reactive ester. In certain aspects, if the desired conjugation site is a cysteine residue, the reactive moiety is a maleimide.

[00166] If the chelator is desferroxamine (DFO), suitable reactive moieties include, but are not limited to, an isothiocyanatobenzyl group, n-hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, n-succinimidyl-S-acetylthioacetate, and moieties described in BioMed Research International, Vol 2014, Article ID 203601, incorporated herein by reference in its entirety. In certain embodiments, the molecule comprising the positron emitter chelator and the conjugation site-reactive moiety is p-isothiocyanatobenzyldesferroxamine (p-SCN-Bn-DFO):

[00167] Loading of a positron emitter is accomplished by incubating an anti-MUC16 antibody or a conjugate of a chelator and an anti-MUC16/CD3 bispecific antigen-binding molecule with the positron emitter for a time sufficient to ensure the formation of a coordination bond between said positron emitter and the chelator, for example by implementing the methods described in the examples provided herein, or a substantially similar method.

Exemplary Embodiments of Conjugates

[00168] The present invention includes radiolabeled antibody conjugates comprising an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule and a positron emitter. Also included in the present invention are radiolabeled antibody conjugates comprising an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule, a chelating moiety, and a positron emitter. (Tavare et al. Cancer Res. 2016, 76(1): 73-82, and Azad et al. Oncotarget. 2016, 7(11): 12344-58.)

[00169] In some embodiments, the chelating moiety includes a chelator capable of complexing with ⁸⁹ Zr. In certain embodiments, the chelating moiety comprises desferroxamine. In certain embodiments, the chelating moiety is p-isothiocyanatobenzyldesferroxamine.

[00170] In some embodiments, the positron emitter is ⁸⁹ Zr. In some embodiments, less than 1.0% of the anti-MUC16 antibody or bispecific antigen-binding molecule to MUC16/CD3 is conjugated to the positron emitter, less than 0.9% of the anti-MUC16 antibody or bispecific antigen-binding molecule to MUC16/CD3 is conjugated to the positron emitter, less than 0.8 % of anti-MUC16 antibody or bispecific antigen-binding molecule to MUC16/CD3 conjugated to a positron emitter, less than 0.7% of anti-MUC16 antibody or bispecific antigen-binding molecule to MUC16/CD3 conjugated to a positron emitter, less than 0.6% of anti-MUC16 antibody or bispecific antigen-binding molecule molecules to MUC16/CD3 conjugated to a positron emitter, less than 0.5% of an antibody to MUC16 or a bispecific antigen binding molecule to MUC16/CD3 is conjugated to a positron emitter, less than 0.4% of an antibody to MUC16 or a bispecific antigen binding molecule to MUC16/CD3 is conjugated to a positron emitter emitter, less than 0.3% anti-MUC16 antibody or bispecific antigen-binding molecule anti-MUC16/CD3 conjugated to a positron emitter, less than 0.2% anti-MUC16 antibody or bispecific antigen binding molecule anti-MUC16/CD3 conjugated to a positron emitter or less than 0.1% antibody to MUC16 or bispecific antigen-binding molecule to MUC16/CD3 conjugated to a positron emitter.

[00171] In some embodiments, the ratio of chelating moiety to antibody in the conjugate is from 1.0 to 2.0. As used herein, the term “chelator-to-antibody ratio” is the average ratio of chelator-to-antibody and is an indication of chelator loading on the antibody. This ratio is similar to “DAR”, i.e. drug-to-antibody ratio, which is used by those skilled in the art to measure drug loading on an antibody in the case of antibody-drug conjugates (ADCs); wherein, in the context of the iPET imaging conjugates described herein, the ratio of the chelating moiety to the antibody can be determined using the methods described herein and other DAR determination methods known in the art, such as those described in Wang et al ., Antibody-Drug Conjugates, The ^21st Century Magic Bullets for Cancer (2015). In some embodiments, the ratio of chelating moiety to antibody is approximately 1.7. In some embodiments, the ratio of chelating moiety to antibody is from 1.0 to 2.0. In some embodiments, the ratio of chelating moiety to antibody is approximately 1.7.

[00172] In a specific embodiment, the chelating moiety is p-isothiocyanatobenzyldesferroxamine and the positron emitter is ⁸⁹ Zr. In another specific embodiment, the chelating moiety is p-isothiocyanatobenzyldesferroxamine, and the positron emitter is ⁸⁹ Zr, and the ratio of the chelating moiety to the antibody in the conjugate is from 1 to 2.

[00173] In some embodiments, provided herein are anti-MUC16 antibodies or anti-MUC16/CD3 bispecific antigen-binding molecules, wherein said antigen-binding molecules are covalently linked to one or more moieties characterized by the following structure:

wherein L is a chelating moiety, M is a positron emitter, and z is independently in each case 0 or 1, and wherein at least one of z is 1. In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (I ):

A is an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[00174] In some embodiments, L is:

[00175] In some embodiments, M is ⁸⁹ Zr.

[00176] In some embodiments, k is an integer between 1 and 2. In some embodiments, k is 1. In some embodiments, k is 2.

[00177] In some embodiments, -L-M is:

where Zr represents the positron emitter ⁸⁹ Zr.

[00178] Also included in the present invention are methods for synthesizing a radiolabeled antibody conjugate comprising contacting a compound of formula (III):

with ⁸⁹ Zr, where A is an anti-MUC16 antibody or a bispecific antigen-binding molecule to MUC16/CD3. In certain embodiments, a compound of formula (III) is synthesized by contacting an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule with p-SCN-Bn-DFO.

[00179] Also provided herein is the reaction product of a compound of formula (III) with ⁸⁹ Zr.

[00180] Presented herein are compounds of formula (III):

wherein A is an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule, and k is an integer from 1 to 30. In some embodiments, k is 1 or 2.

[00181] This document covers axis antibody conjugates containing: (i) an anti-MUC16 antibody or a bispecific antigen-binding molecule to MUC16/CD3 and (ii) one or more chelates about forming fragments.

[00182] In some embodiments, the chelating moiety includes:

is a covalent bond with an antibody or its antigen binding fragment

[00183] In some aspects, the antibody conjugate has a chelating moiety to antibody ratio of from about 1.0 to about 2.0. In some aspects, the antibody conjugate has a chelating moiety to antibody ratio of approximately 1.7.

[00184] In some embodiments, provided herein are compositions containing a conjugate having the following structure:

wherein A is an anti-MUC16 antibody or an anti-MUC16/CD3 bispecific antigen-binding molecule, L is a chelating moiety, and k is an integer from 1 to 30, wherein the conjugate chelates a positron emitter in an amount sufficient to provide specific activity , suitable for clinical PET imaging. In some embodiments, the amount of the chelated positron emitter is an amount sufficient to provide a specific activity of about 1 to about 50 mCi per 1 to 50 mg of anti-MUC16 antibody or anti-MUC16/CD3 bispecific antigen-binding molecule.

[00185] In some embodiments, the amount of chelated positron emitter is an amount sufficient to provide a specific activity of up to 50 mCi, up to 45 mCi, up to 40 mCi, up to 35 mCi, up to 30 mCi, up to 25 mCi, or up to 10 mCi per 1- 50 mg of bispecific antigen binding molecule to MUC16/CD3, for example, in the range of about 5 to about 50 mCi, from about 10 to about 40 mCi, from about 15 to about 30 mCi, from about 7 to about 25 mCi, from about 20 to about 50 mCi or about 5 to about 10 mCi.

Methods of using radiolabeled immunoconjugates

[00186] In certain aspects of the present invention, diagnostic and therapeutic methods of using radiolabeled antibody conjugates of the present invention are provided.

[00187] In one aspect of the present invention, methods are provided for detecting MUC16 in tissue, the methods comprising administering to the tissue a radiolabeled anti-MUC16 antibody conjugate or an anti-MUC16/CD3 bispecific antigen-binding molecule conjugate provided herein and visually observation of MUC16 expression by positron emission tomography (PET) imaging. In certain embodiments, the tissue comprises cells or cell lines. In certain embodiments, the tissue is present in a subject, where the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject has a disease or disorder selected from the group consisting of a cancer that expresses the MUC16 antigen, such as ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer , intrahepatic cholangiocarcinoma of the mass-forming type, adenocarcinoma of the cervix and adenocarcinoma of the gastric tract. In one embodiment, the subject has ovarian cancer.

[00188] In one aspect, the present invention provides methods for imaging tissue that expresses MUC16, comprising administering to the tissue a radiolabeled anti-MUC16 antibody conjugate or a bispecific antigen-binding molecule conjugate to MUC16/CD3 of the present invention, and visually observing the expression of MUC16 through positron emission tomography (PET) imaging. In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained in a culture of tumor cells or a tumor cell line. In one embodiment, the tissue is contained within a tumor site in a subject. In one embodiment, the tissue is intratumoral lymphocytes in the tissue. In one embodiment, the tissue contains MUC16-expressing cells.

[00189] In accordance with one aspect of the present invention, methods are provided for determining whether a subject who has a tumor is suitable for anticancer therapy, the methods comprising administering a radiolabeled conjugate based on an antibody of the present invention and localizing the administered radiolabeled conjugate to antibody in the tumor via PET imaging, wherein the presence of a radiolabeled antibody conjugate in the tumor allows the subject to be identified as suitable for antitumor therapy.

[00190] According to one aspect of the present invention, methods are provided for predicting the response of a subject who has a solid tumor to anticancer therapy, the methods comprising determining whether the tumor is MUC16 positive, wherein a positive response of the subject is predicted if the tumor is MUC16 positive. In certain embodiments, a tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present invention and localizing the radiolabeled antibody conjugate to the tumor via PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that that the tumor is MUC16 positive.

[00191] In one aspect of the present invention, methods are provided for detecting a MUC16-positive tumor in a subject. The methods of this aspect include administering to a subject a radiolabeled antibody conjugate of the present invention and determining the localization of the radiolabeled antibody conjugate by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MUC16 positive.

[00192] Provided herein are methods for predicting a positive response to antitumor therapy comprising administering to a subject a radiolabeled anti-MUC16 antibody conjugate or a bispecific antigen-binding molecule conjugate to MUC16/CD3 to determine the presence of MUC16-positive cells in a solid tumor. The presence of MUC16-positive cells predicts a positive response to antitumor therapy.

[00193] As used herein, the phrase “subject in need” means a human or non-human mammal that exhibits one or more symptoms or signs of cancer, and/or has been diagnosed with cancer, including solid tumor, and who needs his treatment. 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 signs, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal bloating, chest pain/pressure, enlarged spleen, and increased levels of a cancer-related biomarker (eg, CA125). This expression includes subjects with primary or established tumors. In specific embodiments, the expression includes human subjects who have or are in need of treatment for a solid tumor, for example, ovarian cancer, breast cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass-forming type, cervical adenocarcinoma and gastric tract adenocarcinoma. The term includes subjects with primary or metastatic tumors (advanced cancers). In certain embodiments, the expression “subject in need thereof” includes subjects with a solid tumor that is resistant, refractory to, or inadequately controlled by prior therapy (eg, treatment with an anticancer agent). For example, the expression includes subjects who have been treated with one or more lines of prior therapy, such as chemotherapy (eg, carboplatin or docetaxel). In certain embodiments, the expression "subject in need thereof" includes subjects with a solid tumor who have been treated with one or more lines of prior therapy but who subsequently relapse or metastasize.

[00194] In certain embodiments, the methods of the present invention are used in a subject with a solid tumor. The terms “tumour”, “cancer” and “malignancy” are used interchangeably in this document. As used herein, the term “solid tumor” refers to an abnormal mass of tissue that typically does not contain cysts or areas of fluid. Solid tumors can be benign (not cancerous) or malignant (cancerous). For the purposes of the present invention, the term “solid tumor” means malignant solid tumors. The term includes various types of solid tumors, named for the types of cells that form them, namely sarcomas, carcinomas and lymphomas.

[00195] In certain embodiments, the cancer or tumor is selected from the group consisting of astrocytoma, anal cancer, bladder cancer, blood cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, clear cell renal cell carcinoma , colorectal cancer, colorectal cancer with moderate levels of microsatellite instability, squamous cell carcinoma of the skin, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fallopian tube cancer, mesothelioma, fibrosarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, hepatic cell carcinoma, intrahepatic mass-forming cholangiocarcinoma, leukemia, liver cancer, leiomyosarcoma, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngeal cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary and/or or recurrent cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, squamous cell cancer, gastric cancer, synovial sarcoma, testicular cancer, thyroid cancer, triple negative breast cancer, uterine cancer, including adenocarcinoma of the cervix, and Wilms tumor. In some aspects, the cancer is a primary cancer. In some aspects, the cancer is metastatic and/or recurrent cancer.

[00196] In certain embodiments, the cancer or tumor is selected from a MUC16-positive tumor, such as a tumor originating from the ovary, breast, bronchi, endometrium, cornea, gastric epithelium, pancreas, or colon. In some aspects, the cancer is breast cancer, ovarian cancer, corneal cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, uterine cancer, cervical cancer, gastric cancer, or colorectal carcinoma . In some aspects, the cancer is ovarian cancer. In some aspects, the cancer is a metastatic cancer originating from a primary ovarian tumor.

[00197] As used herein, the terms “treat,” “treat,” or the like. means to relieve symptoms, eliminate the causes of symptoms either temporarily or permanently, delay or suppress tumor growth, reduce tumor cell load or tumor mass, promote tumor regression, promote shrinkage, necrosis and/or disappearance of a tumor, prevent tumor recurrence, prevent or suppressing metastasis, inhibiting metastatic tumor growth and/or increasing the survival time of the subject.

[00198] In certain embodiments, a radiolabeled anti-MUC16 antibody conjugate or a bispecific antigen-binding molecule conjugate to MUC16/CD3 is administered to a subject intravenously, intraperitoneally, or subcutaneously. In certain embodiments, the radiolabeled antibody conjugate is administered intratumorally. After administration, the radiolabeled antibody conjugate is localized to the tumor. The localized radiolabeled antibody conjugate is visualized by PET imaging, and tumor uptake of the radiolabeled antibody conjugate is measured by methods known in the art. In certain embodiments, imaging is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of the radiolabeled conjugate. In certain embodiments, imaging is performed on the same day after administration of the radiolabeled antibody conjugate.

[00199] In certain embodiments, a radiolabeled anti-MUC16 antibody conjugate or a bispecific antigen-binding molecule conjugate to MUC16/CD3 can be administered at a dose ranging from about 0.1 mg/kg body weight to about 100 mg/kg body weight body of the subject, for example, from about 0.1 mg/kg to about 50 mg/kg, or from about 0.5 mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 1.0 mg/kg body weight.

Formulation and administration of a therapeutic drug

[00200] Pharmaceutical compositions containing antigen binding molecules as used herein are useful in accordance with the present invention. In some aspects, the pharmaceutical composition further comprises a 4-1BB agonist. Pharmaceutical compositions are formulated with suitable carriers, excipients and other means that provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in a reference book well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, USA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN™, Life Technologies, Carlsbad, California, USA), DNA-based conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions based on carbowax (polyethylene glycols with different molecular weights), semi-liquid gels and semi-liquid mixtures containing carbowax. See also Powell et al. Compendium of excipients for parenteral formulations) PDA (1998) J Pharm Sci Technol 52:238–311.

[00201] The dose of antigen binding molecule administered to a patient may vary depending on the age and body size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is generally calculated according to body weight or body surface area. If the bispecific antigen binding molecule is used for therapeutic purposes in an adult patient, it is advantageous to administer the bispecific antigen binding molecule intravenously at a normally single dose of from about 0.01 to about 20 mg/kg body weight, more preferably from about 0.02 to about 7 , from about 0.03 to about 5 or from about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency of administration and duration of treatment can be adjusted. Effective dosages and schedules of administration of a bispecific antigen-binding molecule can be determined empirically; for example, through periodic assessment, the patient's progress can be monitored and the dose adjusted accordingly. Moreover, cross-species dosage scaling can be performed using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[00202] The dose of 4-1BB agonist administered to a patient may vary depending on the age and body size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is generally calculated according to body weight or body surface area. When a 4-1BB agonist is used for therapeutic purposes in an adult patient, it is advantageous to administer the agonist intravenously, typically at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7. from about 0.03 to about 5 or from about 0.05 to about 3 or about 2.5 mg/kg body weight.

[00203] Various delivery systems are known and can be used to administer pharmaceutical compositions as used herein, for example, encapsulation in liposomes, microparticles, microcapsules, 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. 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. Administration may be systemic or local.

[00204] The pharmaceutical composition useful herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to subcutaneous delivery, when delivering the pharmaceutical composition used herein, a pen-type delivery device can be freely used. This pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge has been emptied, the empty cartridge can be immediately discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen-type delivery device can then be reused. In the case of a disposable pen-type delivery device, there is no replaceable cartridge. Instead, the disposable pen-type delivery device is provided pre-filled with a pharmaceutical composition contained in a reservoir within the device. After the reservoir is emptied of the pharmaceutical composition, the device is disposed of as a whole.

[00205] Subcutaneous delivery of a pharmaceutical composition as used herein uses a variety of reusable pen and auto-injector delivery devices. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, syringe -HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana, USA), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe -BD™ pen (Becton Dickinson, Franklin Lakes, New Jersey, USA), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices used for subcutaneous administration of a pharmaceutical composition useful herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly) , SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA, USA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park, IL, USA) , but only a few of them are mentioned.

[00206] In certain situations, the pharmaceutical composition can be delivered in the form of a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FL, CILLA. In yet another embodiment, the controlled release system can be placed near the target of the composition, requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra vol. 2, pp. 115-138 ). Other controlled release systems are discussed in Langer, 1990, Science 249:1527-1533.

[00207] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared using generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium conventionally used for injection. As the aqueous injection medium, for example, physiological saline solution, isotonic solution containing glucose and other auxiliary agents, etc. are used, which can be used in combination with a suitable solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil and the like are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol and the like. The injectable composition obtained in this manner is preferably filled into a suitable ampoule.

[00208] Advantageously, the pharmaceutical compositions described above for oral or parenteral administration are prepared in dosage forms with a unit dose adjusted to match the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injectable formulations (ampoules), suppositories, etc. The amount of the above antibody contained is generally from about 1 to about 500 mg per unit dose dosage form; in particular in the form of an injectable composition; in the case of other dosage forms, it is preferable that the content of the above antibody is from about 1 to about 300 mg and from about 10 to about 500 mg.

Means and compositions for combination therapy

[00209] The present invention provides methods that provide for the administration of a pharmaceutical composition containing any of the illustrative monospecific or bispecific antigen binding molecules described herein in combination with a 4-1BB agonist and one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined or administered in combination with a 4-1BB agonist and a bispecific antigen binding molecule as used herein include, for example, an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule EGFR inhibitor [e.g., gefitinib or erlotinib]), an antagonist of another member of the EGFR family such as Her2/ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2 antibody, an anti-ErbB3 antibody, or an anti-ErbB4 antibody or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an antagonist EGFRvIII (eg, an antibody that specifically binds EGFRvIII), cMET antagonist (eg, anti-cMET antibody), IGF1R antagonist (eg, anti-IGFIR antibody), B-raf inhibitor (eg, vemurafenib, sorafenib, GDC-0879, PLX-4720 ), PDGFR-α inhibitor (eg, anti-PDGFR-α antibody), PDGFR-β inhibitor (eg, anti-PDGFR-β antibody), VEGF antagonist (eg, VEGF decoy, see, for example, US Pat. No. 7,087,411 (also referred to herein as “VEGF inhibitory fusion protein”), anti-VEGF antibody (e.g., bevacizumab), small molecule VEGF receptor kinase inhibitor (e.g., sunitinib, sorafenib, or pazopanib)), DLL4 antagonist (e.g., anti-DLL4 antibody, disclosed in US Patent Application No. 2009/0142354, such as REGN421), Ang2 antagonist (eg, the anti-Ang2 antibody disclosed in US Patent Application No. 2011/0027286, such as H1H685P), FOLH1 antagonist (PSMA), PRLR antagonist (eg , anti-PRLR antibody), STEAP1 or STEAP2 antagonist (eg, anti-STEAP1 antibody or anti-STEAP2 antibody), TMPRSS2 antagonist (eg, anti-TMPRSS2 antibody), MSLN antagonist (eg, anti-MSLN antibody), CA9 antagonist (eg, anti-CA9 antibody ), uroplakin antagonist (for example, anti-uroplakin antibody), etc. Other agents that may preferably be administered in combination with the compositions provided herein include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL -4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors. The pharmaceutical compositions used herein (e.g., pharmaceutical compositions comprising the anti-CD3/MUC16 bispecific antigen-binding molecule disclosed herein) may also be administered as part of a therapeutic regimen comprising a 4-1BB agonist and one or more therapeutic combinations selected from " ICE": ifosfamide (eg Ifex®), carboplatin (eg Paraplatin®), etoposide (eg Etopophos®, Toposar®, VePesid®, VP-16); "DFLAP": dexamethasone (eg Decadron®), cytarabine (eg Cytosar-U®, cytosine arabinoside, aga-C), cisplatin (eg Platinol®-AQ), and "ESHAP": etoposide (eg Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (eg Medrol®), high dose cytarabine, cisplatin (eg Platinol®-AQ).

[00210] Also included in the present invention are combinations of therapeutics comprising any of the antigen binding molecules mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B -raf, PDGFR-α, PDGFR-β, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the above cytokines, where the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment ( for example, Fab fragment; F(ab') _fragment ; Fv fragment; dAb fragment; or other engineered molecules, such as diabodies, tribodies, minibodies and minimal recognition units) . The antigen-binding molecules disclosed herein can also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The antigen-binding molecules disclosed herein can also be administered as part of a treatment regimen that also includes radiation therapy and/or standard chemotherapy.

[00211] The additional therapeutically active component(s) may be administered immediately prior to, concurrently with, or shortly after administration of the antigen binding molecule as used herein (for purposes of the present invention, such administration schedules are referred to as administration of the antigen binding molecule " in combination with an additional therapeutically active component).

[00212] The present invention includes pharmaceutical compositions in which an antigen binding molecule as used herein is formulated in conjunction with one or more additional therapeutically active components described elsewhere herein.

Administration regimens

[00213] In certain embodiments of the present invention, multiple doses of an antigen binding molecule (eg, an anti-MUC16 antibody or a bispecific antigen binding molecule against CD3/MUC16) may be administered to a subject over a period of time. In addition, multiple doses of the 4-1BB agonist may be administered to the subject over a period of time. Methods according to this aspect include sequentially administering to a subject one or more doses of each therapeutic drug, i.e. one or more doses of an antigen binding molecule and one or more doses of a 4-1BB agonist. As used herein, “sequential administration” means that each dose of a therapeutic drug, such as an antigen binding molecule, is administered to a subject at a separate point in time, such as on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that comprise sequentially administering to a patient a single initial dose of an antigen binding molecule, referred to as a loading dose, followed by one or more secondary doses of the antigen binding molecule, and optionally then one or more tertiary doses of the antigen binding molecule. Included in the present invention are methods that comprise sequentially administering to a patient a single initial dose of a 4-1BB agonist, referred to as a loading dose, followed by one or more secondary doses of a 4-1BB agonist, and optionally then one or more tertiary doses of a 4-1BB agonist.

[00214] The terms "initial dose", "secondary doses" and "tertiary doses" refer to the time sequence of administration of the antigen binding molecule and/or 4-1BB agonist as used herein. Thus, the "starting dose" is the dose that is administered at the beginning of the treatment regimen (also called the "starting dose"); “secondary doses” are doses that are administered after the initial dose; and “tertiary doses” are doses that are administered after secondary doses. Each of the initial, secondary and tertiary doses may contain the same amount of antigen binding molecule (or 4-1BB agonist), but may generally differ from each other in the frequency of administration. However, in certain embodiments, the amount of antigen binding molecule (or 4-1BB agonist) contained in the initial, secondary, and/or tertiary doses varies from each other (eg, adjusted upward or downward as necessary) throughout the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" and then subsequent doses are administered at less frequent intervals (eg, "maintenance doses").

[00215] In one illustrative embodiment of the present invention, each secondary and/or tertiary dose is administered after 1-26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½ or more) weeks after the immediately preceding dose. As used herein, the phrase “immediately preceding dose” means a dose of an antigen binding molecule (or 4-1BB agonist) in a sequence of administrations that is administered to a patient before the administration of the next next dose in the sequence, without intervening doses.

[00216] Methods according to this aspect of the present invention may include administering to the patient any number of secondary and/or tertiary doses of a 4-1BB agonist, an anti-MUC16 antibody, or a bispecific antigen-binding molecule that specifically binds MUC16 and CD3. 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, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[00217] 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 may be administered to the patient 12 weeks after the immediately preceding 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 to 4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to the patient may vary throughout the treatment regimen. The physician may adjust the frequency of administration during the course of treatment depending on the needs of the individual patient after clinical evaluation.

Ways to use antibodies in diagnostics

[00218] The bispecific antibodies of the present invention can also be used to detect and/or measure the content of MUC16 or MUC16-expressing cells in a sample, for example for diagnostic purposes. For example, an anti-MUC16 antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of MUC16. Exemplary MUC16 diagnostic assays may involve, for example, contacting a sample obtained from a subject with a kMUC16 antibody or an anti-MUC16xCD3 bispecific antibody, wherein the antibody is labeled with a detectable label or reporter molecule. Alternatively, in diagnostic applications, an unlabeled anti-MUC16 antibody or a bispecific anti-MUC16xCD3 antibody may be used in combination with a secondary antibody that is labeled as such to enable detection. The detectable label or reporter molecule may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Another exemplary diagnostic use of the MUC16xCD3 bispecific antibodies useful herein includes ⁸⁹ Zr-labeled antibodies, such as ⁸⁹ Zr-desferoxamine-labeled, for the purpose of non-invasively identifying and tracking tumor cells in a subject (eg, via positron emission tomography imaging). PET)). (See, for example, Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82; and Azad, B.B. et al. Oncotarget. 2016 Mar 15; 7(11): 12344- 58.) Specific exemplary assays that can be used to detect or measure MUC16 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[00219] Samples that can be used in diagnostic assays for MUC16 according to the present invention include a sample of any tissue or fluid that can be obtained from a patient and that contains detectable amounts of MUC16 protein or fragments thereof in normal or pathological conditions. Typically, to initially determine a baseline or standard MUC16 level, MUC16 levels will be measured in a specific sample obtained from a healthy patient (eg, a patient not affected by a disease or condition associated with abnormal MUC16 levels or activity). This baseline MUC16 level can then be compared with MUC16 levels measured in samples obtained from individuals suspected of having a disease or condition associated with MUC16 (eg, a tumor containing MUC16-expressing cells).

EXAMPLES

[00220] The following examples are presented to provide those of ordinary skill in the art with complete disclosure and description of how to make and use the methods and compositions applicable herein, and are not intended to limit the scope of what the present inventors contemplate. as his own invention. Efforts have been made to ensure accuracy in the numbers used (e.g. quantities, temperatures, etc.), but some experimental errors and variations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1: Preparation of bispecific antibodies that bind MUC16 and CD3

[00221] The present invention provides antibodies to MUC16, useful according to the methods disclosed herein. Antibodies were prepared according to the disclosure provided in U.S. 2018/0112001. Exemplary antibodies useful herein include the H1H8767P antibody, as well as the CDR, HCVR, and LCVR sequences covered by this antibody. Thus, an exemplary anti-MUC16 antibody or antigen binding fragment thereof comprises HCVR of SEQ ID NO: 18 (SEQ ID NO: 1, herein) and LCVR of SEQ ID NO: 26 (SEQ ID NO: 3, herein) disclosed in the U.S. 2018/0112001.

[00222] The present invention also provides bispecific antigen binding molecules that bind CD3 and mucin 16 (MUC16); such bispecific antigen-binding molecules are also referred to herein as “MUC16/CD3 bispecific molecules.” The MUC16 binding portion of the MUC16/CD3 bispecific molecule is useful for targeting tumor cells that express MUC16, and the CD3 binding portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of MUC16 on the tumor cell and CD3 on the T cell facilitates targeted killing (cell lysis) of the targeted tumor cell by the activated T cell.

[00223] Bispecific antibodies containing a MUC16-specific binding domain and a CD3-specific binding domain were constructed using standard techniques, wherein the anti-MUC16 antigen binding domain and the anti-CD3 antigen binding domain each contain different, distinct HCVRs paired with a common LCVR. For bispecific antibodies, molecules were constructed using an anti-CD3 heavy chain, an anti-MUC16 heavy chain, and a common anti-MUC16 light chain. In other cases, bispecific antibodies can be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody or an antibody light chain that is known to be “promiscuous” or effectively pair with multiple arms heavy chains.

[00224] A brief description of the constituents of an exemplary anti-MUC16xCD3 bispecific antibody construct is set forth in Table 1.

Example 2. Model of xenogeneic ascites with OVCAR-3 cells expressing luciferase

[00225] The in vivo efficacy of a bispecific anti-MUC16/CD3 antibody in combination with 4-1BB costimulation was initially assessed in a xenogeneic tumor model. In the xenogeneic ascites model, cells from the OVCAR-3/Luc cell line, previously passaged in vivo (day 0), were intraperitoneally injected into NSG (Nod scid gamma) mice thirteen days after inoculation with human PBMC. Five mice in each group were treated intraperitoneally with MUC16XCD3 at 12.5 μg/mouse or a CO3-binding control antibody alone or in combination with anti-human 4-1BB antibody at 100 μg/mouse (clone LOB12.3 from BioXcell, Lebanon , New Hampshire) on days 5 and 8. Tumor burden was assessed by bioluminescence imaging (BLI; a measurement of luminescence) on days 4, 8, 12, 15, 20, and 25 after tumor implantation.

[00226] Xenograft tumor growth calculation and suppression: BLI was used to measure tumor burden. IP mice were injected with 150 mg/kg (as determined by body weight values at baseline) of the luciferase substrate D-luciferin suspended in PBS. Ten minutes after dosing, YOU imaging was performed on mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with a field of view D, an object height of 1.5 cm, and an average binning level for an exposure time of 0.5 min. BLI signals were acquired using Living Image software. Regions of interest were delineated around each tumor mass and photon emission intensity values were recorded as f/s/cm ² /sr. Statistical analysis was performed using GraphPad Prism software (version 6). Statistical significance of BLI results was determined using the unpaired nonparametric Mann-Whitney t test.

[00227] The data shown are tumor burden assessed by BLI on day 4 after tumor implantation (Table 2 and Figure 1). Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. There was little difference in tumor burden between groups at day 4 (demonstrating that all groups had similar tumor loads at baseline).

[00228] Table 3 shows the tumor burden assessed by BLI at day 25 after tumor implantation (see also Figure 2). Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Groups were compared with a control CD3-binding antibody (*p=0.0159 for MUC16XCD3, **p<0.0079 for MUC16XCD3 and anti-4-1BB combination). The combination of MUC16XCD3 and anti-4-1BB was also compared with MUC16XCD3 alone (#p=0.0159). MUC16XCD3 significantly reduced tumor burden at 12.5 μg compared with tumor burden after treatment with CD3-binding control antibody, and addition of anti-4-1BB antibody increased antitumor efficacy compared with MUC16XCD3 alone.

Example 3. Treatment with MUC16xCD3+4-1BB on a model of syngeneic ascites

[00229] To evaluate efficacy in the immunocompetent model, the mouse CD3 gene was replaced with human CD3 and a portion of the mouse MUC16 gene was replaced with the human sequence. As a result, mice have T cells that express human CD3 and the mice express a chimeric MUC16 molecule containing part of human MUC16, with bispecific antibodies binding to MUC16xCD3. Mice were implanted with ID8-VEGF/huMUC16 cells (a mouse ovarian tumor cell line engineered to express a portion of human MUC16) intraperitoneally and antibody treatment was administered intravenously, starting on day 3 after tumor implantation. Mice were treated with either MUC16XCD3 (1 mg/kg IV) or control CD3-binding antibody (1 mg/kg IV) with isotype control on days 3, 6, and 10 postimplantation or with mouse anti-4-1BB antibody (2.5 mg/kg IV). kg IV) on day 3 postimplantation, followed by two additional doses of MUC16XCD3 (1 mg/kg IV) on days 6 and 10. There were 8-12 mice in each group. Mice were sacrificed if they exhibited a weight gain of more than 30% due to ascites-induced abdominal distension. Statistical significance was determined using the Gehan-Breslow-Wilcoxon method.

[00230] Treatment with MUC16XCD3 significantly increased median survival in the ID8-VEGF/huMUC16 ascites model, and the addition of 4-1BB co-stimulation allowed several mice to survive. For statistical analysis, groups were compared to a control CD3-binding antibody (**p=0.0026 for MUC16XCD3, ****p<0.0001 for MUC16XCD3 with anti-4-1BB). Additionally, to determine whether the addition of anti-4-1BB produced any benefit compared to MUC16XCD3 alone, anti-4-1BB was compared with MUC16XCD3 alone (#p=0.0168). MUC16XCD3 increased the median survival of the control CD3-binding antibody-treated group, as well as the % of mice surviving (from 0 to 27%). The median survival for the MUC16XCD3 + anti-4-1BB combination group could not be determined because more than 50% of the mice survived. Overall survival in the combination-treated group was 55%) (Table 4 and Figure 3). These effects were observed without any significant weight loss during the administration period, which was used as an indicator of toxicity (Table 5 and Figure 4).

[00231] In addition to baseline antitumor efficacy, mice were also assessed for any response with memory cell formation and epitope spreading. At day 116 postimplantation, tumor-free mice were reintroduced to parental ID8-VEGF via subcutaneous implantation. Nine untreated mice were also included as controls for tumor growth. Data shown are the number of tumor-free mice after repeated administration (Table 6). In two of three mice treated with MUC16XCD3, tumors disappeared after secondary administration of the original tumor cell line that did not express MUC16. In addition, all of the 5 mice treated with the MUC16XCD3 + anti-4-1BB combination had tumor resolution. This shows that it was possible to shape a response to form memory cells using these two processing modes.

Example 4: Treatment with MUC16XCD3 + 4-1BB on Model ID8-VEGF/huMUC16

[00232] As in Example 3, mice expressing human CD3 instead of mouse CD3 and a chimeric MUC16 molecule were implanted with a mouse ovarian tumor cell line expressing a portion of human MUC16. Three dosing regimens were tested with 9-10 mice per treatment in each administration regimen.

A) Combination of either MUC16XCD3 (1 mg/kg IV) or CD3-binding control antibody (1 mg/kg IV) with either isotopic control (2.5 mg/kg IV) or mouse anti-4-1BB antibody (2 .5 mg/kg intravenously) on days 3, 7 and 10 after implantation.

B) One dose of the combination MUC16XCD3 (1 mg/kg IV) plus mouse anti-4-1BB (2.5 mg/kg IV) on day 3 post-implantation, followed by doses of MUC16XCD3 (1 mg/kg IV) on days 7 and 10 after implantation.

C) One dose of a combination of MUC16XCD3 (1 mg/kg IV) or CD3-binding control antibody (1 mg/kg IV) plus mouse anti-4-1BB (2.5 mg/kg IV) on day 3 postimplantation without additional treatments .

[00233] Data shown are median survival at day 67 after tumor implantation. See Table 7 and Figure 5. Mice were sacrificed before day 67 if they exhibited greater than 30% weight gain due to ascites-induced abdominal distension. Statistical significance was determined using the Gehan-Breslow-Wilcoxon method. For statistical analysis, groups were compared with a control CD3-binding antibody (**p=0.002 for MUC16XCD3, ****p<0.0001 for all three groups consisting of MUC16XCD3 and combination with anti-4-1BB antibody). Additionally, to determine whether the combination with anti-4-1BB resulted in any benefit compared with MUC16XCD3 alone, all groups were compared to this group (#p=0.011 for Gp4 (3 doses of anti-CD3+ bispecific antibody) 3 doses of antibody to 4-1BB), #p=0.027 for Gp5 (3 doses of bispecific antibody to CD3 + only 1 dose of antibody to 4-1BB), #p=0.011 for Gp7 (one dose of bispecific antibody to CD3 + antibody to 4 -1ВВ)). Mice treated with MUC16xCD3+anti-4-1BB showed efficacy and long-term survival. These effects were observed without any significant weight loss during the administration period, which was used as an indicator of toxicity (Table 8 and Figure 6).

conclusions

[00234] A bispecific anti-CD3 antibody targeting the tumor antigen MUC16 (MUC16) demonstrates preclinical efficacy in several mouse models. MUC16xCD3 in combination with anti-4-1BB provided robust antitumor activity resulting in long-term survival of mice, demonstrating that co-stimulation can enhance the efficacy of bispecific anti-MUC16xCD3 antibodies against advanced solid tumors. These effects were observed in the absence of weight loss in mice; weight loss was used as an indicator of toxicity. Additionally, a memory cell response occurred upon reinjection of tumor cells lacking MUC16 antigen, demonstrating a robust antitumor response that was independent of responses to MUC16 antigen.

[00235] The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art based on the foregoing description. Such modifications are intended to be within the scope of the appended claims.

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LIST OF SEQUENCES

<110> Regeneron Pharmaceuticals, Inc.

Kirshner, Jessica R.

Crawford, Alison

Chiu, Danica

<120> USE OF BISPECIFIC ANTIGEN-BINDING MOLECULES, WHICH

BIND MUC16 AND CD3, IN COMBINATION WITH 4-1BB COSTIMULATION

<130> 10604WO01

<140> TBA

<141> 2020-06-19

<150> 62/864,960

<151> 2019-06-21

<160> 6

<170> PatentIn version 3.5

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<---

Claims (30)

1. The use of a bispecific antigen-binding molecule to CD3/MUC16 and a 4-1BB agonist for the treatment of MUC16-positive cancer or suppression of the growth of a MUC16-positive tumor, wherein the bispecific antigen-binding molecule to CD3/MUC16 contains: a first antigen binding domain that specifically binds human CD3; And a second antigen binding domain that specifically binds human MUC16 and contains three heavy chain complementarity determining region amino acid sequences (HCDR1-2, HCDR2-2 and HCDR3-2) within the heavy chain variable region (HCVR-2) amino acid sequence of SEQ ID NO: 1 and three light chain complementarity determining region amino acid sequences (LCDR1, LCDR2 and LCDR3) within the light chain variable region (LCVR) amino acid sequence comprising SEQ ID NO: 3; And wherein the 4-1BB agonist is an anti-4-1BB antibody. 2. Use according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, corneal cancer, pancreatic cancer, endometrial cancer, fallopian tube cancer, mesothelioma, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, uterine cancer , cervical cancer, stomach cancer or colorectal carcinoma. 3. Use according to claim 2, wherein the cancer is ovarian cancer. 4. Use according to claim 2, wherein the cancer is breast cancer. 5. Use according to claim 1, wherein the anti-CD3/MUC16 bispecific antibody and the 4-1BB agonist are administered separately. 6. Use according to claim 1, wherein the anti-CD3/MUC16 bispecific antibody and the 4-1BB agonist are co-administered. 7. Use according to claim 1, wherein the anti-CD3/MUC16 bispecific antibody is administered before, simultaneously with, or after administration of the 4-1BB agonist. 8. Use according to claim 7, wherein the bispecific antibody to CD3/MUC16 is administered before administration of the 4-1BB agonist. 9. Use according to claim 7, wherein the bispecific antibody to CD3/MUC16 is administered on the same day as the 4-1BB agonist. 10. Application according to any one of paragraphs. 1-9, wherein the anti-CD3/MUC16 bispecific antibody is administered in combination with a 4-1BB agonist. 11. Use according to claim 1, wherein the anti-4-1BB antibody is an antibody selected from the group consisting of urelumab and utomilumab. 12. Application according to any one of paragraphs. 1-11, wherein the first antigen binding domain that specifically binds human CD3 comprises the heavy chain variable region (HCVR-1) amino acid sequence of SEQ ID NO: 2. 13. Application according to any one of paragraphs. 1-12, wherein the second antigen binding domain, which specifically binds human MUC16, contains the amino acid sequence of HCVR-2 of SEQ ID NO: 1. 14. Application according to any one of paragraphs. 1-13, wherein the first antigen binding domain that specifically binds CD3 contains the amino acid sequence of HCVR-1 of SEQ ID NO: 2 and the second antigen binding domain that specifically binds MUC16 contains the amino acid sequence of HCVR-2 of SEQ ID NO: 1. 15. Application according to any one of paragraphs. 1-14, wherein the bispecific antigen binding molecule comprises the light chain common variable region (LCVR) amino acid sequence of SEQ ID NO: 3. 16. The use of claim 1, wherein the tumor volume is reduced compared to the tumor volume in a subject administered the CD3/MUC16 bispecific antigen-binding molecule in the absence of the 4-1BB agonist. 17. The use of claim 1, wherein tumor-free survival of the subject is increased compared to tumor-free survival of the subject administered the CD3/MUC16 bispecific antigen-binding molecule in the absence of the 4-1BB agonist. 18. Use according to claim 17, wherein the increase in tumor-free survival occurs without loss of weight in the subject. 19. The use of claim 1, wherein subsequent exposure to tumor cells initiates a memory cell response in a subject treated with a CD3/MUC16 bispecific antigen-binding molecule in the presence of a 4-1BB agonist. 20. Pharmaceutical combination for the treatment of MUC16-positive cancer or suppression of the growth of MiC16-positive tumor, containing: (a) a bispecific antigen binding molecule to CD3/MUC16, comprising: (i) a first antigen binding domain that specifically binds human CD3, and (ii) a second antigen binding domain that specifically binds human MUC16 and contains three heavy chain complementarity determining region amino acid sequences ( HCDR1-2, HCDR2-2 and HCDR3-2) within the amino acid sequence of the heavy chain variable region (HCVR-2) of SEQ ID NO: 1 and three amino acid sequences of the light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) within the amino acid light chain variable region (LCVR) sequence containing SEQ ID NO: 3; (b) a 4-1BB agonist, wherein the 4-1BB agonist is an anti-4-1BB antibody, and (c) a pharmaceutically acceptable carrier or diluent. 21. The pharmaceutical combination according to claim 20, wherein the bispecific antigen binding molecule of part (a) contains the amino acid sequence of the general LCVR of SEQ ID NO: 3. 22. The pharmaceutical combination according to claim 20, wherein the first antigen binding domain that specifically binds human CD3 comprises the amino acid sequence of the heavy chain variable region (HCVR-1) of SEQ ID NO: 2. 23. The pharmaceutical combination according to claim 20, wherein the second antigen binding domain, which specifically binds human MUC16, contains the amino acid sequence of HCVR-2 under SEQ ID NO: 1. 24. The pharmaceutical combination according to claim 22 or 23, where the first antigen binding domain, which specifically binds CD3, contains the amino acid sequence of HCVR-1 under SEQ ID NO: 2 and the second antigen binding domain, which specifically binds MUC16, contains the amino acid sequence of HCVR-2 under SEQ ID NO: 1.