Methods of treating disease using Anti-CD47 x Anti-mesothelin antibodies in combination with chemotherapy

Abstract

The invention relates method of treatment of cancer using novel bispecific antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, where one of the binding sites is specific for CD47 and the second is specific for mesothelin (MSLN). In addition, the disclosure contains methods of combination treatment using novel bispecific antibodies in combination with paclitaxel.

Description

METHODS OF TREATING DISEASE USING ANTI-CD47 X ANTI-MESOTHELIN ANTIBODIES IN COMBINATION WITH CHEMOTHERAPY RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 709,317, filed on October 18, 2024, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (NOVI- 061_001WO_SEQLIST_ST26.xml; size: 23,533bytes; and Date of Creation: October 16, 2025) are herein incorporated by reference in its entirety. BACKGROUND

[0003] CD47 or Integrin-Associated-Protein (IAP) is a ubiquitous 50 kDa transmembrane glycoprotein with multiple functions in cell-cell communication. It interacts with multiple ligands, such as integrins, SIRPα (Signal Regulatory Protein alpha), SIRPγ and thrombospondins (Oldenborg, P.A., CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease, ISRN Hematol. 2013; 2013:614619; Soto-Pantoja DR, et al., Therapeutic opportunities for targeting the ubiquitous cell surface receptor CD47 (2012), Expert Opin Ther Targets. 2013 Jan;17(1):89-103; Sick E, et al., CD47 Update: a multifaced actor in the tumor microenvironment of potential therapeutic interest, Br J Pharmacol.2012 Dec;167(7):1415-30).

[0004] The widespread expression of CD47 in healthy tissues brings the question of treatment safety and efficacy: first, targeting CD47 with a neutralizing monoclonal antibody (Mab) could affect healthy cells, resulting in severe toxicities as shown in preclinical studies with mice and cynomolgus monkeys (Willingham SB, et al., Proc Natl Acad Sci U S A.2012 Apr 24;109(17):6662-7; Weiskopf K, et al., Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies, Science.2013 Jul 5;341(6141):88-91). Second, even if severe toxicities could be avoided or mitigated by using alternative formats (Weiskopf K, et al., Science.2013 Jul 5;341(6141):88-91), broad expression of CD47 could still cause a rapid elimination of CD47-binding molecules through target-mediated drug disposition resulting in poor pharmacokinetics and decreased efficacy.

[0005] Accordingly, there exists a need for antibodies and therapeutics that enable targeting of CD47 and overcome these obstacles. Furthermore, there exists a need to determine the therapeutically effective dose and schedule of particular anti-CD47 targeting antibodies and their potential use as a combination therapy with chemotherapy. Moreover, there exists a need to develop therapies that can treat cancers that are resistant to the standard of care treatment alone. SUMMARY

[0006] The present disclosure relates to the therapeutic methods, uses, and compositions comprising bispecific antibodies that recognize CD47 and mesothelin. The present disclosure relates to the bispecific antibody is Ka3 x O38 or a fragment thereof and the clinical use thereof. Ka3 x O38 refers to “NI-1801”.

[0007] The disclosure also provides bispecific antibodies that include at least a first arm that is specific for CD47. In some embodiments, the first arm is specific for at least human CD47. In some embodiments, the first arm recognizes human CD47 and is also cross-reactive for at least one other non-human CD47 protein, such as, by way of non-limiting example, non- human primate CD47, e.g., cynomolgus monkey CD47, and / or rodent CD47. In some embodiments, these anti-CD47 monoclonal antibodies inhibit the interaction between CD47 and signal-regulatory protein alpha (SIRPα). In some embodiments, these bispecific antibodies inhibit the interaction between human CD47 and human SIRPα. The invention also includes antibodies that bind to the same epitope as a bispecific antibody disclosed herein and inhibits the interaction between CD47 and SIRPα, e.g., between human CD47 and human SIRPα.

[0008] The disclosure also provides bispecific antibodies that recognize CD47 and a second target. The disclosure allows for the identification, production, and purification of bispecific antibodies that are undistinguishable in sequence from standard antibodies and where one of the binding sites is specific for CD47 and the second binding site is specific for another target, for example, a tumor-associated antigen (TAA). In some embodiments, the TAA is an antigen expressed on a cancer cell's cell surface. In some embodiments, the cancer cell is selected from a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian cell, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and / or bladder cancer cell, a renal cancer cell, an oral cavity cancer cell, a pharyngeal cancer cell, a uterine cancer cell, and / or a melanoma cancer cell.

[0009] The bispecific antibodies of the disclosure that bind at least CD47 and fragments thereof serve to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the functional activity of CD47. Functional activities of CD47 include, by way of non- limiting example, interaction with SIRPα. The antibodies are considered to completely modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the CD47- SIRPα interaction when the level of CD47-SIRPα interaction in the presence of the antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of CD47-SIRPα interaction in the absence of binding with an antibody described herein. The antibodies are considered to partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the CD47-SIRPα interaction when the level of CD47-SIRPα interaction in the presence of the antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of CD47- SIRPα interaction in the absence of binding with an antibody described herein.

[0010] In some embodiments, the bispecific antibody exhibits a “balanced” affinity for each of the two targets. In other embodiments, the bispecific antibody exhibits an “unbalanced” affinity for each of the two targets. For example, in an anti-CD47 / MSLN bispecific antibody, the affinity of the anti-MSLN arm is increased. For example, in an anti-CD47 / MSLN bispecific antibody, the affinity of the anti-CD47 arm is decreased. For example, in an anti- CD47 / MSLN bispecific antibody, the affinity of the anti-MSLN arm is increased and the affinity of the anti-CD47 arm is decreased. These unbalanced affinity bispecific antibodies are useful, for example, to improve selectivity for a target cell or group of target cells.

[0011] In some embodiments, the affinity of the anti-MSLN arm is increased by at least 100- fold following affinity maturation. In some embodiments, the affinity of the anti-CD47 arm is decreased by at least 2-fold following affinity dematuration. For example, in some embodiments, the anti-CD47 arm exhibits an affinity for CD47 that is between about 2-fold and 100-fold lower following affinity dematuration.

[0012] In some embodiments, the first arm amino acid sequence includes a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 1, a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 2, a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 3, a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 4, a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence of SEQ ID NO: 5, and avariable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence of SEQ ID NO: 6.

[0013] In some embodiments, the first arm amino acid sequence includes a variable heavy chain domain comprising the acid sequence of SEQ ID NO: 13 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 17.

[0014] In some embodiments, the first arm amino acid sequence includes a heavy chain polypeptide comprising the acid sequence of SEQ ID NO: 11 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0015] In some embodiments, the second arm amino acid sequence includes a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 13 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21.

[0016] In some embodiments, the second arm amino acid sequence includes a heavy chain polypeptide comprising the acid sequence of SEQ ID NO: 11 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 19.

[0017] In some embodiments, the bispecific antibody includes two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

[0018] In some embodiments, at least a portion of the first light chain is of the Kappa type and at least a portion of the second light chain is of the Lambda type. In some embodiments, the first light chain includes at least a Kappa constant region. In some embodiments, the first light chain further includes a Kappa variable region. In some embodiments, the first light chain further includes a Lambda variable region. In some embodiments, the second light chain includes at least a Lambda constant region. In some embodiments, the second light chain further includes a Lambda variable region. In some embodiments, the second light chain further includes a Kappa variable region. In some embodiments, the first light chain includes a Kappa constant region and a Kappa variable region, and wherein the second light chain includes a Lambda constant region and a Lambda variable region.

[0019] In some embodiments, the constant and variable framework region sequences are human.

[0020] The disclosure also relates to the method of treating or preventing a disease by the administration of a bispecific antibody in combination with a chemotherapy.

[0021] The bispecific antibodies of the invention are generated using any methods known in the art such as, by way of non-limiting example, the use of cross-linked fragments, quadromas, and / or any of a variety of recombinant formats such as, by way of non-limitingexamples, linked antibody fragments, forced heterodimers, and or recombinant formats based on single domains. Examples of Bispecific formats include but are not limited to bispecific IgG based on Fab arm exchange (Gramer et al., 2013 MAbs. 5(6)); the CrossMab format (Klein C et al., 2012 MAbs 4(6)); multiple formats based on forced heterodimerization approaches such as SEED technology (Davis JH et al., 2010 Protein Eng Des Sel.23(4):195- 202), electrostatic steering (Gunasekaran K et al., J Biol Chem.2010285(25):19637-46.) or knob-into-hole (Ridgway JB et al., Protein Eng.19969(7):617-21.) or other sets of mutations preventing homodimer formation (Von Kreudenstein TS et al., 2013 MAbs. 5(5):646-54.); fragment based bispecific formats such as tandem scFv (such asBiTEs) (Wolf E et al., 2005 Drug Discov. Today 10(18):1237-44.); bispecific tetravalent antibodies (Pörtner LM et al., 2012 Cancer Immunol Immunother. 61(10):1869-75.); dual affinity retargeting molecules (Moore PA et al., 2011 Blood.117(17):4542-51), diabodies (Kontermann RE et al., Nat Biotechnol.199715(7):629-31).

[0022] In some embodiments, the bispecific antibodies carry a different specificity in each combining site and including two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different. In some embodiments, at least a first portion of the first light chain is of the Kappa type and at least a portion of the second light chain is of the Lambda type. In some embodiments, the first light chain includes at least a Kappa constant region. In some embodiments, the first light chain further includes a Kappa variable region. In some embodiments, the first light chain further includes a Lambda variable region. In some embodiments, the second light chain includes at least a Lambda constant region. In some embodiments, the second light chain further includes a Lambda variable region. In some embodiments, the second light chain further includes a Kappa variable region. In some embodiments, the first light chain includes a Kappa constant region and a Kappa variable region, and the second light chain includes a Lambda constant region and a Lambda variable region. In some embodiments, the constant and variable framework region sequences are human.

[0023] The bispecific antibodies of the invention that recognize MSLN and CD47 are generated using any methods known in the art such as, by way of non-limiting example, the use of cross-linked fragments, quadromas, and / or any of a variety of recombinant formats such as, by way of non-limiting examples, linked antibody fragments, forced heterodimers, and or recombinant formats based on single domains. The invention allows for the identification, production and purification of bispecific antibodies that are undistinguishable in sequence from standard antibodies and where one of the binding sites is specific for MSLNand the second binding site is specific for another target, for example a tumor-associated antigen (TAA). The unmodified nature of the antibodies of the invention provides them with favorable manufacturing and biochemical characteristics similar to standard monoclonal antibodies.

[0024] In some embodiments, the bispecific antibodies carry a different specificity in each combining site and including two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

[0025] In some embodiments, at least a first portion of the first light chain is of the Kappa type and at least a portion of the second light chain is of the Lambda type. In some embodiments, the first light chain includes at least a Kappa constant region. In some embodiments, the first light chain further includes a Kappa variable region. In some embodiments, the first light chain further includes a Lambda variable region. In some embodiments, the second light chain includes at least a Lambda constant region. In some embodiments, the second light chain further includes a Lambda variable region. In some embodiments, the second light chain further includes a Kappa variable region. In some embodiments, the first light chain includes a Kappa constant region and a Kappa variable region, and the second light chain includes a Lambda constant region and a Lambda variable region. In some embodiments, the constant and variable framework region sequences are human.

[0026] The monoclonal, monovalent and / or bispecific antibodies of the invention can be used for therapeutic intervention or as a research or diagnostic reagent. For example, the monoclonal, monovalent and / or bispecific antibodies of the invention are useful in methods of treating, preventing and / or delaying the progression of pathologies associated with aberrant CD47 and / or aberrant CD47-SIRPα expression and / or activity or alleviating a symptom associated with such pathologies, by administering an antibody of the invention to a subject in which such treatment or prevention is desired. The subject to be treated is, e.g., human. The monoclonal, monovalent and / or bispecific antibody is administered in an amount sufficient to treat, prevent, delay the progression or alleviate a symptom associated with the pathology.

[0027] In some embodiments, the monoclonal, monovalent and / or bispecific antibodies of the disclosure are useful in methods of treating, preventing and / or delaying the progression of, or alleviating a symptom of cancer or other neoplastic condition by administering an antibody of the invention to a subject in which such treatment or prevention is desired. For example, the monoclonal, monovalent and / or bispecific antibodies described herein are usefulin treating hematological malignancies and / or solid tumors. For example, the monoclonal, monovalent and / or bispecific antibodies described herein are useful in treating CD47+tumors, mesothelin+tumors, and combinations thereof. By way of non-limiting example, the monoclonal, monovalent and / or bispecific antibodies described herein are useful in treating non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, including pancreatic adenocarcinoma, lung cancer, including lung adenocarcinoma, leiomyoma, leiomyosarcoma, kidney cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary gastric cancer, and prostate cancer. Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.

[0028] In certain embodiments, the monoclonal, monovalent and / or bispecific antibodies of the disclosure are useful in methods of treating, preventing and / or delaying the progression of, or alleviating a symptom of ovarian cancer.

[0029] In some embodiments, the monoclonal, monovalent and / or bispecific antibodies described herein are used in conjunction with one or more additional agents or a combination of additional agents. Suitable additional agents include current pharmaceutical and / or surgical therapies for an intended application, such as, for example, cancer, inflammation and / or autoimmune diseases. In some embodiments, the monoclonal, monovalent and / or bispecific antibodies can be used in conjunction with rituximab.

[0030] In some embodiments, the monoclonal, monovalent and / or bispecific antibodies described herein are used in conjunction with one or more additional agents or a combination of additional agents. In certain embodiments, the agent is a chemotherapeutic agent. In certain embodiments, the agent is paclitaxel.

[0031] In certain embodiments, the monoclonal, monovalent and / or bispecific antibodies of the disclosure and combination paclitaxel are useful in methods of treating, preventing and / or delaying the progression of, or alleviating a symptom of ovarian cancer. In certain embodiments, the patient has ovarian cancer that is resistant to a standard of care therapy alone.

[0032] In some embodiments, the monoclonal, monovalent and / or bispecific antibodies and the additional agent are formulated into a single therapeutic composition, and the monoclonal,monovalent and / or bispecific antibody and additional agent are administered simultaneously. Alternatively, the monoclonal, monovalent and / or bispecific antibodies and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the monoclonal, monovalent and / or bispecific antibody and the additional agent are administered simultaneously, or the monoclonal, monovalent and / or bispecific antibodies and the additional agent are administered at different times during a treatment regimen. For example, the monoclonal, monovalent and / or bispecific antibody is administered prior to the administration of the additional agent, the monoclonal, monovalent and / or bispecific antibody is administered subsequent to the administration of the additional agent, or the monoclonal, monovalent and / or bispecific antibody and the additional agent are administered in an alternating fashion. As described herein, the monoclonal, monovalent and / or bispecific antibody and additional agent are administered in single doses or in multiple doses.

[0033] Pathologies treated and / or prevented using the antibodies of the invention include, for example, cancer or any other disease or disorder associated with aberrant CD47 expression and / or activity.

[0034] Pharmaceutical compositions according to the invention can include an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

[0035] Any of the above embodiments, or any other embodiment described herein, can be combined with any other embodiment.

[0036] 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 this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0037] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materialsare described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG.1 illustrates schedule of events for Part A, Part B, and combination with paclitaxel alone and in combination with NI-1801 of the clinical trails described in the experimental examples herein. ADA, anti-drug antibodies; AE, adverse event; C, cycle; CT, computed tomography; D, day; ECG, electrocardiogram; ECHO, echocardiography; ECOG, Eastern Cooperative Oncology Group; EOT, end of treatment; LVEF, left ventricular ejection fraction; IV, intravenous; MUGA, multi-gated acquisition; PD, pharmacodynamic; PK, pharmacokinetic; PS, performance status. Note: All cycles are 28 days in duration, there are no rest periods between cycles.*For patients in the dose escalation phase (Part A) only.#If not done within the last 30 days as part of a treatment period, tumor assessment should also be performed at the EOT visit.aThe informed consent consists of three parts.bNI-1801: In Cycle 1, administer loading dose on Day1 and cohort dose on Days 8 and 22. In Cycles 2-12, administer cohort dose on Days 1 and 15. Fourteen days must elapse between C1D22 and C2D1. Patients enrolled in the pembrolizumab sub-study will be treated with NI-1801 in combination with pembrolizumab. Pembrolizumab will be administered at the dosage of 400 mg every 6 weeks, in 4 cycles. Pembrolizumab will be administered for the first time on Cycle 1, Day 8 (C1D8), and subsequently every 6 weeks thereafter. Pembrolizumab will be administered as first drug; later, NI-1801 will be infused after 30 minutes. Patients enrolled in the randomized cohort will be treated with NI-1801 in combination with paclitaxel or paclitaxel alone. Paclitaxel is administered weekly at a dose of 80 mg / m2 at least 30 minutes after completing the NI-1801 infusion, over a 1-hour period..cMust include pulse and systolic / diastolic blood pressure as well as body temperature. Blood pressure has to be measured after resting for 5 minutes in a sitting position.dSerum β-hCG (for all female of childbearing potential [FCBP]) is performed at screening; remaining pregnancy tests may be serum or urine at the Investigator’s discretion. Pregnancy testing (for all FCBP) must be done within 72 hours prior to the firstadministration of NI-1801 and prior to dosing on Day 1 of every cycle, and at the EOT visit. If the serum screening pregnancy test is performed > 72 hours before first dose, a serum or urine pregnancy test should be performed (Investigator’s discretion). The subject may not receive NI-1801 until the Investigator has verified that the result of the pregnancy test is negative.fTriplicate ECGs (i.e., 3 recordings within 2 [± 1] minute intervals) will be performed at Screening and during NI-1801 treatment.gFor patients with relevant tumor types, tumor marker (CA-125 or CYFRA 21-1 as relevant) will be checked during screening and thereafter as indicated.hCT scans or MRI to include chest, abdomen, and pelvis. Measurements using revised RECIST 1.1. At screening and thereafter until intolerable toxicity or progressive disease as indicated. Tumor measurements and evaluation of tumor response should be performed every 8 weeks.iOnly for patients in single agent dose escalation and combination with pembrolizumab cohorts.jTesting for MSLN expression necessary for study entrance can be performed using archival tumor tissue and is foreseen to be performed at the institution’s pathology. Mandatory slice for centralized assessment for validation analysis.kPatients treated in either the single agent recommended dose expansion cohort or in the combination with pembrolizumab cohort should have accessible lesions at screening for baseline and on treatment biopsies. If in a patient treated in any of these two cohorts, biopsy is considered by the investigator and / or the sponsor’s medical monitor to be risky or not feasible, the patient can be treated in any of the above cohorts without undergoing the study protocol required biopsies.lWith separate informed consent. DETAILED DESCRIPTION

[0039] The present disclosure provides, inter alia, methods of treating cancer comprising administering a combination of an anti-CD47 x anti-MSLN bispecific antibody and paclitaxel. Without wishing to be bound by theory, the addition of paclitaxel to the administration of the anti-CD47 x anti-MSLN bispecific antibody is believed to prolong the durability of treatment response and increase progression-free survival (PFS) and median overall survival (OS) without adding any relevant additional toxicity to the standard of care safety profile.

[0040] CD47 or Integrin-Associated-Protein (IAP) is a ubiquitous 50 kDa transmembrane glycoprotein with multiple functions in cell-cell communication. It interacts with multiple ligands, such as, for example, integrins, and / or SIRPα. In the context of the innate immunesystem, CD47 functions as a marker of self, transmitting an inhibitory “don’t kill me” signal through binding to SIRPα expressed by myeloid cells, such as macrophages, neutrophils, and dendritic cells. The role of widespread expression of CD47 in the physiological situation is therefore to protect healthy cells against the elimination by the innate immune system (Oldenborg PA, et al., CD47-Signal Regulatory Protein α (Sirpα) Regulates Fcγ and Complement Receptor–Mediated Phagocytosis, J Exp Med. 2001 Apr 2;193(7):855-62; Mattias Olsson, Role of the CD47 / SIRPα-interaction in regulation of macrophage phagocytosis, Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden, Thesis; Oldenborg PA., Role of CD47 in erythroid cells and in autoimmunity, Leuk Lymphoma. 2004 Jul;45(7):1319-27; Oldenborg PA, et al., Role of CD47 as a Marker of Self on Red Blood Cells., Science. 2000 Jun 16;288(5473):2051-4; Brown EJ, Frazier WA., integrin-associated protein (CD47) and its ligands., Trends Cell Biol.2001 Mar;11(3):130-5).

[0041] Tumor cells hijack this immunosuppressive mechanism by overexpressing CD47, which efficiently helps them to escape immune surveillance and killing by innate immune cells. (Majeti R, Chet al., CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells, Cell.2009 Jul 23;138(2):286-99; S. Jaiswal et al., CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis., Cell. 2009 Jul 23;138(2):271-85). CD47 expression is upregulated in most human cancers (e.g., NHL, AML, breast, colon, glioblastoma, glioma, ovarian, bladder and prostate cancers) and increased levels of CD47 expression clearly correlate with aggressive disease and poor survival. (Majeti R, et al., Cell.2009 Jul 23;138(2):286-99; S. Jaiswal et al., Cell.2009 Jul 23;138(2):271-85; Willingham SB, et al., The CD47-signal regulatory protein alpha (SIRPα) interaction is a therapeutic target for human solid tumors, Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6662-7; Chao MP, et al., Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia., Cancer Res. 2011 Feb 15;71(4):1374-84).

[0042] The widespread expression of CD47 in healthy tissues brings the question of treatment safety and efficacy: First, targeting CD47 with a neutralizing monoclonal antibody (Mab) could affect healthy cells, resulting in severe toxicities as shown in preclinical studies with mice and cynomolgus monkeys (Willingham SB, et al., Proc Natl Acad Sci U S A.2012 Apr 24;109(17):6662-7; Weiskopf K, et al., Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies, Science.2013 Jul 5;341(6141):88-91). Second, even if severe toxicities could be avoided or mitigated by using alternative formats (Weiskopf K, etal., Science.2013 Jul 5;341(6141):88-91), broad expression of CD47 could still cause a rapid elimination of CD47-binding molecules through target-mediated drug disposition resulting in poor pharmacokinetics and decreased efficacy.

[0043] The bispecific antibody compositions were described in U.S.11,260,117 (the contents of which are incorporated by reference in their entireties).

[0044] CD47 (Cluster of Differentiation 47) functions as a “don’t eat me” signal for phagocytic cells and is known to be over-expressed by many tumors (immune escape). CD47 interacts with SIRPα, which is expressed on phagocytic cells. CD47 down-regulates phagocytic activity. CD47 inhibits dendritic cell (DC) maturation and activation. CD47 has also been implicated in processes such as, for example, apoptosis, survival, proliferation, adhesion, migration, and regulation of angiogenesis, blood pressure, tissue perfusion, and / or platelet homeostasis.

[0045] CD47 has also been implicated in cancer. For example, CD47 is overexpressed in various hematological and solid malignancies. CD47 is a documented cancer stem cell / tumor initiating cell marker. It is thought that CD47 overexpression may help tumor cells escape immune surveillance and killing by innate immune cells. High levels of CD47 are also associated with poor clinical outcomes in cancers such as for example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, and / or glioma. Thus, targeting CD47 would be useful in treating, delaying the progression of, or otherwise ameliorating a symptom of cancer.

[0046] Mesothelin (MSLN) is expressed in normal tissues at relatively low levels. In contrast to normal tissues, mesothelin is highly expressed in several types of solid tumors such as malignant mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, as well as endometrial, biliary gastric, and prostate cancers. Tumor mesothelin expression has often been correlated with increased tumor aggressiveness and poor clinical outcome. Thus, targeting mesothelin would be useful in treating, delaying the progression of, or otherwise ameliorating a cancer symptom.

[0047] Mesothelin (MSLN) is a 40 kDa glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein that is generated proteolytically from a 71 kDa precursor. In normal tissues, mesothelin is expressed – at relatively low levels – only in mesothelial cells lining serosal membranes such as the pleura, peritoneum, and pericardium. The normal physiologic function of mesothelin remains unclear but it seems dispensable, since mesothelin deficient mice grow and reproduce normally and display no obvious abnormalities.

[0048] In contrast to normal tissues, mesothelin is highly expressed in several types of solidtumors such as malignant mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, as well as endometrial, biliary gastric and prostate cancers. Tumor mesothelin expression has often been correlated with increased tumor aggressiveness and poor clinical outcome. Mesothelin binding to ovarian cancer antigen MUC16 (CA-125) has been shown to mediate cell-to-cell adhesion, possibly contributing metastatic dissemination. In addition, mesothelin-mediated intracellular signaling was reported to promote tumor cell proliferation, as well as resistance to chemotherapy and to anoikis (programmed cell death resulting from loss of normal cell–matrix interactions).

[0049] Similar to most other GPI-anchored proteins, mesothelin is shed from the membrane, and soluble mesothelin has been reported in sera of tumor patients. Soluble mesothelin is therefore a useful biomarker, for diagnosis of mesothelin-positive tumors, but also for monitoring disease progression and response to treatment. Soluble mesothelin is also considered as a negative prognostic biomarker for patients with ovarian cancer, lung or pancreatic adenocarcinoma, and triple-negative breast cancer. Last but not least, serum mesothelin levels are a predictive biomarker in mesothelioma, as they have been found to positively correlate with therapeutic responses to mesothelin-targeting therapies.

[0050] Most tumor-associated antigens used to therapeutically target solid tumors are also expressed in essential normal tissues. In contrast, expression of mesothelin is generally low- level and limited to mesothelial cells (which seem dispensable). On the other hand, cell- surface expression of mesothelin is high in many solid tumors, which makes mesothelin a particularly attractive target of therapeutic intervention. Accordingly, numerous mesothelin- directed therapies, using monoclonal antibodies, recombinant immunotoxins, antibody-drug conjugates, cancer vaccines, and chimeric antigen receptor T cells, are currently under development, including clinical evaluation at late-stage trials for MPM and pancreatic adenocarcinoma.

[0051] The invention also provides bispecific antibodies that recognize CD47 and mesothelin.

[0052] The bispecific antibodies of the invention allow for simultaneous binding of the two antibody arms to two antigens on the surface of the cell (termed co-engagement), which results in additive or synergistic increase of affinity due to avidity mechanism. As a consequence, co-engagement confers high selectivity towards cells expressing both antigens as compared to cells that express just one single antigen. In addition, the affinities of the two arms of a bispecific antibody to their respective targets can be set up in a way that binding to target cells is principally driven by one of the antibody arms. In some embodiments, thebispecific antibody includes a first arm that binds CD47 and a second arm that binds mesothelin, where the second arm binds to mesothelin with high affinity, and the first arm binds to CD47 with low affinity, i.e., an affinity that is sufficient to inhibit CD47 / SIRPα upon mesothelin co-engagement. This design allows the bispecific antibodies of the disclosure to preferentially inhibit CD47 in cancer versus normal cells. In the examples provided herein, a bispecific antibody with a first arm that binds CD47 with low affinity and a second arm that binds MSLN with high affinity (termed a CD47xMSLN bispecific) allow preferential inhibition of CD47 in cancer versus normal cells. Besides the two antigen-binding arms, the CD47 x MSLN bispecific antibody requires a functional Fc portion to recruit macrophages and / or other immune effector cells. A fully human bispecific IgG format (such as the κλ-body format described herein) is well suited for the generation of dual targeting CD47 x MSLN bispecific antibodies. The ability of dual targeting bispecific antibodies to co-engage CD47 and MSLN results in efficient and selective cancer cell killing mediated by the CD47xMSLN bispecific antibody, as demonstrated in the ADCC and ADCP experiments provided herein.

[0053] In some embodiments, exemplary bispecific antibodies of the invention that include at least a first arm that binds CD47 include a combination of heavy chain and light chain complementarity determining regions (CDRs) selected from the CDR sequences shown in Tables 1, 2 and 3, where the CDRs shown in Tables 1, 2 and 3 are defined according to the IMGT nomenclature.

[0054] In some embodiments, exemplary bispecific antibodies of the invention that include at least a first arm that binds CD47 include the combination of heavy chain CDR sequences from Table 1 and two sets of light chain CDRs selected from the CDRL1, CDRL2 and CDRL3 sequences shown in Tables 2 and 3.

[0055] In some embodiments, exemplary bispecific antibodies of the invention that include at least a first arm that binds CD47 include the combination of heavy chain CDR sequences from Table 1 and a first set of light chain CDRs selected from the CDRL1, CDRL2 and CDRL3 sequences shown in Table 2 and a second set of light chain CDRs selected from the CDRL1, CDRL2 and CDRL3 sequences shown in Table 3.

[0056] In some embodiments, exemplary bispecific antibodies of the invention that include a first arm that binds CD47 and a second arm that binds MSLN, wherein the first arm includes the combination of heavy chain complementarity determining regions (CDRs) shown in Table 1 and a combination of the light chain CDRs selected from the CDR sequences shown in Table 2, and wherein the second arm includes the combination of heavy chaincomplementarity determining regions (CDRs) shown in Table 1 and a combination of the light chain CDRs selected from the CDR sequences shown in Table 3.

[0057] Table 1: Common Heavy Chain CDRs CDRH1 CDRH2 CDRH3 GFTFSSYA ISGSGGST AKSYGAFDY (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3)

[0058] Table 2: Anti-CD47 Kappa Light Chain CDRs CDRL1 CDRL2 CDRL3 QSISSY AAS QQMHPRAPKT (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)

[0059] Table 3: Anti-MSLN Lambda Light Chain CDRs CDRL1 CDRL2 CDRL3 SGINVRDYR YKSASDK MIWHHDSEGHAFV (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 9)

[0060] Each of the exemplary anti-CD47, anti-MSLN, monovalent and bispecific antibodies described herein include a common heavy chain (HC), one kappa chain or one lambda chain for anti-CD47 and anti-MSLN antibodies, one kappa and one lambda light chains (LC) for monovalent and bispecific antibodies, as shown in the amino acid and corresponding nucleic acid sequences listed below. Each of the exemplary anti-CD47, anti-MSLN, monovalent and bispecific antibodies described below includes a common variable heavy domain (VH), one kappa variable light domain or one lambda variable light domain for anti-CD47 and anti- MSLN antibodies, one kappa and one lambda variable light domains (VL) for monovalent and bispecific antibodies, as shown in the amino acid and corresponding nucleic acid sequences listed below.

[0061] While antibody sequences below are provided herein as examples, it is to be understood that these sequences can be used to generate bispecific antibodies using any of a variety of art-recognized techniques. Examples of bispecific formats include but are not limited to bispecific IgG based on Fab arm exchange (Gramer et al., 2013 MAbs.5(6)); the CrossMab format (Klein C et al., 2012 MAbs 4(6)); multiple formats based on forced heterodimerization approaches such as SEED technology (Davis JH et al., 2010 Protein Eng Des Sel. 23(4):195-202), electrostatic steering (Gunasekaran K et al., J Biol Chem. 2010285(25):19637-46.) or knob-into-hole (Ridgway JB et al., Protein Eng.19969(7):617-21.) or other sets of mutations preventing homodimer formation (Von Kreudenstein TS et al., 2013 MAbs.5(5):646-54.); fragment based bispecific formats such as tandem scFv (such asBiTEs) (Wolf E et al., 2005 Drug Discov. Today 10(18):1237-44.); bispecific tetravalent antibodies (Pörtner LM et al., 2012 Cancer Immunol Immunother. 61(10):1869-75.); dual affinity retargeting molecules (Moore PA et al., 2011 Blood.117(17):4542-51), diabodies (Kontermann RE et al., Nat Biotechnol.199715(7):629-31).

[0062] The exemplary anti-CD47, anti-MSLN, monovalent and bispecific antibodies include a common heavy chain (SEQ ID NO: 11) encoded by the nucleic acid sequence shown in SEQ ID NO: 10. >COMMON-HC-NT (SEQ ID NO: 10) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAAAGTTATGGTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACAGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACAGTCTCGT GGAACTCAGGAGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACTGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACA CATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCTCGGGAGGAGATGA CCAAGAACCAGGTCAGCCTGACTTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG GGAGAGCAACGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGTCCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAA >COMMON-HC-AA (SEQ ID NO: 11) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0063] The anti-CD47, anti-MSLN, monovalent and bispecific antibodies include a common variable heavy domain (SEQ ID NO: 13) encoded by the nucleic acid sequence shown in SEQ ID NO: 12. >COMMON-VH-NT (SEQ ID NO: 12) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAAAGTTATGGTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACAGTCTCGAGC >COMMON-VH-AA (SEQ ID NO: 13) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSS

[0064] ANTI-CD47 ANTIBODIES

[0065] The Ka3 antibody includes a common heavy chain (SEQ ID NO: 11) encoded by the nucleic acid sequence shown in SEQ ID NO: 10 and includes a kappa light chain (SEQ ID NO: 15) encoded by the nucleic acid sequence shown in SEQ ID NO: 14. >KA3-LC-NT (SEQ ID NO: 14) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTT GCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA GCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGC AGATGCACCCGCGCGCCCCGAAGACCTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGC TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA>KA3-LC-AA (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQMHPRAPKTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

[0066] The Ka3 antibody includes a common variable heavy domain (SEQ ID NO: 13) encoded by the nucleic acid sequence shown in SEQ ID NO: 12 and includes a kappa variable light domain (SEQ ID NO: 17) encoded by the nucleic acid sequence shown in SEQ ID NO: 16. >KA3-VL-NT (SEQ ID NO: 16) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTT GCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA GCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGC AGATGCACCCGCGCGCCCCGAAGACCTTCGGCCAAGGGACCAAGGTGGAAATCAAA >KA3-VL-AA (SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQMHPRAPKTFGQGTKVEIK

[0067] ANTI-MESOTHELIN (ANTI-MSLN) ANTIBODIES

[0068] The O38 antibody includes a common heavy chain (SEQ ID NO: 11) encoded by the nucleic acid sequence shown in SEQ ID NO: 10 and includes a lambda light chain (SEQ ID NO: 19) encoded by the nucleic acid sequence shown in SEQ ID NO: 18. The variable region of the lambda light chain is bolded in the amino acid sequence below. >O38-LC-NT (SEQ ID NO: 18) CAGCCTGTGCTGACTCAGCCGGCTTCCCTCTCTGCATCTCCTGGGGCATCAGCCAGTCTCACCTGCA CCTTGCGCAGTGGCATCAACGTTAGAGATTACAGGATATTCTGGTACCAGCAGAAGCCAGGGAGTCC TCCCCAGTATCTCCTGAGGTACAAAAGCGCATCAGATAAGCAGCAGGGCTCTGGAGTCCCCAGCCGC TTCTCTGGGTCCAAAGATGCTTCGGCCAATGCAGGGATTTTACTCATCTCTGGGCTCCAGTCTGAGG ATGAGGCTGACTATTACTGTATGATTTGGCACCACGATTCGGAGGGGCATGCTTTTGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCC TCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCG TGACAGTGGCTTGGAAAGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAA ACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCAC AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCATAA >O38-LC-AA (SEQ ID NO: 19) QPVLTQPASLSASPGASASLTCTLRSGINVRDYRIFWYQQKPGSPPQYLLRYKSASDKQQGSGVPSR FSGSKDASANAGILLISGLQSEDEADYYCMIWHHDSEGHAFVFGGGTKLTVLGQPKAAPSVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH RSYSCQVTHEGSTVEKTVAPTECS

[0069] The O38 antibody includes a common variable heavy domain (SEQ ID NO: 13) encoded by the nucleic acid sequence shown in SEQ ID NO: 12 and includes a lambda variable light domain (SEQ ID NO: 21) encoded by the nucleic acid sequence shown in SEQ ID NO: 20. >O38-VL-NT (SEQ ID NO: 20) CAGCCTGTGCTGACTCAGCCGGCTTCCCTCTCTGCATCTCCTGGGGCATCAGCCAGTCTCACCTGCA CCTTGCGCAGTGGCATCAACGTTAGAGATTACAGGATATTCTGGTACCAGCAGAAGCCAGGGAGTCC TCCCCAGTATCTCCTGAGGTACAAAAGCGCATCAGATAAGCAGCAGGGCTCTGGAGTCCCCAGCCGC TTCTCTGGGTCCAAAGATGCTTCGGCCAATGCAGGGATTTTACTCATCTCTGGGCTCCAGTCTGAGG ATGAGGCTGACTATTACTGTATGATTTGGCACCACGATTCGGAGGGGCATGCTTTTGTGTTCGGCGG AGGGACCAAGCTGACCGTCCTA >O38-VL-AA (SEQ ID NO: 21) QPVLTQPASLSASPGASASLTCTLRSGINVRDYRIFWYQQKPGSPPQYLLRYKSASDKQQGSGVPSR FSGSKDASANAGILLISGLQSEDEADYYCMIWHHDSEGHAFVFGGGTKLTVL

[0070] BISPECIFIC ANTIBODY Ka3 x O38 (NI-1801)

[0071] In some embodiments, the bispecific antibody Ka3 x O38 (NI-1801) comprises a common heavy chain polypeptide, a light chain polypeptide comprising anti-CD47 CDR sequences (“anti-CD47 light chain polypeptide”), and a light chain comprising anti-MSLN CDR sequences (“anti-MSLN light chain polypeptide”). Accordingly, in some embodiments the bispecific antibody Ka3 x O38 comprises two copies of the common heavy chainpolypeptide, one copy of the anti-CD47 light chain polypeptide, and one copy of the anti- MSLN light chain polypeptide. In some embodiments, the anti-CD47 light chain is a kappa light chain polypeptide (i.e. comprises a kappa constant region)—such a light chain is referred to as an anti-CD47 kappa light chain polypeptide. In some embodiments, the anti-MSLN light chain polypeptide is a lambda light chain polypeptide (i.e. comprises a lambda constant region)—such a light chain is referred to as an anti-MSLN lambda light chain polypeptide. In the descriptions below, the common heavy chain polypeptide is also referred to as “a common heavy chain”, the anti-CD47 kappa light chain polypeptide is also referred to as “a kappa light chain” and the anti-MSLN lambda light chain polypeptide is also referred to as “a lambda light chain”.

[0072] In some embodiments, the bispecific antibody Ka3 x O38 includes a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9.

[0073] Accordingly, in some embodiments, the bispecific antibody Ka3 x O38 comprises a common heavy chain polypeptide comprising a CDRH1 having the amnio acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and a CDRH3 having the amino acid sequence of SEQ ID NO: 3; an anti-CD47 kappa light chain polypeptide comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 6; and an anti-MSLN lambda light chain polypeptide comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 7, a CDRL2 having the amino acid sequence of SEQ ID NO: 8, and a CDRL3 having the amino acid sequence of SEQ ID NO: 9.

[0074] In some embodiments, the bispecific antibody Ka3 x O38 includes a common heavy chain variable region (SEQ ID NO: 13) encoded by the nucleic acid sequence shown in SEQ ID NO: 12, a kappa light chain variable region (SEQ ID NO: 17) encoded by the nucleic acid sequence shown in SEQ ID NO: 16, and a lambda light chain variable region (SEQ ID NO: 21) encoded by the nucleic acid sequence shown in SEQ ID NO: 20.

[0075] Accordingly, in some embodiments, the bispecific antibody Ka3 x O38 comprises: a common heavy chain polypeptide comprising a variable heavy domain (VH) comprising the amino acid sequence of SEQ ID NO: 13; an anti-CD47 kappa light chain polypeptide comprising a variable light domain (VL) comprising the amino acid sequence of SEQ ID NO: 17; and an anti-MSLN lambda light chain polypeptide comprising a VL comprising the amino acid sequence of SEQ ID NO: 21.

[0076] Accordingly, in some embodiments, the bispecific antibody Ka3 x O38 comprises: a common heavy chain polypeptide comprising a VH having the amino acid sequence of SEQ ID NO: 13; an anti-CD47 kappa light chain polypeptide comprising a VL comprising the amino acid sequence of SEQ ID NO: 17; and an anti-MSLN lambda light chain polypeptide comprising a VL having the amino acid sequence of SEQ ID NO: 21.

[0077] In some embodiments, the bispecific antibody Ka3 x O38 includes a common heavy chain (SEQ ID NO: 11) encoded by the nucleic acid sequence shown in SEQ ID NO: 10, a kappa light chain (SEQ ID NO: 15) encoded by the nucleic acid sequence shown in SEQ ID NO: 14, and a lambda light chain (SEQ ID NO: 19) encoded by the nucleic acid sequence shown in SEQ ID NO: 18.

[0078] Accordingly, in some embodiments, the bispecific antibody Ka3 x O38 comprises: a common heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11; an anti-CD47 kappa light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 15; and an anti-MSLN lambda light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 19.

[0079] Accordingly, in some embodiments, the bispecific antibody Ka3 x O38 comprises: a common heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 11; an anti-CD47 kappa light chain polypeptide having the amino acid sequence of SEQ ID NO: 15; and an anti-MSLN lambda light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 19.

[0080] METHODS OF TREATMENT USING Ka3 x O38 (NI-1801)

[0081] The disclosure herein provides for a method of method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a bispecific antibody comprising a first arm that comprises a first amino acid sequence that binds CD47 and a second arm that comprises a second amino acid that binds mesothelin (MSLN).

[0082] In some embodiments, the bispecific antibody is administered as a single agent, that is, without an additional combination therapy. In some embodiments, the bispecific antibodyis administered in combination with another therapeutic agent, such as a chemotherapeutic. In some embodiments, the bispecific antibody and the chemotherapeutic are administered at different therapeutically effective doses and at different schedules, respectively.

[0083] In some embodiments, the patient is treated with a therapeutically effective amount of an anti-CD47 x anti-MSLN bispecific antibody and a therapeutically effective amount of a chemotherapeutic (i.e., a combination therapy).

[0084] In certain embodiments, the bispecific antibody (i.e. the anti-CD47 x anti-MSLN bispecific antibody) is Ka3 x O38, or a fragment thereof. Ka3 x O38 is also referred to herein as “NI-1801”. In some embodiments, the Ka3 x O38 fragment is an antigen-binding fragment of Ka3 x O38.

[0085] In typical embodiments, the bispecific antibody comprises a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9, wherein the therapeutically effective amount of the bispecific antibody is about 15 mg to about 1200 mg.

[0086] In some embodiments, the bispecific antibody comprises a variable heavy chain comprising the amino acid of SEQ ID NO: 13 and a variable kappa light chain comprising the amino acid sequence of SEQ ID NO: 17 and a variable lambda light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0087] In some embodiments, the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, a kappa light chain comprising the amino acid sequence of SEQ ID NO: 15, and a lambda light chain comprising the amino acid sequence of SEQ ID NO: 19.

[0088] In some embodiments, the patient is further administered a therapeutically effective amount of a chemotherapeutic. In typical embodiments, the chemotherapeutic is paclitaxel.

[0089] The present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject an anti-CD47 x anti-MSLN bispecific antibody and at least one chemotherapy.

[0090] The present disclosure provides a method of treating cancer in the subject, the method comprising administering to the subject a combination comprising an anti-CD47 x anti- MSLN bispecific antibody and at least one chemotherapy. The present disclosure provides a combination comprising an anti-CD47 x anti-MSLN bispecific antibody and at least one chemotherapy for use in treating cancer in a subject. The present disclosure provides the use of a combination comprising an anti-CD47 x anti-MSLN bispecific antibody and at least one chemotherapy in the manufacture of a medicament for the treatment of cancer in a subject.

[0091] The present disclosure provides an anti-CD47 x anti-MSLN bispecific antibody for use in a method of treating cancer in a subject, wherein the method further comprises administering to the subject at least one chemotherapy. The present disclosure provides use of an anti-CD47 x anti-MSLN bispecific antibody in the manufacture of a medicament for the treatment of cancer in a subject, wherein the treatment further comprises administering to the subject at least one chemotherapy.

[0092] The present disclosure provides at least one chemotherapy for use in a method of treating cancer in a subject, wherein the method further comprises administering to the subject an anti-CD47 x anti-MSLN bispecific antibody. The present disclosure provides use of at least one chemotherapy in the manufacture of a medicament for the treatment of cancer in a subject, wherein the treatment further comprises administering to the subject an anti-CD47 x anti-MSLN bispecific antibody.

[0093] The present disclosure provides kits comprising an anti-CD47 x anti-MSLN bispecific antibody and at least one chemotherapy. The present disclosure provides a method of treating cancer in a subject comprising administering at least one amount of the preceding kits to the subject. The present disclosure provides the preceding kits for use in treating cancer in a subject. The present disclosure provides the use of the preceding kits in the manufacture of a medicament for the treatment of cancer in a subject.

[0094] In some embodiments of the preceding methods, uses, and kits, the anti-CD47 x anti- MSLN bispecific antibody and the at least one chemotherapy can be administered in temporal proximity.

[0095] In some embodiments of the preceding methods, uses and kits, the anti-CD47 x anti- MSLN bispecific antibody can be the Ka3 x O38 bispecific antibody (NI-1801) described herein, or an antigen-binding fragment thereof. In some embodiments of the preceding methods, uses and kits, the at least one chemotherapy can comprise paclitaxel. Accordingly, in some embodiments of the preceding methods, uses, and kits, the anti-CD47 x anti-MSLNbispecific antibody can be the Ka3 x O38 bispecific antibody (NI-1801) described herein and the at least one chemotherapy can comprise paclitaxel.

[0096] Accordingly, the present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject NI-1801 and paclitaxel.

[0097] The present disclosure provides a method of treating cancer in the subject, the method comprising administering to the subject a combination comprising NI-1801 and paclitaxel. The present disclosure provides a combination comprising NI-1801 and paclitaxel for use in treating cancer in a subject. The present disclosure provides the use of a combination comprising NI-1801 and paclitaxel in the manufacture of a medicament for the treatment of cancer in a subject.

[0098] The present disclosure provides NI-1801 for use in a method of treating cancer in a subject, wherein the method further comprises administering to the subject paclitaxel. The present disclosure provides use of NI-1801 in the manufacture of a medicament for the treatment of cancer in a subject, wherein the treatment further comprises administering to the subject paclitaxel.

[0099] The present disclosure provides paclitaxel for use in a method of treating cancer in a subject, wherein the method further comprises administering to the subject NI-1801. The present disclosure provides use of paclitaxel in the manufacture of a medicament for the treatment of cancer in a subject, wherein the treatment further comprises administering to the subject NI-1801.

[0100] The present disclosure provides kits comprising NI-1801 and paclitaxel. The present disclosure provides a method of treating cancer in a subject comprising administering at least one amount of the preceding kits to the subject. The present disclosure provides the preceding kits for use in treating cancer in a subject. The present disclosure provides the use of the preceding kits in the manufacture of a medicament for the treatment of cancer in a subject.

[0101] In some embodiments, paclitaxel is administered at an FDA approved dose. In some embodiments, the therapeutically effective amount of paclitaxel is about 80 mg / m2.

[0102] In some embodiments, the bispecific antibody is administered by intravenous injection. In some embodiments, paclitaxel is administered by intravenous injection. In some embodiments, the bispecific antibody and paclitaxel are administered by intravenous injection.

[0103] In some embodiments, the bispecific antibody or paclitaxel are administered to the patient over the course of a 1-hour or 2-hour IV infusion. In some embodiments, the bispecific antibody is administered over a 1-hour IV infusion, and then paclitaxel is administered withinat least 30 minutes. In some embodiments, the paclitaxel is administered over a 1-hour IV infusion period.

[0104] In some embodiments, the bispecific antibody is administered as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. In some embodiments, the paclitaxel is administered as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

[0105] In certain embodiments, the bispecific antibody is administered once every two weeks.

[0106] In certain embodiments, the bispecific antibody is administered once every two weeks for at least one cycle, wherein a cycle is 28 days. In some embodiments, the bispecific antibody is administered once every two weeks for at least six cycles, wherein a cycle is 28 days.

[0107] In some embodiments, the bispecific antibody is administered once every two weeks, and paclitaxel is administered, in combination, every week for at least four weeks.

[0108] In some embodiments, the therapeutically effective amount of the bispecific antibody is about 15 mg to about 50 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 50 mg to about 150 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 150 mg to about 300 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 300 mg to about 450 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 450 mg to about 600 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 600 mg to about 750 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 750 mg to about 900 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 900 mg to about 1200 mg.

[0109] In some embodiments, the therapeutically effective amount of the bispecific antibody is about 900 mg. In some embodiments, the therapeutically effective amount of the bispecific antibody is about 900 mg and is administered once every two weeks.

[0110] In some embodiments, the therapeutically effective amount of the bispecific antibody is about 900 mg and is administered once every two weeks and the therapeutically effective amount of paclitaxel is about 80 mg / m2and is administered once every week.

[0111] In the methods, uses, and kits described above, the subject can be administered one or more amounts of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801). In someembodiments, the amount of an anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) is a therapeutically effective amount.

[0112] In some embodiments of the methods, uses, and kits described above, the subject can be administered a loading dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI- 1801). In some embodiments of the methods, uses, and kits described above the subject can be administered a treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI- 1801).

[0113] In some embodiments of the methods, uses, and kits described above, the subject can be administered a loading dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI- 1801). In some embodiments, a loading dose can be about 100 mg to about 400 mg. In some embodiments a loading dose can be about 300 mg. In some embodiments, a loading dose can be about 500 mg to about 700 mg. In some embodiments, a loading dose can be about 600 mg. In some embodiments, a loading dose can be administered about one week (7 days) before the administration of a treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801). In some embodiments, a loading dose can be administered about 7 days (± 1 day) before the administration of a treatment dose.

[0114] A treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) can be any of the therapeutically effective amounts described above for the bispecific antibody, e.g. about 15 mg to about 50 mg, about 50 mg to about 150 mg, about 150 mg to about 300 mg, about 300 mg to about 450 mg, about 450 mg to about 600 mg, about 600 mg to about 750 mg, about 750 mg to about 900 mg, about 900 mg to about 1200 mg, or about 900 mg. In some embodiments, a treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) is about 900 mg. In some embodiments, the first treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) can be administered to the subject about 7 days after the administration of the loading dose. In some embodiments, the first treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) can be administered about 7 days (± 1 day) after the administration of the loading dose.

[0115] Following the administration of a loading dose and a first treatment dose of the anti- CD47 x anti-MSLN bispecific antibody, a subject can be administered additional treatment doses about every two weeks (Q2W).

[0116] Accordingly, in a non-limiting example, a subject can be administered a loading dose of about 300 mg of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) and then receive a first treatment dose of about 900 mg about 7 days after administration of the loadingdose. Subsequent treatment doses of 900 mg are then administered to the subject every two weeks.

[0117] In another non-limiting example, a subject can be administered a loading dose of about 300 mg of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) and the receive a first treatment dose of about 900 mg 7 days (± 1 day) after the administration of the loading dose. Subsequent treatment doses of 900 mg can then be administered to the subject every 14 days (± 1 day) or ever 14 days (± 2 days).

[0118] In some embodiments, the anti-CD47 x anti-MSLN bispecific antibody can be administered as part of a treatment cycle. In some embodiments, a treatment cycle is about 4 weeks (28 days). In some embodiments, the subject is treated for at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about seven, or at least about eight, or at least about nine, or at least about 10 treatment cycles, or at least about 11 treatment cycles, or at least about 12 treatment cycles. In some embodiments, when a subject receives more than cycle of treatment with the anti-CD47 x anti-MSLN bispecific antibody, only the first cycle comprises the administration of a loading dose.

[0119] Accordingly, an exemplary dosing regimen can be as follows: a first treatment cycle (wherein the treatment cycle is 28 days in length) can comprise the administration of a loading dose of the anti-CD47 x anti-MSLN bispecific antibody on day 1 of the cycle, the administration of the first treatment dose of the anti-CD47 x anti-MSLN bispecific antibody can be on day 8 of the cycle, and the administration of the second treatment dose of the anti- CD47 x anti-MSLN bispecific antibody can be on day 22 of the cycle. Following this exemplary first treatment cycle, an exemplary second treatment cycle can be initiated about two weeks after the administration of the second treatment dose of the anti-CD47 x anti- MSLN bispecific antibody.

[0120] Accordingly, another exemplary dosing regimen can be as follows: a first treatment cycle (wherein the treatment cycle is 28 days in length) can comprise the administration of a loading dose of the anti-CD47 x anti-MSLN bispecific antibody on day 1 of the cycle, the administration of the first treatment dose of the anti-CD47 x anti-MSLN bispecific antibody can be on day 8 (± 1 day) of the cycle, and the administration of the second treatment dose of the anti-CD47 x anti-MSLN bispecific antibody can be on day 22 (± 1 day) of the cycle. Following this exemplary first treatment cycle, an exemplary second treatment cycle can be initiated about 14 days (± 2 days) after the administration of the second treatment dose of the anti-CD47 x anti-MSLN bispecific antibody.

[0121] In some embodiments, following a first treatment cycle that comprises the administration of a loading dose, a second treatment cycle can be initiated about 14 days (± 2 days) after the administration of the second treatment dose of the anti-CD47 x anti-MSLN bispecific antibody (i.e. the administration of the anti-CD47 x anti-MSLN bispecific antibody on day 22 (± 1 day) of the first treatment cycle).

[0122] In some embodiments, for a second treatment cycle, the subject can be administered a treatment dose of the anti-CD47 x anti-MSLN bispecific antibody on day 1 of the second treatment cycle and on day 15 (± 1 day) of the second treatment cycle.

[0123] In some embodiments, for a third or subsequent (i.e. fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, etc.), the subject can be administered a treatment dose of the anti-CD47 x anti-MSLN bispecific antibody on day 1 of the second treatment cycle and on day 15 (± 2 days) of the second treatment cycle.

[0124] For third and subsequent treatment cycles, the treatment cycle can be initiated about 14 days (± 2 days) after the administration of the second treatment dose of the anti-CD47 x anti-MSLN bispecific antibody in the previous cycle (i.e. the administration of the anti-CD47 x anti-MSLN bispecific antibody on day 15 (± 1 day or ±2 days) of the previous treatment cycle).

[0125] The following table is an exemplary schematic of the administration schedule for the anti-CD47 x anti-MSLN bispecific antibody in the methods, uses, and kits of the present disclosure:

[0126] Table A Dose # Dose Type of anti-CD47 x anti-MSLN bispecific antibody Day of Cycle Treatment Cycle 1 1.1 Loading 1 1.2 Treatment 8 (±1 day) 1.3 Treatment 22 (± 1 day) Treatment Cycle 2 (started 14 days (± 2 days) after administration of dose #1.3) 2.1 Treatment 1 2.2 Treatment 15 (± 1 day) Treatment Cycles 3 (started 14 days (± 2 days) after administration of dose #2.2) 3.1 Treatment 1 3.2 Treatment 15 (± 2 days) Treatment Cycles 4 and beyond (started 14 days (± 2 days) after administration of second dose in previous cycle) X.1 Treatment 1 X.2 Treatment 15 (± 2 days)

[0127] In some embodiments, a loading dose or a treatment dose (e.g. therapeutically effective amount) of the anti-CD47 x anti-MSLN bispecific antibody is administered over the course of one hour. In some embodiments, a therapeutically effective amount of anti-CD47 x anti-MSLN bispecific antibody is administered intravenously.

[0128] Accordingly, in some embodiments of the methods, uses, and kits of the present disclosure, the subject is administered an anti-CD47 x anti-MSLN bispecific antibody (e.g. NI-1801) and paclitaxel, wherein the subject is first administered a loading dose of the anti- CD47 x anti-MSLN bispecific antibody and on the same day is administered a therapeutically effective amount of paclitaxel.

[0129] After the administration of this loading dose and a therapeutically effective amount of paclitaxel, subsequent doses of paclitaxel comprising a therapeutically effective amount of paclitaxel are administered to the subject weekly.

[0130] In some embodiments, a therapeutically effective amount of paclitaxel is about 70 mg / m2to about 90 mg / m2. In some embodiments, a therapeutically effective amount of paclitaxel is about 80 mg / m2.

[0131] Accordingly, in some embodiments of the methods, uses, and kits of the present disclosure, the subject is administered paclitaxel one weekly during a treatment cycle. Accordingly, in some embodiments, the subject is administered paclitaxel on day 1, day 7, and day 15 of a treatment cycle. In some embodiments, the subject is administered paclitaxel on day 1, day 7, day 15, and day 28 of a treatment cycle.

[0132] In some embodiments, the administration of paclitaxel is performed at least about 30 minutes after the administration of the anti-CD47 x anti-MSLN bispecific antibody (e.g. NI- 1801).

[0133] In some embodiments, a therapeutically effective amount of paclitaxel is administered over the course of one hour. In some embodiments, a therapeutically effective amount of paclitaxel is administered intravenously.

[0134] Diseases and Subjects

[0135] In typical embodiments, the patient in need of a therapeutically effective amount of the bispecific antibody and paclitaxel has cancer.

[0136] In some embodiments, the cancer is a solid tumor. In some embodiments, the tumor is platinum resistant. In some embodiments, the cancer is resistant to the standard of care treatment alone. In some embodiments, the cancer ovarian cancer, wherein the ovarian cancer is platinum-resistant.

[0137] In some embodiments, the cancer expresses mesothelin (MSLN). In some embodiments, the solid tumor is or is derived from ovarian cancer, triple-negative breast cancer (TNBC), non-squamous non-small cell lung cancer (NCSLC), head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, leiomyoma, leiomyosarcoma, kidney cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary gastric cancer, prostate cancer, or combinations thereof.

[0138] In certain embodiments, the subject is an adult greater than 18 years of age.

[0139] In some embodiments, before administration of any one of the antibodies disclosed herein and paclitaxel (i.e., in combination) to a patient, said patient has a platinum-resistant cancer. In some embodiments, before administration of any one of the antibodies disclosed herein and paclitaxel (i.e., in combination) to a patient, said patient has previously received at least one platinum-based chemotherapy. In some embodiments, before administration of any one of the antibodies disclosed herein and paclitaxel (i.e., in combination) to a patient, said patient has previously received at least one platinum-based chemotherapy and has relapsed from the last platinum-based chemotherapy.

[0140] In some embodiments, before administration of any one of the antibodies and / or chemotherapy disclosed herein to a patient, said patient has a histologically or cytologically confirmed diagnosis of any one of epithelial ovarian cancer, triple negative breast cancer, or non-squamous non-small cell lung cancer. In certain embodiments, the patient has a MSLN positive tumor, wherein MSLN expression is confirmed with a staining intensity of >2+ as per immunohistochemistry (IHC) in >40% of tumor cells.

[0141] In some embodiments, prior to administration of any one of the antibodies disclosed herein to a patient, said patient has a confirmed diagnosis of advanced, metastatic, or recurrent cancer.

[0142] In some embodiments, the cancer is a solid cancer expressing MSLN. In some embodiments, the cancer of the subject exhibits a MSLN expression with a staining intensity of greater than or equal to about 2+ per immunohistochemistry in greater than or equal to about 40% of tumor cells.

[0143] In some embodiments, the subject has been previously treated with at least one anti- cancer therapy. In some embodiments, the subject has been previously treated with at least one chemotherapy agent. In some embodiments, the subject has been previously treated with platinum-based chemotherapy.

[0144] In some embodiments, the cancer is resistant to platinum-based chemotherapy.

[0145] In some embodiments, the cancer is advanced.

[0146] In some embodiments, the cancer is metastatic.

[0147] In some embodiments, the cancer is recurrent.

[0148] In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is high-grade serous epithelial ovarian cancer (HGSEOC). In some embodiments, the ovarian cancer is endometrioid ovarian cancer. In some embodiments, the ovarian cancer of the subject is resistant to treatment with at least one platinum-based therapy (platinum-resistant ovarian cancer).

[0149] Accordingly, in some embodiments, the subject is a subject having ovarian cancer who has been previously treated with at least one platinum-based therapy (e.g. platinum-based chemotherapeutic).

[0150] In some embodiments, the subject has been previously treated with at least one but no more than 3 prior systemic lines of anticancer therapy.

[0151] In some embodiments, the subject has previously received one line of platinum- based therapy. In some embodiments, the subject has previously receive one line of platinum- based therapy, wherein the subject as received at least four cycles of the platinum-based therapy. In some embodiments, the subject demonstrated a response (either complete response or partial response) and then progressed between about 3 months and about 6 months after the date of the last dose of the platinum-based therapy.

[0152] In some embodiments, the subject has previously received at least two lines of platinum-based therapy (e.g. two lines or three lines). In some embodiments, the subject who has received at least two lines of platinum-based therapy progressed on or within about 6 months after the date of the last dose of the platinum-based therapy.

[0153] As used herein, the term “platinum-based therapy” or “platinum-based chemotherapy” or “platinum-based chemotherapeutic” refers to any anticancer drug whose active moiety contains a platinum complex and includes, by way of non-limiting example, cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, heptaplatin, and satraplatin.

[0154] In some embodiments, the cancer is triple negative breast cancer.

[0155] In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is non-squamous NSCLC.

[0156] In some embodiments, the cancer is pancreatic cancer.

[0157] Definitions:

[0158] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0159] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0160] As used herein, the term “temporal proximity” refers to that administration of one therapeutic agent (e.g., an anti-CD47 x anti-MSLN bispecific antibody disclosed herein) occurs within a time period before or after the administration of another therapeutic agent (e.g., paclitaxel), such that the therapeutic effect of the one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of the one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, “temporal proximity” means that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent. “Temporal proximity” may vary according to various factors, including but not limited to, the age, gender, weight, genetic background, medical condition, disease history, and treatment history of the subject to which the therapeutic agents are to be administered; the disease or condition to be treated or ameliorated; the therapeutic outcome to be achieved; the dosage, dosing frequency, and dosing duration of the therapeutic agents; the pharmacokinetics and pharmacodynamics ofthe therapeutic agents; and the route(s) through which the therapeutic agents are administered. In some embodiments, “temporal proximity” means within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks. In some embodiments, multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may change during a treatment cycle or within a dosing regimen.

[0161] The terms “effective amount” and “therapeutically effective amount” of an agent or compound are used in the broadest sense to refer to a nontoxic but sufficient amount of an active agent or compound to provide the desired effect or benefit.

[0162] The term "benefit" is used in the broadest sense and refers to any desirable effect and specifically includes clinical benefit as defined herein. Clinical benefit can be measured by assessing various endpoints, e.g., inhibition, to some extent, of disease progression, including slowing down and / or complete arrest; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and / or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; relief, to some extent, of one or more symptoms associated with the disorder; increase in the length of disease-free presentation following treatment, e.g., progression-free survival; increased overall survival; higher response rate; decreased mortality at a given point of time following treatment; and / or improvement in one of the following symptoms: congestion, fluid / edema, wheezing, coughing, hypoxemia, low oxygen saturation, lung stiffness, shortness of breath, shortness of breath during exercise, dry hacking cough, fast shallow breathing, weight loss, tiredness, aching joints, aching muscles and clubbing.

[0163] As used herein, the term “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0164] As used herein, the term “combination therapy” or “co-therapy” includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.

[0165] It is to be understood that the present disclosure also provides pharmaceutical compositions comprising any compound described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0166] It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the compounds to provide such treatment or prophylaxis as is described herein, as well as use of the compounds to prepare a medicament to treat or prevent such condition. The treatment includes treatment of human or non-human animals including rodents and other disease models.

[0167] As used herein, the term “subject” is interchangeable with the term “subject in need thereof”, both of which refer to a subject having a disease or having an increased risk of developing the disease. A “subject” includes a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In one embodiment, the mammal is a human.

[0168] As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

[0169] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd> 10-6). Antibodies include,but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab’and F(ab')2fragments, scFvs, and an Fabexpression library.

[0170] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0171] The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0172] The term “antigen-binding site,” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0173] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide. An antibody is the to specifically bind an antigen when the dissociation constant is ≤ 1 µM; e.g., ≤ 100 nM, preferably ≤ 10 nM and more preferably ≤ 1 nM.

[0174] As used herein, the terms “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kdrepresents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Konenables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is the to specifically bind to its target, when the equilibrium binding constant (Kd) is ^1 ^M, e.g., ^ 100 nM, preferably ^ 10 nM, and more preferably ^ 1 nM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0175] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to apolynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the invention include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules, and nucleic acid molecules encoding the light chain immunoglobulin molecules described herein.

[0176] The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

[0177] The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the invention comprise the heavy chain immunoglobulin molecules, and the light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0178] The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0179] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0180] The term “control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0181] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D- amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε -N- acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, ^-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0182] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0183] Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0184] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.

[0185] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0186] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).

[0187] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y,99Tc,111In,125I,131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0188] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw- Hill, San Francisco (1985)).

[0189] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[0190] Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0191] The term patient includes human and veterinary subjects.

[0192] Antibodies

[0193] Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a given target, such as, for example, CD47, a tumor associated antigen or other target, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0194] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).

[0195] In some embodiments, the antibodies of the invention are monoclonal antibodies. Monoclonal antibodies are generated, for example, by using the procedures set forth in the Examples provided herein. Antibodies are also generated, e.g., by immunizing BALB / c mice with combinations of cell transfectants expressing high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the selected target.

[0196] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method,a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0197] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0198] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0199] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover,in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0200] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0201] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0202] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No.4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0203] Monoclonal antibodies of the invention include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization is performed, e.g., by following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No.5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, e.g., residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody includes substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0204] Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Monoclonal antibodies can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0205] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.1365-93 (1995).

[0206] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94 / 02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. An example of such a nonhuman animal is a mouse termed the XenomouseTMas disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.

[0207] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No.5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0208] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expressionvector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0209] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds specifically to the relevant epitope with high affinity are disclosed in PCT publication WO 99 / 53049.

[0210] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0211] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA. gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93 / 64701, which has targeting moiety (e.g., a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vector (e.g., a DNA or RNA viral vector), fusion proteins such as described in PCT / US 95 / 02140 (WO 95 / 22618) which is a fusion protein containing a target moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

[0212] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

[0213] Pox viral vectors introduce the gene into the cells cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the conditionbeing treated. The introduction can be by standard techniques, e.g., infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO4precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0214] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g., adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

[0215] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a target such as CD47 or any fragment thereof. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

[0216] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0217] Bispecific and / or monovalent antibodies of the invention can be made using any of a variety of art-recognized techniques, including those disclosed in co-pending application WO 2012 / 023053, filed August 16, 2011, the contents of which are hereby incorporated by reference in their entirety. The methods described in WO 2012 / 023053 generate bispecific antibodies that are identical in structure to a human immunoglobulin. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and second light chain variable region fused to aconstant Lambda domain. Each combining site displays a different antigen specificity to which both the heavy and light chain contribute. The light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively. This is preferred in order to avoid the generation of non-natural polypeptide junctions. However, it is also possible to obtain bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgGκλ antibodies or “κλ bodies,” a new fully human bispecific IgG format. This κλ-body format allows the affinity purification of a bispecific antibody that is undistinguishable from a standard IgG molecule with characteristics that are undistinguishable from a standard monoclonal antibody and, therefore, favorable as compared to previous formats.

[0218] An important step of the method is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequence of both the light chain and the heavy chain, including the CDRs 1 and 2, arise from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0219] Monoclonal antibodies are generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encodingthe target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No.6,075,181 and No.6,150,584, which is hereby incorporated by reference in its entirety.

[0220] Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”).

[0221] Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the target antigen. Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0222] Although not strictly impossible, the serendipitous identification of different antibodies having the same heavy chain variable domain but directed against different antigens is highly unlikely. Indeed, in most cases the heavy chain contributes largely to the antigen binding surface and is also the most variable in sequence. In particular the CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost invariably carry different heavy chain variable domains.

[0223] The methods disclosed in co-pending application WO 2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies having the same heavy chain variable domain by the use of antibody libraries in which the heavy chain variable domain is the same for all the library members and thus the diversity is confined to the light chain variable domain. Such libraries are described, for example, in co-pending applications WO 2010 / 135558 and WO 2011 / 084255, each of which is hereby incorporated by reference in its entirety. However, as the light chain variable domain is expressed in conjunction with the heavy variable domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing the same heavy chain variable domain and either a diversity of Lambda variable light chains or Kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach enablesthe identification of two antibodies having a common heavy chain but one carrying a Lambda light chain variable domain and the other a Kappa light chain variable domain that can be used as building blocks for the generation of a bispecific antibody in the full immunoglobulin format of the invention. The bispecific antibodies of the invention can be of different Isotypes and their Fc portion can be modified in order to alter the bind properties to different Fc receptors and in this way modify the effectors functions of the antibody as well as it pharmacokinetic properties. Numerous methods for the modification of the Fc portion have been described and are applicable to antibodies of the invention. (see for example Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Pat. No.6,528,624; PCT / US2009 / 0191199 filed Jan 9, 2009). The methods of the invention can also be used to generate bispecific antibodies and antibody mixtures in a F(ab’)2 format that lacks the Fc portion.

[0224] The common heavy chain and two different light chains are co-expressed into a single cell to allow for the assembly of a bispecific antibody of the invention. If all the polypeptides get expressed at the same level and get assembled equally well to form an immunoglobulin molecule then the ratio of monospecific (same light chains) and bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, a means to modulate the relative expression of the different polypeptides is used to compensate for their intrinsic expression characteristics or different propensities to assemble with the common heavy chain. This modulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) featuring different efficiencies or other types of regulatory elements that can act at transcriptional or translational levels as well as acting on mRNA stability. Different promoters of different strength could include CMV (Immediate- early Cytomegalovirus virus promoter); EF1-1α (Human elongation factor 1α-subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simian virus 40 promoter). Different IRES have also been described from mammalian and viral origin. (See e.g., Hellen CU and Sarnow P. Genes Dev 200115: 1593–612). These IRES can greatly differ in their length and ribosome recruiting efficiency. Furthermore, it is possible to further tune the activity by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). The modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions. The Examples provided herein demonstrate that controlling the relative expression of the different chains is critical for maximizing the assembly and overall yield of the bispecific antibody.

[0225] The co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter has to be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the invention. This is in sharp contrast to specific purification methods that have to be developed and optimized for each bispecific antibodies derived from quadromas or other cell lines expressing antibody mixtures. Indeed, if the biochemical characteristics of the different antibodies in the mixtures are similar, their separation using standard chromatography technique such as ion exchange chromatography can be challenging or not possible at all.

[0226] Other suitable purification methods include those disclosed in co-pending application PCT / IB2012 / 003028, filed on October 19, 2012, published as WO2013 / 088259, the contents of which are hereby incorporated by reference in their entirety.

[0227] In other embodiments of producing bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0228] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface includes at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine orthreonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0229] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0230] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VHand VLdomains of one fragment are forced to pair with the complementary VLand VHdomains of another fragment, thereby forming two antigen- binding sites. Another strategy for making bispecific antibody fragments by the use of single- chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994).

[0231] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.147:60 (1991).

[0232] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0233] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No.4,676,980), and for treatment of HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0234] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer and / or other diseases and disorders associated with aberrant CD47 expression and / or activity. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0235] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0236] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include212Bi,131I,131In,90Y, and186Re.

[0237] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94 / 11026).

[0238] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the invention. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0239] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0240] Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0241] The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

[0242] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.

[0243] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

[0244] Use of anti-CD47 x anti-MSLN antibodies

[0245] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol.32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0246] Therapeutic formulations of the invention, which include an antibody of the invention, are used to treat or alleviate a symptom associated with a cancer, such as, by way of non-limiting example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung & bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity & pharynx cancer, uterine corpus cancer, and / or melanoma The present invention also provides methods of treating or alleviating a symptom associated with a cancer. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods.

[0247] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers a clinical benefit.

[0248] Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0249] Antibodies directed against a target such as CD47, mesothelin, or a combination thereof (or a fragment thereof), may be used in methods known within the art relating to the localization and / or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific any of thesetargets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).

[0250] An antibody of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies of the invention (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H.

[0251] Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target. Administration of the antibody may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds. For example, the antibody binds to the target and neutralizes or otherwise inhibits the interaction between CD47 and SIRPα.

[0252] A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subjectto which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

[0253] Antibodies or a fragment thereof of the invention can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York.

[0254] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0255] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0256] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0257] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymerscontaining the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0258] An antibody according to the invention can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyteprotein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Pharmaceutical compositions

[0259] The antibodies of the invention (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0260] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0261] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0262] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0263] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / oradjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0264] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0265] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0266] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0267] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.4,522,811.

[0268] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated;each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0269] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0270] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0271] EXAMPLE 1: Nonclinical Pharmacokinetics and Drug Metabolism

[0272] The nonclinical pharmacokinetics and drug metabolism of NI-1801 (i.e., Ka3 x O38) was determined. The PK of NI-1801 was investigated in both a non-cross-reactive species, mouse (C57BL / 6J), and in a cross-reactive species, monkey (cynomolgus). While NI-1801 can bind both human and cynomolgus monkey CD47 and MSLN with similar affinities, no cross-reactivity was reported in mice.

[0273] The PK profile of NI-1801 was evaluated in C57BL / 6J mice following a single IV injection of 5 mg / kg. As a comparator, a separate group of mice was administered a single IV injection of 5 mg / kg of a control human IgG1 bsAb, which has been shown to have a liner pharmacokinetic profile in the same non-cross-reactive species.

[0274] The terminal half-life (t1 / 2) of NI-1801 was 329 hours, i.e., 13.7 days. Previous reports have shown that IgGs bind to the neonatal Fc receptor (FcRn) which reduces their catabolism by receptor-mediated endocytosis (pinocytosis) leading to a long half-life in vivo. Moreover, human IgG1 has been described to bind mouse FcRn with superior affinity than mouse IgG, thus explaining the relative long elimination half-life of human IgG1 in this species.

[0275] In summary, NI-1801 bsAb has a PK profile like that of human IgG1 bsAb control and exhibited a biphasic and linear elimination profile. No unexpected off-target binding was identified and the ability to bind mouse FcRn was equivalent to the control human IgG1 bsAb.

[0276] Single and Repeat-Dose Pharmacokinetics

[0277] The PK profile of NI-1801 was investigated in male and female cynomolgus monkeys. NI-1801 was administered as a single-dose to one male and one female per groupat doses of 0.5 or 10 mg / kg and as repeat doses (on Days 1, 8, 15, and 22) to one male and two females at 10 mg / kg. NI-1801 was administered via IV bolus at a dose of 1 mL / kg.

[0278] Following single dose administration of NI-1801, T1 / 2of NI-1801 was estimated in females to be approx.140 hours (5.8 days) at 0.5 mg / kg and 99 hours (4.1 days) at 10 mg / kg, respectively. For male animals (both dose-levels), due to the limited timepoints in the terminal phase, T1 / 2could not be considered as appropriately estimated and reliable.

[0279] Differences between male and female monkeys were mainly seen in the terminal phase, and to a lesser extent in the distribution phase. Results showed comparable AUC0-168hand a lower AUC0-lastin males. After repeated dosing at 10 mg / kg, a slight to moderate decrease in AUC was observed in week 4 in comparison to week 1 for the three animals. This possible decrease in exposure after repeated dosing may be due to anti-CD47 / MSLN antibodies (ADA).

[0280] Toxicokinetics Dose range finding study

[0281] A dose range finding (DRF) study was performed in cynomolgus monkeys to evaluate the toxicity and toxicokinetic profile of NI-1801. NI-1801 was administered bi- weekly by IV bolus over four weeks (i.e., seven administrations in total) to two male and two female monkeys at doses of 15 or 45 mg / kg, respectively.

[0282] NI-1801 exposure in treated animals was comparable in male and female cynomolgus monkeys. Systemic exposure to NI-1801 increased in a dose-proportional manner in the range of administered doses following Day 1 administration.

[0283] NI-1801 was quantifiable in serum samples on Day 1 from 0.25 hours to last timepoint (96 hours) prior to the subsequent dosing at both doses of 15 and 45 mg / kg. On Day 1, concentration profiles were similar between animals for the same dose and between sexes.

[0284] During week 4, the overall toxicokinetic profile was consistent between animals, with one exception, that is a female from the 15 mg / kg treatment group which had notably lower exposure (mainly from 4-8 hours post-dosing onwards) compared to the three remaining animals from the same group.

[0285] Following administration of NI-1801 at 15 mg / kg, the mean exposure values (males / females) for Cmaxand AUC0-168hwere 481 / 490 µg / mL and 24’571 / 24’213 hours * µg / mL respectively, while at 45 mg / kg NI-1801, the mean exposure values (males / females) for Cmaxand AUC0-168hwere 1’757 / 1’743 µg / mL and 169’725 / 144’727 hours * µg / mL, respectively, at the end of the treatment period.

[0286] 4-week repeat-dose toxicokinetics from GLP toxicity study

[0287] The toxicokinetic profile of NI-1801 was investigated during the 4-week GLP toxicity study conducted in cynomolgus monkeys. During this study, NI-1801 was administered bi-weekly as a slow IV bolus, to three animals / sex / group (at 15 mg / kg dose) or five animals / sex / group (at 45 mg / kg dose). Animals received a total of seven injections over four weeks (i.e., Day 1, 5, 9, 13, 17, 21, and 25).

[0288] On Day 1, NI-1801 was quantifiable over the sampling interval (from 15 min to 96 hours post-dose) and at all doses. At 15 and 45 mg / kg / administration, the profiles showed a constant decline of the serum concentrations of NI-1801.

[0289] Following repeated administrations (Day 25), a significant decrease in exposure was observed after the 7thand last dose in two females and two males (15 mg / kg / administration) which were also reported to be ADA positive. The high dose group animals (45 mg / kg / administration) showed quantifiable serum concentrations of NI-1801 until 96 hours post-dose after the last dose on Day 25. Serum concentrations in the 45 mg / kg / administration recovery animals remained quantifiable for up to 25 days (i.e., 4 weeks of recovery period or Study Day 50) after the end of treatment; except for one female where the last quantifiable concentration was detected on Day 36. This female was tested ADA positive on Day 50. Overall, no gender differences were seen between the male and females on the parameters measured. The AUC increased in a dose proportional manner. For ADA negative animals, NI-1801 was quantifiable up to 96 hours post-dose on both Day 1 and Day 25, with a slight increase in systemic exposure observed by Day 25, with accumulation ratios ranging from 1.40 to 3.13 for both males and females.

[0290] Comparative Systemic Exposure Margins

[0291] Two population PK models were developed using data originating from studies in cynomolgus monkeys: One after the first injection to avoid the impact of ADAs on NI-1801 PK and a second one also including data after the last administration to describe NI-1801 PK in the presence of ADAs. The first model was used to simulate NI-1801 PK profiles in humans using allometric scaling.

[0292] The systemic exposure margins (or margins of safety) have been calculated using the exposures (Cmaxand AUC) in the 4-week repeat-dose toxicity studies in cynomolgus monkeys compared to predicted exposures in humans. Due to different designs between preclinical and clinical studies, the following comparisons were made: For Cmax, in monkeys the first observed NI-1801 concentration after the last injection was considered as Cmax(t+0.25h after bolus administration), while in humans, NI-1801 concentration at the end of the infusion was considered as Cmax(t+1h after the start of the last infusion). For AUC in humans, AUC0-168hwas computed based on simulated NI-1801 clearance after the last injection, while in monkeys the predicted NI-1801 clearance after the first injection was used to derive AUCinf. This approach was considered as the more conservative in terms of exposures, avoiding the implication of immunogenicity in cynomolgus, which significantly decrease NI-1801 exposure in some animals.

[0293] Based on this comparison. the Cmaxand AUC values in patients dosed weekly for 4 weeks with NI-1801 at e.g., 150 mg are predicted to be at least 32-fold and 10.2-fold below those measured in cynomolgus monkeys at the 45 mg / kg / administration NOAEL dose. While at 600 mg, for example, the predicted human Cmaxand AUC values in patients should remain at least 8- and 2.5-fold below those measured in monkeys. Please refer to the Investigator’s Brochure for a tabulated summary of the exposure margins.

[0294] EXAMPLE 2: Nonclinical Toxicology

[0295] The following section describes the nonclinical toxicology of NI-1801. The cynomolgus monkey was identified as the relevant species to investigate in vivo the safety of NI-1801 and therefore two studies were conducted in this species.

[0296] 4-Week DRF Study in Cynomolgus Monkeys

[0297] NI-1801 was administered twice weekly by IV (bolus) over four weeks (seven administrations in total) to two male and two female cynomolgus monkeys at doses of 15 or 45 mg / kg / administration. Animals were observed daily for clinical signs, local tolerance, body weight, food consumption. Blood samples were collected for toxicokinetics, hematology, coagulation, and blood chemistry. Organ weights, gross pathology and histopathology were investigated upon sacrifice of the animals.

[0298] Following twice weekly administration of NI-1801 for up to four weeks, there were no test item-related effects on clinical signs, body weight or food consumption.

[0299] Clinical pathology findings were limited to decreases in white blood cell, neutrophil and lymphocyte counts observed in high-dose (45 mg / kg) females and one high-dose male, observed from Day 15 and at the end of the treatment period (Day 28) for almost all animals. Decreased platelet count was also noted in all animals at 45 mg / kg / administration at Day 28. None of these changes was considered as adverse. No effects were observed on coagulation and blood chemistry parameters.

[0300] There were no organ weight differences or related macroscopic findings. NI-1801- related non-adverse microscopic findings were noted in the lymphoid organs (spleen, lymph nodes and GALT), and consisted of decreased lymphoid cellularity (in germinal centers mainly).

[0301] The no-observed-adverse-effect level (NOAEL) was therefore set at 45 mg / kg / administration. At this dose level, the mean exposure values (males / females), namely Cmaxand AUC0-168h, were 1’757 / 1’743 μg / mL and 169’725 / 144’727 h*μg / mL at the end of the treatment period.

[0302] 4-Week GLP Toxicology Study in Cynomolgus Monkeys

[0303] A 4-week study GLP repeat-dose study was conducted in cynomolgus monkeys to investigate the toxicity of NI-1801 following IV administrations at 15 or 45 mg / kg for four weeks, with an assessment of the reversibility of potential findings after a 6-week recovery (treatment free) period.

[0304] In this study, NI-1801 or vehicle were administered twice weekly on study days 1, 5, 9, 13, 17, 21, and 25 (every 96 hours) to 26 monkeys of Mauritian origin by slow bolus IV injections. Three animals / sex / group were terminated after four weeks of treatment and two animals / sex in the control and high-dose (i.e., 45 mg / kg) groups were terminated at the end of the recovery period.

[0305] Assessment of toxicity was based on clinical observations, body weights, food consumption, examination of injection sites, clinical pathology (hematology, clinical chemistry, and coagulation), ophthalmoscopies, urinalysis, and anatomic pathology examinations.

[0306] Following twice weekly administrations of NI-1801 for up to four weeks, no unscheduled deaths occurred, and no NI-1801-treatment related clinical signs were observed. Body weight, body weight gains, and food intake remained unaffected. No NI-1801-treatment related abnormalities were reported at ophthalmic examinations, urinalysis, and blood biochemistry investigations. Overall, NI-1801 treatment was clinically well tolerated.

[0307] Hematological changes were noted in males at >15mg / kg / administration and females at 45mg / kg / administration. They appeared from Day 2 onward and were limited to a minimal decrease in white blood cell counts compared to pre-study values, mainly triggered by neutrophils and lymphocytes decreased counts. These changes were aligned to those noted by immunophenotyping, at 45mg / kg / administration, which consisted of decreases in absolute and relative B- and NK-cell counts. Fibrinogen levels were minimally increased in several individuals at ≥15mg / kg / administration at the end of the treatment period. All changes were reversible following the recovery period except for the NK-cells counts, which tended towards recovery, but remained below pre-dose levels and 1.5 to 1.8-fold lower than in control animal.

[0308] There were no NI-1801-related macroscopic or organ weight changes at any dose level and microscopic findings were non-adverse and limited to those observed at 45 mg / kg / administration. This included a non-adverse decreased lymphoid cellularity in the lymph nodes (partially to fully reversible), spleen, and gut associated lymphoid tissue (fully reversible) and a non-adverse multisystemic arterial inflammation / necrosis and / or perivascular inflammatory cell infiltrate (minimal or slight). The latter was observed in a single male at the end of the treatment period and in both recovery females at the end of the dose-free period in the 45mg / kg dose group and considered to be likely secondary to immunogenicity of NI-1801.

[0309] Microscopic findings observed in the injection sites in NI-1801-treated animals were similar to those seen in control animals and / or were as expected after repeated IV injection. Similarly, reactions observed at the IV injection sites after dosing (i.e., swelling of the injected cephalic vein, abnormal color and scabs around the injected area) were present in all groups, including vehicle control group and were, therefore, attributed to the IV injection procedure rather than NI-1801 treatment.

[0310] There were no acute or chronic NI-1801 related effects on hemodynamics, ECG, and respiratory parameters and no abnormalities were reported at neurobehavioral examinations at any dose-level.

[0311] Based on the absence of adverse findings at any dose level, the NOAEL for this study was identified at 45mg / kg / administration.

[0312] Human Tissue Cross-Reactivity Study

[0313] A GLP-compliant tissue cross-reactivity study was conducted to investigate the potential off-target cross-reactivity of NI-1801.

[0314] A validated immunohistochemistry (IHC) method using biotin-conjugated NI-1801 was applied for testing of 42 frozen human tissues and blood smears (three donors per tissue). Sections of ovarian carcinoma (endometrioid adenocarcinoma) were used as positive controls.

[0315] In human tissues, biotinylated NI-1801 produced staining in cells whose morphology and distribution were generally consistent with mesothelial cells (duodenum, jejunum, ileum, cecum, lung, ovary, spleen), in epithelial cells of the oviduct and in a subset of squamous epithelial cells in the tonsil, described in the literature to express MSLN. Staining was also seen in some other epithelial cells (breast, parotid gland, prostate, urinary bladder), where expression of one or both targets is plausible based on the ubiquitous expression of CD47, inrare capillary blood vessel walls of the spleen and of thyroid, and in cytoplasmic granules consistent with lipofuscin (adrenal, thyroid).

[0316] CD47 is ubiquitously expressed, although levels are heterogenous on various tissues and cells according to the literature. As such, the staining pattern observed in this study is assumed to represent on-target binding on tissues expressing both mesothelin and CD47.

[0317] The staining in breast was not deemed relevant as it was mainly cytoplasmic and may be due to overexpression of MSLN secondary to breast cancer. The staining in urinary bladder, adrenal, and thyroid (follicular cells), mucous cells was also not relevant as it was exclusively cytoplasmic or represented artefacts. For the results observed in the remaining tissues (i.e., capillary vessels in thyroid and spleen, parotid gland), for which published data on MSLN expression is currently insufficient or limited to mRNA expression, staining could not be definitively attributed to NI-1801 binding or artifacts, and as such, non-specific binding could not be ruled out.

[0318] EXAMPLE 3: Clinical study of NI-1801 in combination with paclitaxel

[0319] Given non-clinical data showing significant anti-tumor effects (Example 1 and 2 above) NI-1801 (i.e., Ka3 x 038) as new IMP has a strong biological rationale for the treatment of subjects with advanced, metastatic, or recurrent solid malignancies expressing MSLN.

[0320] In a randomized, open-label cohort, the experimental arm of the study will receive NI-1801 at 900 mg administered once every two weeks (i.e., 900 mg Q2W) in combination with weekly administration of paclitaxel at a dose of 80 mg / m2over four-week cycles. The control arm will be treated with weekly paclitaxel at the same regimen representing one of the standard of care treatments in this population. This cohort specifically targets patients with platinum-resistant ovarian cancer, wherein the ovarian cancer expresses MSLN.

[0321] The cohort is made up of 20 evaluable patients, 10 evaluable patients per arm. An evaluable patient is defined as one who has received at least the loading dose (300 mg) and the first dose (P2RD to be defined by the SRC) of NI-1801, in addition to two doses of weekly paclitaxel. Discontinuing from the study due to related adverse events is considered progressive disease and patients are evaluable for the efficacy analysis.

[0322] The study demonstrates the efficacy and safety of NI-1801 in combination with weekly paclitaxel, compared to chemotherapy alone.

[0323] Combination Treatment Arm: Dosing Details

[0324] Cycle 1, Day 1 (C1D1): Participants in the combination arm initiate treatment with a loading dose of 300 mg of NI-1801, followed by paclitaxel at a dose of 80 mg / m^2. Theloading dose of NI-1801 is designed to enhance the treatment's tolerability profile. Starting from the second cycle (C2) and beyond, the NI-1801 loading dose is not administered again.

[0325] Cycle 1, Day 8 (C1D8) and onwards: On C1D8, participants receive the NI-1801 recommended dose (i.e., 900 mg) along with paclitaxel (80 mg / m^2). Subsequently, paclitaxel is administered weekly at the same dose, and NI-1801 at the recommended dose is given every two weeks.

[0326] NI-1801 administration involves a 1-hour infusion with standard premedication protocols. Chemotherapy is administered at least 30 minutes after completing the NI-1801 infusion, over a 1-hour period.

[0327] Control Arm

[0328] Participants in the control arm receive only the standard weekly chemotherapy regimen without the addition of NI-1801.

[0329] Study Objectives

[0330] The primary objectives for the randomized cohort are:

[0331] 1.) Determining the safety and tolerability of NI-1801 in combination with weekly paclitaxel in subjects with advanced first-line recurrent platinum-resistant ovarian cancer expressing MSLN.

[0332] 2.) Evaluating PFS of weekly paclitaxel (SOC) + NI-1801 vs paclitaxel alone in this subset of patients.

[0333] The secondary objective for the randomized cohort are:

[0334] 1) To evaluate ORR: objective response includes best of complete response (CR) or partial response (PR).

[0335] 2) To evaluate OS: time from date of randomization until the date of death.

[0336] 3) To evaluate DOR: time from initial response until Investigator-assessed progressive disease for all patients who achieve a confirmed objective response.

[0337] 4) To evaluate biomarker CA-125 response: serum CA-125 response determined using GCIG criteria.

[0338] Study endpoints:

[0339] Primary endpoints:

[0340] The primary endpoints for the randomized cohort are:

[0341] 1) AEs according to NCI CTCAE version 5.0.

[0342] 2) PFS defined as the time from date of randomization until Investigator-assessed progressive disease or death, whichever occurs first.

[0343] Secondary endpoints:

[0344] 1) Time to response, DOR, DCR, BOR, ORR.

[0345] 2) OS defined as the time from date of randomization until the date of death.

[0346] 3) Biomarker CA-125 response: Serum CA-125 response determined using the GCIG criteria.

[0347] Exploratory Endpoints:

[0348] The exploratory endpoints for this trial are:

[0349] 1.) Evaluate measures of tumor sensitivity / resistance to NI-1801, i.e., response according to revised RECIST guideline version 1.1 (3), in relation to e.g., CD47 and MSLN expression levels in tumor tissue, the immune cell infiltration status, as well as NI-1801 tissue penetration.

[0350] 2.) PD markers of NI-1801 biological activity potentially including, but not limited to, levels of serum MSLN.

[0351] 3.) Tumor circulating DNA in peripheral blood.

[0352] Study Population:

[0353] Approximately 20 patients diagnosed with first recurrence platinum-resistant ovarian cancer who are eligible to be treated with weekly paclitaxel as standard of care will be treated in the randomized arm comparing weekly paclitaxel (SOC) plus NI-1801 versus paclitaxel in a 1:1 ratio to evaluate the safety of this combination and its preliminary efficacy as primary endpoints of this cohort.

[0354] Inclusion criteria

[0355] 1. Female patients ≥ 18 years of age.

[0356] 2. Patients must have a confirmed diagnosis of high-grade serous epithelial ovarian cancer.

[0357] 3. Patients must have platinum-resistant disease:

[0358] Patients who have only had 1 line of platinum-based therapy must have received at least 4 cycles of platinum, must have had a response (CR or PR) and then progressed between >3 months and ≤ 6 months after the date of the last dose of platinum.

[0359] Patients who have received 2 or 3 lines of platinum therapy must have progressed on or within 6 months after the date of the last dose of platinum Note: Progression should be calculated from the date of the last administered dose of platinum therapy to the date of the radiographic imaging showing progression. Patients who are platinum-refractory during front-line treatment are excluded.

[0360] 4. Patients must have progressed radiographically on or after their most recent line of therapy.

[0361] 5. Patients must be willing to provide an archival tumor tissue block or slides, or undergo procedure to obtain a new biopsy using a low risk, medically routine procedure for IHC confirmation of MSLN expression.

[0362] 6. MSLN expression with staining intensity of ≥ 2+ as per IHC in ≥ 40% of tumor cells. Staining for MSLN expression can be performed using archival tumor tissue and can be done at the institution’s pathology. A slice for centralized IHC assessment for validation and biomarker analysis is mandatory.

[0363] 7. Patients must have at least one lesion that meets the definition of measurable disease by RECIST v1.1 (radiologically measured by the Investigator).

[0364] 8. Patients must have received at least 1 but no more than 3 prior systemic lines of anticancer therapy, and for whom single-agent therapy is appropriate as the next line of treatment:

[0365] Adjuvant ± neoadjuvant considered one line of therapy

[0366] Maintenance therapy (e.g., bevacizumab, PARP inhibitors) will be considered as part of the preceding line of therapy (i.e., not counted independently)

[0367] Therapy changed due to toxicity in the absence of progression will be considered as part of the same line (i.e., not counted independently)

[0368] Hormonal therapy will be counted as a separate line of therapy unless it was given as maintenance

[0369] 9. ECOG PS of 0 or 1

[0370] 10. Time from prior therapy:

[0371] Systemic antineoplastic therapy (5 half-lives or 4 weeks, whichever is shorter)

[0372] Focal radiation completed at least 2 weeks prior to first dose of study drug

[0373] 11. Patients must have stabilized or recovered (Grade 1 or baseline) from all prior therapy-related toxicities.

[0374] 12. Major surgery must be completed at least 4 weeks prior to first dose and have recovered or stabilized from the side effects of prior surgery.

[0375] 13. Patients must have adequate hematologic, liver and kidney functions defined as: Absolute neutrophil count (ANC) ≥ 1.5 x 10^9 / L (1,500 / μL) without G-CSF in the prior 10 days or long-acting WBC growth factors in the prior 20 days

[0376] Platelet count ≥ 100 x 10^9 / L (100,000 / μL) without platelet transfusion in the prior 10 days

[0377] Hemoglobin ≥ 9.0 g / dL without packed red blood cell (PRBC) transfusion in the prior 21 days

[0378] Serum creatinine ≤ 1.5 x upper limit of normal (ULN)

[0379] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3.0 x ULN

[0380] Serum bilirubin ≤ 1.5 x ULN (patients with documented diagnosis of Gilbert syndrome are eligible

[0381] if total bilirubin < 3.0 x ULN

[0382] Serum albumin ≥ 2 g / dL

[0383] 14. Patients or their legally authorized representative must be willing and able to sign the informed consent form (ICF) and to adhere to the protocol requirements.

[0384] 15. Negative pregnancy test at inclusion (female of childbearing potential [FCBP]). For FCBP:

[0385] Patients must be willing and capable to use adequate contraception during the complete therapy.

[0386] Females of childbearing potential (FCBP) must have two negative urine or serum pregnancy tests as verified by the Investigator prior to starting NI-1801; the subject may not receive NI-1801 until the Investigator has verified that the result of the pregnancy test is negative.

[0387] A urine or serum pregnancy test is required at screening and within 72 hours prior to dosing on C1D1, and within 72 hours prior to Day 1 of every subsequent cycle. Note: C1D1 pregnancy test does not need to be repeated if the screening pregnancy test was done within 72 hours prior to dosing. A serum or urine pregnancy test (Investigator’s discretion) must also be performed at the end of study for each FCBP; and agree to use and be able to comply with a highly effective birth control method, i.e., one that can achieve a failure rate of less than 1% per year when used consistently and correctly, from signing the ICF, throughout the study, and for up to and for up to 209 days following the last dose of NI-1801, according to CTFCG guidelines*.

[0388] An FCBP is defined as a sexually mature woman who has:

[0389] -Not undergone a hysterectomy or bilateral oophorectomy, and

[0390] -Not been naturally postmenopausal (amenorrhea following cancer therapy does not rule out childbearing potential) for at least 24 consecutive months (e.g., has had menses at any time in the preceding 24 consecutive months).

[0391] Highly effective birth control methods according to the Clinical Trials Facilitation and Coordination Group guideline are:

[0392] - combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation: oral, intravaginal or transdermal

[0393] - progestogen-only hormonal contraception associated with inhibition of ovulation: oral, injectable or implantable

[0394] - intrauterine device (IUD)

[0395] - intrauterine hormone-releasing system (IUS)

[0396] - bilateral tubal occlusion

[0397] - vasectomized partner if partner is the sole sexual partner of the FCBP study participant and that the vasectomized partner has received medical assessment of the surgical success.

[0398] - sexual abstinence

[0399] Males must be willing to use 1 effective method of contraception during the study, e.g., agree to use a condom (a latex condom is recommended) during sexual contact with a pregnant female or

[0400] a FCBP, and to avoid conceiving from signing the ICF, while participating in the study, during dose interruptions, and for at least 119 days following NI-1801 discontinuation, even if he has undergone a successful vasectomy*. * = The expected NILK-1801 half-life in humans based on pre-clinical studies and modeled from the population-based PK is approximately 139 h (6 days). This results in 119 days for males (approx.4 months) and 209 days for women (approx.7 months) according to Clinical Trial

[0401] Facilitation Group (CTFG) guidelines. Moreover, the duration of contraception after the end of treatment should be 6 months for paclitaxel.

[0402] Exclusion criteria

[0403] 1. Patients with clear cell, mucinous, or sarcomatous histology, mixed tumors containing any of the above histology, or low-grade or borderline ovarian tumor.

[0404] 2. Patients with primary platinum-refractory disease, defined as disease that did not respond to (CR or PR) or has progressed within 3 months of the last dose of first line platinum- containing chemotherapy.

[0405] 3. Patients with prior wide-field radiotherapy (RT) affecting at least 20% of the bone marrow.

[0406] 4. Patients with serious concurrent illness or clinically relevant active infection, including, but not limited to the following:

[0407] Active hepatitis B or C infection (whether or not on active antiviral therapy)

[0408] HIV infection

[0409] Active cytomegalovirus infection

[0410] Any other concurrent infectious disease requiring IV antibiotics within 2 weeks before starting study drug. Note: Testing at screening is not required for the above infections unless clinically indicated.

[0411] Patients with history of multiple sclerosis or other demyelinating disease and / or Lambert-Eaton syndrome (paraneoplastic syndrome)

[0412] 5. Patients with clinically significant cardiac disease including, but not limited to, any one of the

[0413] following:

[0414] Myocardial infarction ≤ 6 months prior to first dose

[0415] Unstable angina pectoris

[0416] Uncontrolled congestive heart failure (New York Heart Association > class II)

[0417] Uncontrolled ≥ Grade 3 hypertension (per CTCAE)

[0418] Uncontrolled cardiac arrhythmias

[0419] Patients assigned to PLD stratum only: Left ventricular ejection fraction (LVEF) below the institutional limit of normal as measured by echocardiography (ECHO) or multigated acquisition (MUGA) scan

[0420] History of hemorrhagic or ischemic stroke within six months prior to randomization

[0421] 6. History of cirrhotic liver disease (Child-Pugh Class B or C)

[0422] 7. Previous clinical diagnosis of non-infectious interstitial lung disease (ILD), including noninfectious pneumonitis

[0423] 8. Patients with prior hypersensitivity to monoclonal antibodies.

[0424] 9. Women who are pregnant or lactating.

[0425] 10. Patients with prior treatment with a CD47, signal regulatory protein (SIRP) alpha, or MSLN targeting agent.

[0426] 11. Patients with central nervous system (CNS) metastases.

[0427] 12. Patients with a history of other malignancy within 3 years prior to randomization. Note: does not include tumors with a negligible risk for metastasis or death (e.g., adequately controlled basal-cell carcinoma or squamous-cell carcinoma of the skin, or carcinoma in situ of the cervix or breast).

[0428] 13. Prior known hypersensitivity reactions to study drugs and / or any of their excipients.

[0429] 14. People who are detained through a court or administrative decision, receiving psychiatric care against their will, adults who are the subject of a legal protection order (undertutorship / curatorship), people who are unable to express their consent, and people who are subject to a legal guardianship order.

[0430] 15. Simultaneous participation in another research study, in countries or localities where this is the health authority guidance.

[0431] Schedule of events

[0432] The procedures conducted for each patient enrolled in the study are outline in FIG. 1. More specifically, the below schedule of events illustrates the “randomized arm” which is the NI-1801 + paclitaxel treatment arm of the study.

[0433] Response Evaluation

[0434] Tumor response and progression will be evaluated according to the revised RECIST 1.1, as provided here: Response Tumor response Response Tumor response Non- Category Target lesions Category target legions Complete response Disappearance of Complete response Disappearance of all (CR) all target lesions. (CR) non-target lesions and Any pathological normalization of tumor lymph nodes marker level. All (whether target or lymph nodes must be non-target) must non-pathological in have reduction in size (<10 mm short short axis to <10 axis). mm. Partial response At least a 30% Non- Unequivocal (PR) decrease in the sum CR / Nonprogressive progression (see (3) for of diameters of disease details) of existing target lesions, non-target lesions. taking as reference (Note: the appearance the baseline sum of one or more new diameters. lesion(s) is also considered progression). Progressive disease At least a 20% Progressive disease Unequivocal increase in the sum progression of existing of diameters of non-target lesions. target lesions, (Note: the appearance taking as reference of one or more new the smallest sum on lesion(s) is also study (this includes considered the baseline sum if progression). that is the smallest on study). In addition to the relative increase of20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesion(s) is also considered progression). Stable disease (SD Neither sufficient shrinkage to qualify for progressive disease nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum diameters while on study.

[0435] EXAMPLE 4: Preliminary Results from Clinical study of NI-1801 in combination with paclitaxel

[0436] Preliminary clinical data of the randomized cohort (12 patients) exploring the combination of NI-1801 and weekly paclitaxel vs weekly paclitaxel in mesothelin expressing platinum resistant ovarian cancer patients that have received 1 to 3 prior chemotherapy lines, showed that both the combination of paclitaxel and NI-1801 and the paclitaxel monotherapy (standard of care) have been safe and well tolerated and that there are no new or unexpected AEs reported related to the combination arm treatment. The safety profiles of the two randomized arms treatments are consistent with the well stablished safety profile of weekly paclitaxel reported by the manufacturer. That is, adding NI-1801 in the experimental arm did not modify the above in the first 6 patients dosed with this combination.

[0437] In the control arm, out of the 4 patients that have at least one tumor assessment (CT scan), one patient experienced progressive disease (PD) after two months of treatment, another two patients experienced PD a week before completing 6 months of treatment and one patient is currently ongoing on month 5. Without wishing to be bound by theory, this data is consistent with the expected clinical benefit that has been reported with weekly paclitaxel in this population of ovarian cancer.

[0438] In the experimental arm, one patient experience PD a week before completing 6 months, another patient experience PD a week before completing 10 months of treatment, and two patients are ongoing at months 8 and 9.

[0439] At this point in the study, only one patient in the control arm died after experiencing progressive disease.

[0440] Without wishing to be bound by theory, this preliminary data suggests improved clinical benefit to the patients in the combination arm vs the patients in the control arm with a longer stabilization of their disease, well beyond the 4.34 months median PFS reported with weekly paclitaxel in this population (see K. N. Moore et al. MIRASOL study.2023 ASCO Annual Meeting).

[0441] Accordingly, the data described in this example improves further the benefit / risk assessment of NI-1801 in this ovarian cancer population. NUMBERED EMBODIMENTS

[0442] Embodiment 1. A method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient about 15 mg to about 1200 mg of a bispecific antibody comprising a first arm that comprises a first amino acid sequence that binds CD47 and a second arm that comprises a second amino acid that binds mesothelin (MSLN); wherein the bispecific antibody comprises a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of AAS, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9.

[0443] Embodiment 2. A method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a bispecific antibody and a therapeutically effective amount of a chemotherapeutic, wherein the bispecific antibody comprises a first arm that comprises a first amino acid sequence that binds CD47 and a second arm that comprises a second amino acid that binds mesothelin (MSLN); wherein the bispecific antibody comprises a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ IDNO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of AAS, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9.

[0444] Embodiment 3. The method of embodiment 1 or 2, wherein the bispecific antibody comprises a variable heavy chain comprising the amino acid of SEQ ID NO: 13 and a variable kappa light chain comprising the amino acid sequence of SEQ ID NO: 17 and a variable lambda light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0445] Embodiment 4. The method of any one of embodiments 1-3, wherein the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, a kappa light chain comprising the amino acid sequence of SEQ ID NO: 15, and a lambda light chain comprising the amino acid sequence of SEQ ID NO: 19.

[0446] Embodiment 5. The method of embodiment 1, further administering to the patient a therapeutically effective amount of a chemotherapeutic.

[0447] Embodiment 6. The method of any one of embodiments 2-5, wherein the chemotherapeutic is paclitaxel.

[0448] Embodiment 7. The method of any one of embodiments 5-6, wherein the therapeutically effective amount of the paclitaxel is about 80 mg / m2.

[0449] Embodiment 8. The method of any one of embodiments 1-4, wherein the bispecific antibody is administered by intravenous injection.

[0450] Embodiment 9. The method of embodiment 6 or 7, wherein the paclitaxel is administered by intravenous injection.

[0451] Embodiment 10. The method of embodiment 8, wherein the bispecific antibody is administered as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

[0452] Embodiment 11. The method of embodiment 9, wherein the paclitaxel is administered as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

[0453] Embodiment 12. The method of embodiment 10, wherein the bispecific antibody is administered once every two weeks.

[0454] Embodiment 13. The method of embodiment 12, wherein the bispecific antibody is administered once every two weeks for at least one cycle, wherein a cycle is 28 days.

[0455] Embodiment 14. The method of embodiment 13, wherein the bispecific is administered once every two weeks for at least six cycles.

[0456] Embodiment 15. The method of embodiment 11, further comprising administering the paclitaxel every week for at least four weeks.

[0457] Embodiment 16. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 15 mg to 50 mg.

[0458] Embodiment 17. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 50 mg to 150 mg.

[0459] Embodiment 18. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 150 mg to 300 mg.

[0460] Embodiment 19. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 300 mg to 450 mg.

[0461] Embodiment 20. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 450 mg to 600 mg.

[0462] Embodiment 21. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 600 mg to 750 mg.

[0463] Embodiment 22. The method of any one of embodiments 1-15, the bispecific antibody is administered in an amount of 750 mg to 900 mg.

[0464] Embodiment 23. The method of any one of embodiments 1-15, wherein the bispecific antibody is administered in an amount of 900 mg to 1200 mg.

[0465] Embodiment 24. The method of embodiment 8, wherein the bispecific antibody is administered in an amount of 900 mg and is administered once every two weeks.

[0466] Embodiment 25. The method of embodiment 24, further comprising administering the paclitaxel every week for at least four weeks.

[0467] Embodiment 26. The method of any one of embodiments 1-25, wherein the cancer is a solid tumor.

[0468] Embodiment 27. The method of embodiment 26, wherein the cancer expresses MSLN.

[0469] Embodiment 28. The method of embodiment 27, wherein the solid tumor is or is derived from ovarian cancer, triple-negative breast cancer (TNBC), non-squamous non-small cell lung cancer (NCSLC), head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, leiomyoma, leiomyosarcoma, kidney cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary gastric cancer, prostate cancer, or combinations thereof.

[0470] Embodiment 29. The method of embodiment 26, wherein the cancer is a platinum- resistant cancer.

[0471] Embodiment 30. A method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a bispecific antibody and a therapeutically effective amount of paclitaxel, wherein the bispecific antibody comprises a first arm that comprises a first amino acid sequence that binds CD47 and a second arm that comprises a second amino acid that binds mesothelin (MSLN); wherein the bispecific antibody comprises a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of AAS, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9. OTHER EMBODIMENTS

[0472] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A combination comprising: i) a bispecific antibody comprising: a) a common heavy chain polypeptide comprising a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amnio acid sequence of SEQ ID NO: 1, a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 2, and a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 3; b) a first light chain polypeptide comprising a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 4, a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence of SEQ ID NO: 5, and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence of SEQ ID NO: 6; c) a second light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9; and ii) paclitaxel, for use in treating cancer in a subject.

2. A method of treating cancer in a patient in need thereof, the method comprising administering to the subject: i) a bispecific antibody comprising: a) a common heavy chain polypeptide comprising a CDRH1 comprising the amnio acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b) a first light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6;c) a second light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9; and ii) paclitaxel.

3. A bispecific antibody comprising: a) a common heavy chain polypeptide comprising a CDRH1 comprising the amnio acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b) a first light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; c) a second light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9; and for use in a method of treating cancer in a subject, wherein the method further comprises administration of paclitaxel.

4. Paclitaxel for use in a method of treating cancer in a subject, wherein the method further comprises the administration of a bispecific antibody comprising: a) a common heavy chain polypeptide comprising a CDRH1 comprising the amnio acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; b) a first light chain polypeptide comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6.

5. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein: a) the common heavy chain polypeptide comprises a variable heavy domain (VH) comprising the amino acid sequence of SEQ ID NO: 13; b) the first light chain polypeptide comprises a variable light domain (VL) comprising the amino acid sequence of SEQ ID NO: 17; and c) the second light chain polypeptide comprises a VL comprising the amino acid sequence of SEQ ID NO:

21.

6. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein: a) the common heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 11; b) the first light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 15; and c) the second light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

19.

7. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the cancer is a solid tumor, preferably wherein the solid tumor expresses Mesothelin (MSLN).

8. The combination, method, bispecific antibody, or paclitaxel of claim 7, wherein the solid tumor is or is derived from ovarian cancer, triple-negative breast cancer (TNBC), non- squamous non-small cell lung cancer (NCSLC), head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, leiomyoma, leiomyosarcoma, kidney cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary gastric cancer, prostate cancer, or combinations thereof.

9. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims wherein the cancer is: i) advanced; ii) metastatic; iii) recurrent;iv) platinum-resistant; or v) any combination thereof.

10. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the cancer is ovarian cancer, preferably wherein the ovarian cancer is high- grade serous epithelial ovarian cancer (HGSEOC) or endometrioid ovarian cancer, preferably wherein the ovarian cancer is resistant to at least one platinum-based therapy.

11. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the bispecific antibody is administered in an amount of about 900 mg.

12. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the bispecific antibody is administered once every two weeks.

13. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the paclitaxel is administered in an amount of about 80 mg / m2.

14. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the paclitaxel is administered once a week.

15. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the bispecific antibody is administered such that: i) an initial loading dose of about 300 mg is administered to the subject; and ii) an initial treatment dose of about 900 mg is administered to the subject about seven days after the administration of the loading dose, wherein subsequent treatment doses of about 900 mg are administered to the subject every two weeks.

16. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the bispecific antibody and paclitaxel are administered in a first treatment cycle that is about 28 days in length, wherein the first treatment cycle comprises: a) on day 1 of the cycle, administering to the subject a loading dose of about 300 mg of the bispecific antibody;b) on day 8 (±1 day), administering to the subject a first treatment dose of about 900 mg of the bispecific antibody; and c) on day 22 (±1 day), administering to the subject a second treatment dose of about 900 mg of the bispecific antibody; wherein the paclitaxel is administered weekly during the first treatment cycle at a dose of about 80 mg / m2, preferably wherein the paclitaxel is administered to the subject on day 1, day 8, day 15, day 22, and day 28 of the first treatment cycle.

17. The combination, method, bispecific antibody, or paclitaxel of claim 16, further comprising the administration of the bispecific antibody and paclitaxel in a second treatment cycle that is about 28 days in length, wherein the second treatment cycle starts 14 days (±1 day) after the administration of the second treatment dose of the bispecific antibody in the first treatment cycle, wherein the second treatment cycle comprises: a) on day 1 of the cycle, administering to the subject a first treatment dose of about 900 mg of the bispecific antibody; and b) on day 15 (±1 day), administering to the subject a second treatment dose of about 900 mg of the bispecific antibody, wherein the paclitaxel is administered weekly during the second treatment cycle at a dose of about 80 mg / m2, preferably wherein the paclitaxel is administered to the subject on day 1, day 8, day 15, day 22, and day 28 of the first treatment cycle.

18. The combination, method, bispecific antibody, or paclitaxel of claim 17, further comprising the administration of the bispecific antibody and paclitaxel in at least third treatment cycle that is about 28 days in length, wherein the at least third treatment cycle starts 14 days (±1 day) after the administration of the second treatment dose of the bispecific antibody in the preceding treatment cycle, wherein the at least third treatment cycle comprises: a) on day 1 of the cycle, administering to the subject a first treatment dose of about 900 mg of the bispecific antibody; and b) on day 15 (±2 days), administering to the subject a second treatment dose of about 900 mg of the bispecific antibody,wherein the paclitaxel is administered weekly during the at least third treatment cycle at a dose of about 80 mg / m2, preferably wherein the paclitaxel is administered to the subject on day 1, day 8, day 15, day 22, and day 28 of the at least third treatment cycle.

19. The combination, method, bispecific antibody, or paclitaxel of claim 18, wherein the subject is treated for at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least 11, or at least twelve treatment cycles.

20. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the bispecific antibody is administered by intravenous injection and the paclitaxel is administered by intravenous injection, preferably wherein the bispecific antibody is administered as part of a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and the paclitaxel is administered as part of a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

21. The combination, method, bispecific antibody, or paclitaxel of any one of the preceding claims, wherein the subject has been previously treated with at least one line of platinum- based therapy, preferably wherein the subject has been treated with at least one line but no more than three systemic lines of anticancer therapy.

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