Generating multispecific antibody mixtures and methods of uses thereof

Abstract

To provide generation of multispecific antibody mixtures and methods of uses thereof.SOLUTION: The present invention relates to the generation of multispecific antibody mixtures including a subset of antibodies isolated from a mixture of two or more monospecific antibodies and one or more bispecific antibodies, wherein all antibodies in the subset have the same common heavy chain. The invention also relates to methods of isolating, purifying, or otherwise producing such a subset of antibodies by using at least one affinity chromatography step. The invention also relates to methods of using such a subset of antibodies in a variety of therapeutic indications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 535,354, filed July 21, 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to the production of multispecific antibody mixtures comprising a subset of antibodies isolated from a mixture of two or more monospecific antibodies and one or more bispecific antibodies, wherein all of the antibodies in said subset have the same common heavy chain. The present invention also relates to methods for isolating, purifying, or otherwise producing such subsets of antibodies by using at least one affinity chromatography step. The present invention also relates to methods of using such subsets of antibodies in various therapeutic applications. [Background technology]

[0003] Background of the Invention Over the past two decades, monoclonal antibodies (mAbs) have become an important therapeutic modality, providing significant benefit to patients in a variety of indications. The success of mAbs is due, in part, to their high specificity for their target antigens and low inherent toxicity. These properties greatly limit off-target side effects when compared to other classes of drugs. The majority of approved therapeutic mAbs are unmodified antibodies of the IgG isotype.

[0004] However, targeting of a single protein, as enabled by standard mAbs, may not always be sufficient to achieve a significant therapeutic effect (Fischer Expert Opin. Drug Discov. 2008 3(8):833-839).

[0005] The obvious option to increase efficacy is to use two mAbs in combination. This strategy has been clinically pursued, for example, for antibodies targeting immune checkpoint molecules, such as anti-CTLA4 antibody and anti-PD-1 antibody (Larkin et al., N Engl J Med 2015;373:23-34; Harris et al., Cancer Biol Med. 2016 13(2):171-93). However, the development of mAb combinations involves significant costs and development obstacles. In particular, two separate manufacturing processes must be introduced, leading to significant increases in costs (Rasmussen et al., Archives of Biochemistry and Biophysics 2012 526:39-145). These issues become even more important when considering targeting three or more proteins or antigens.

[0006] To date, several approaches have been used to target multiple proteins or antigens. The use of two mAbs in combination has been pursued but is often hindered by significant costs and development obstacles. Bispecific antibodies (BiAbs) are a rapidly evolving alternative to achieve multispecific targeting, with over 60 formats described to date (Spiess et al., Mol. Immunol. 2015 67:95-106; Brinkmann and Kontermann mAbs 2017 9:182-212), another approach to achieve targeting of two or even more proteins is the generation of antibody mixtures or recombinant polyclonal mixtures. However, only a few of these multispecific formats have been approved for therapeutic use. Therefore, there is a need to generate multispecific antibody mixtures that can target multiple proteins, epitopes and / or antigens. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Fischer Expert Opin.Drug Discov.2008 3(8):833-839 [Non-patent document 2] Larkin et al. N Engl J Med 2015;373:23-34 [Non-patent document 3] Harris et al. Cancer Biol Med.2016 13(2):171-93 [Non-patent document 4] Rasmussen et al., Archives of Biochemistry and Biophysics 2012 526:39-145 [Non-Patent Document 5] Spiess et al. Mol Immunol.2015 67:95-106 [Non-patent document 6] Brinkmann and Kontermann mAbs 2017 9:182-212 Summary of the Invention

[0008] The present disclosure provides antibody mixtures comprising a subset of antibodies isolated from a mixture of two or more monospecific antibodies and one or more bispecific antibodies, wherein all of the antibodies in the subset share the same common heavy chain. These mixtures are produced when multiple light chains are co-expressed with a common heavy chain in a single cell. In some embodiments, the purified subset comprises only antibodies that contain at least a kappa light chain. In some embodiments, the purified subset comprises only antibodies that contain at least a lambda light chain. In some embodiments, the purified subset comprises only antibodies that contain a kappa light chain and a lambda light chain.

[0009] The present disclosure also provides methods for isolating, purifying, or otherwise producing a subset of antibodies isolated from a mixture of two or more monospecific antibodies and one or more bispecific antibodies by using at least one affinity chromatography step, wherein all antibodies in the subset have the same common heavy chain. In some embodiments, the purification step is performed using a kappa constant or variable domain-specific affinity chromatography medium. In some embodiments, the purification step is performed using a lambda constant or variable domain-specific affinity chromatography medium. In some embodiments, the purification step is performed using a two-step affinity chromatography process. In some embodiments, the first purification step is performed using a kappa constant or variable domain-specific affinity chromatography medium and the second purification step is performed using a lambda constant-specific affinity chromatography medium. In some embodiments, the first purification step is performed using a lambda constant-specific affinity chromatography medium and the second purification step is performed using a kappa constant or variable domain-specific affinity chromatography medium.

[0010] The multispecific antibody mixtures and methods provided herein are useful in any of a variety of therapeutic, diagnostic, and / or prophylactic indications. For example, the multispecific antibody mixtures are useful in treating, preventing, and / or slowing or alleviating the progression of symptoms of cancer or other neoplastic conditions by administering the antibody mixture to a subject in whom such treatment or prevention is desired. In some embodiments, the multispecific antibody mixtures described herein are useful in treating hematological malignancies and / or solid tumors. For example, the multispecific antibody mixtures described herein are useful in treating CD47 + Tumors, mesothelin +It is useful in the treatment of tumors and combinations thereof. As a non-limiting example, the multispecific antibody mixture described herein is useful in the treatment of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, bile duct cancer, 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, for example, 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.

[0011] In some embodiments, the multispecific antibody mixtures are useful in treating, preventing, and / or slowing or alleviating the progression of symptoms of autoimmune diseases and / or inflammatory disorders by administering the antibody mixture to a subject in whom such treatment or prevention is desired. Autoimmune diseases include, for example, acquired immune deficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac sprue-dermatitis herpetiformis; chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, and the like. pemphigold), cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia anemia), polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS), systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0012] Inflammatory disorders include, for example, chronic inflammatory disorders and acute inflammatory disorders.Examples of inflammatory disorders include Alzheimer's disease, asthma, chronic obstructive pulmonary disease, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy and ventilator-induced lung injury.

[0013] In some embodiments, multispecific antibody mixtures are useful in retargeting T cells.

[0014] Pharmaceutical compositions of the invention may comprise a multispecific antibody mixture according to the invention and a carrier. These pharmaceutical compositions may be included in kits, for example therapeutic and / or diagnostic kits. [Brief explanation of the drawings]

[0015] [Figure 1] Figures 1A, 1B, and 1C are a series of schematic diagrams of various approaches to generating recombinant antibody mixtures. Figure 1A shows a method for mixing independently expressed and purified monoclonal antibodies (mAbs) to produce a mixture of mAbs. Figure 1B shows a method for mixing cell lines, also called a polyclonal cell bank, to produce a mixture of mAbs only in a single fermentation reactor. Figure 1C shows a method for co-expressing multiple antibody chains in a single cell line to produce a mixture of mAbs and bispecific antibodies (BiAbs).

[0016] [Figure 2]Figures 2A, 2B, and 2C are a series of schematic diagrams of methods for co-expressing multiple light chains with a single common heavy chain and the resulting antibody mixtures produced. Figure 2A shows how co-expression of two antibody light chains with a single heavy chain results in the secretion of three different antibodies (two mAbs, each incorporating the same light chain in each Fab, and a single BiAb, each incorporating a different light chain in each Fab). Figure 2B shows how co-expression of three antibody light chains with a single heavy chain results in the secretion of six different antibodies (three mAbs, each incorporating the same light chain in each Fab, and three BiAbs, each incorporating a different light chain in each Fab). Figure 2C shows how co-expression of four antibody light chains with a single heavy chain results in the secretion of ten different antibodies (four mAbs, each incorporating the same light chain in each Fab, and six BiAbs, each incorporating a different light chain in each Fab).

[0017] [Figure 3] 3 is a schematic diagram of a method for co-expressing two lambda light chains, one kappa light chain, and a single common heavy chain in a single cell, resulting in the secretion of a mixture of two IgGλλ mAbs, one IgGκκ mAb, two IgGκλ BiAbs, and one IgGλλ BiAb (Mixture 1). Different affinity chromatography steps can be used to isolate different submixtures from Mixture 1, as shown.

[0018] [Figure 4] 4 is a schematic diagram of a method for co-expressing two lambda light chains, two kappa light chains, and a single common heavy chain in a single cell, resulting in the secretion of two IgGλλ mAbs, two IgGκκ mAbs, four IgGκλ BiAbs, one IgGλλ BiAb, and one IgGκκ BiAb (Mixture 1). Different affinity chromatography steps can be used to isolate different submixtures from Mixture 1, as shown.

[0019] [Figure 5]5 is a schematic diagram of a method for co-expressing three lambda light chains, one kappa light chain, and a single common heavy chain in a single cell, resulting in the secretion of a mixture of three IgGλλ mAbs, one IgGκκ mAb, three IgGκλ BiAbs, three IgGλλ BiAbs, and one IgGκκ BiAb (Mixture 1). Different affinity chromatography steps can be used to isolate different submixtures from Mixture 1, as shown.

[0020] [Figure 6] FIG. 6 is a general schematic diagram of the method of the present disclosure.

[0021] [Figure 7A] FIG. 7A is a schematic diagram of two bispecific antibodies targeting two epitopes on CD47 and a tumor-associated antigen (TAA).

[0022] [Figure 7B] Figure 7B is a schematic diagram of a method for purifying a subset containing two BiAbs (CD47xTAA epitope 1 and CD47xTAA epitope 2) by two-step affinity chromatography using a kappa-specific affinity medium followed by a lambda-specific affinity medium (or vice versa).

[0023] [Figure 8A] FIG. 8A is a schematic diagram of a first bispecific antibody targeting CD47 and a first tumor-associated antigen (TAA1), and a second bispecific antibody targeting CD47 and a second tumor-associated antigen (TAA2).

[0024] [Figure 8B] Figure 8B is a schematic diagram of a method for purifying a subset containing two BiAbs (CD47xTAA1 and CD47xTAA2) by two-step affinity chromatography using a kappa-specific affinity medium followed by a lambda-specific affinity medium (or vice versa).

[0025] [Figure 9A] FIG. 9A is a schematic diagram of a bispecific antibody targeting multiple tumor-associated antigens (TAA1, TAA2 below) and / or multiple epitopes on a TAA.

[0026] [Figure 9B] Figure 9B is a schematic diagram of the method for purifying a subset containing four different BiAbs (CD47xTAA1; CD47xTAA2; PD-L1xTAA1; PD-L1xTAA2) by two-step affinity chromatography using a kappa-specific affinity medium followed by a lambda-specific affinity medium (or vice versa).

[0027] [Figure 10A] FIG. 10A is a schematic diagram of a bispecific antibody for retargeting T cells to multiple tumor-associated antigens (TAA1, TAA2 below) and / or multiple epitopes on a TAA.

[0028] [Figure 10B] Figure 10B is a schematic diagram of a method for purifying a subset containing two different BiAbs (CD3xTAA1; CD3xTAA2) by two-step affinity chromatography using a kappa-specific affinity medium followed by a lambda-specific affinity medium (or vice versa).

[0029] [Figure 11A] FIG. 11A is a schematic diagram of a mixture of mAbs targeting a first TAA in combination with BiAbs targeting a first and second TAA for which bivalent targeting is to be avoided.

[0030] [Figure 11B]Figure 11B is a schematic diagram of a method for purifying the subset containing BiAb (TAA1xTAA2) and anti-TAA1 mAb by two-step affinity chromatography using kappa-specific affinity media followed by lambda-specific affinity media (or vice versa).

[0031] [Figure 12] Figures 12A, 12B, and 12C show a series of graphs depicting the levels of antibody-dependent cellular phagocytosis (ADCP), expressed as the phagocytic index, observed in pancreatic adenocarcinoma HPAC cell lines in the presence of increasing concentrations of various bispecific antibodies and / or monoclonal antibodies and bispecific antibody combinations.

[0032] [Figure 13] 13A, 13B, and 13C show a series of graphs depicting the levels of ADCP, expressed as the phagocytic index, observed in the ovarian adenocarcinoma CaOV cell line in the presence of increasing concentrations of various bispecific antibodies and / or monoclonal antibodies and bispecific antibody combinations.

[0033] [Figure 14] 14A, 14B, and 14C show a series of graphs depicting the levels of ADCP, expressed as phagocytic index, observed in the gastric cancer NCI-N87 cell line in the presence of increasing concentrations of various bispecific antibodies and / or monoclonal antibodies and bispecific antibody combinations.

[0034] [Figure 15] FIG. 15 is a graph showing the levels of ADCP, expressed as phagocytic index, observed in the gastric cancer NCI-N87 cell line in the presence of increasing concentrations of various bispecific antibodies and combinations using different ratios of bispecific antibodies.

[0035] [Figure 16A]Figure 16A is a photograph of an isoelectric focusing gel of a sample of an IgG mixture (lane 1) obtained by co-expression of one heavy chain and three different light chains and purified from the supernatant by protein A chromatography. A K2O25O30 submixture composed of two bispecific antibodies was loaded in lane 2.

[0036] [Figure 16B] Figure 16B is a chromatogram obtained after hydrophobic interaction chromatography of the K2O25O30 submixture. Elution times and peak characteristics are shown in the inset table.

[0037] [Figure 17] FIG. 17 is a graph showing the levels of ADCP, expressed as phagocytic index, observed in the gastric cancer NCI-N87 cell line in the presence of increasing concentrations of K2O25O30 submixtures and an equimolar combination of K2O25 and K2O30.

[0038] [Figure 18] Figure 18A shows the antitumor activity of two CD47xMSLNκλ bodies (K2O38 and K2O41) and their combination (K2O38 + K2O41) in an MSLN-transfected HepG2 mouse model of liver cancer. Figure 18B shows the area under the curve and statistical analysis. Figure 18C is a graph showing the tumor growth inhibition observed in different treatment groups. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description The multispecific antibody mixtures and methods provided herein overcome the limitations of other antibody formats and / or antibody mixtures. Currently, most approved monoclonal antibody formats are unmodified antibodies of the IgG1 isotype. However, targeting a single antigen is not always sufficient to achieve the desired therapeutic effect (Fischer Expert Opin. Drug Discov. 2008 3(8):833-839). To increase efficiency, two mAbs are used in combination. This strategy has been pursued clinically, for example, for antibodies targeting immune checkpoint molecules, such as anti-CTLA4 antibodies and anti-PD-1 antibodies (Larkin et al., N Engl J Med 2015;373:23-34; Harris et al., Cancer Biol Med. 2016 13(2):171-93). However, the development of mAb combinations involves significant costs and development obstacles. In particular, two separate manufacturing processes must be implemented, leading to a significant increase in costs (Rasmussen et al., Archives of Biochemistry and Biophysics 2012 526:39-145). These issues become even more important when considering targeting three or more proteins or antigens.

[0040] Bispecific antibodies (BiAbs) have been developed as an alternative approach to achieving multispecific targeting, with over 60 formats described to date (Spiess et al., Mol Immunol. 2015 67:95-106; Brinkmann and Kontermann mAbs 2017 9:182-212). Because two binding sites are incorporated into the same molecule, BiAbs enable unique modes of action not supported by mAbs or mAb combinations (Fischer and Leger Pathobiology. 2007 74(1):3-14). Examples of such unique modes of action include retargeting of T cells or NK cells to tumor cells, increased transport across the blood-brain barrier for BiAb delivery to the central nervous system, coagulation factor VIII mimetic activity, and selective targeting of receptors expressed on multiple cell types.

[0041] Another approach to achieve the targeting of two or more proteins is to produce antibody mixtures or recombinant polyclonal mixtures.In contrast to the combination of two mAbs, in which each mAb is produced separately, the antibodies in the mixture are produced together as a mixture.Various approaches for producing mixtures have been described (see, for example, Raju and Strohl Expert Opin.Biol.Ther.2013 13(10):1347-1352; Wang et al., 2013 Current Opinion in Chemical Engineering 2013 2:1-11).A common challenge in the production of recombinant polyclonal mixtures is to achieve batch-to-batch consistency, so that each component of the mixture remains constant, thereby ensuring that the overall composition and biological activity of the mixture are consistent.

[0042] One approach involves mixing different stable cell lines, each expressing a single monoclonal antibody, to create a polyclonal cell bank used in a single bioreactor for production. In this case, different antibodies are secreted into the culture medium by different cell lines, and all antibodies are purified together to obtain the final recombinant antibody mixture (Rasmussen et al., Archives of Biochemistry and Biophysics 2012 526:39-145). To ensure batch-to-batch consistency of the recombinant polyclonal mixture, the reproducible growth and productivity characteristics of each individual cell line during fermentation must be highly controlled. Faster growth or increased productivity of one cell line has a direct impact on the composition of the mixture. Achieving this level of control is challenging and represents a major obstacle to this approach. Nevertheless, highly complex mixtures of up to 25 independent antibodies have been developed using polyclonal cell lines and evaluated in clinical trials (Hjelmstroem et al., Blood 2008 112:1987).

[0043] Another strategy is to coexpress multiple antibody heavy and / or light chains in a single cell. In this case, different heavy and light chains can pair to generate a mixture. The complexity of the resulting mixture depends on the number of different chains coexpressed. With this approach, it is important to ensure that the different possible pairings reconstitute functional antigen-binding sites to avoid producing non-functional molecules. This pairing problem can be solved by using a common heavy or light chain or by engineering protein interfaces to preferentially form the desired pairings (see, e.g., Fischer et al., Nat.Comms 20156:6113 doi:10.1038 / ncomms7113). In contrast to previous approaches that rely on the mixing of independent cell lines, this approach leads to the generation of both mAbs and BiAbs. A major advantage is that, since it is similar to the standard mAb process, fermentation and production are simplified once cell lines expressing different antibody chains in a stable manner are identified. However, if all pairings were possible, the complexity of the mixture components (mAbs and BiAbs) could become very large, so antibodies that rely on a common heavy or light chain are preferred.

[0044] The different approaches to generating recombinant antibody mixtures, their advantages and limitations are listed in Table 1 and illustrated in Figures 1A-1C. [Table 1]

[0045] Using some of the above approaches, several mixtures have been developed and have reached the clinical stage, demonstrating the interesting and feasible nature of this therapeutic modality: mixtures allow for unique modes of action that cannot be achieved with a single antibody.

[0046] For example, targeting receptors in the HER / ErbB family is being actively investigated using antibody mixtures. This family of receptor tyrosine kinases includes the growth factor receptors EGFR / ErbB1, HER-2 / ErbB2, HER-3 / ErbB3, and HER-4 / ErbB4. Several monoclonal antibodies targeting EGFR and HER2 have been approved for clinical use. Studies have shown that mixtures of antibodies targeting multiple epitopes, which can simultaneously engage these targets, can lead to increased inhibition of cancer cell growth in vitro and in vivo. This has led to the development of several antibody mixtures directed against members of the HER / ErbB family. One example is Sym004, a mixture of two anti-EGFR mAbs that showed significantly superior activity in preclinical models compared to the approved anti-EGFR mAb cetuximab (Koefoed et al., mAbs 2011 3:6, 584-595). MM-151 is another mixture composed of three human mAbs targeting different, non-overlapping epitopes on EGFR (Arena et al., Science Translational Medicine 2016 8(324),324ra14). This mixture provides more effective receptor blockade and limits the emergence of resistance when compared with single mAb therapy. A more complex mixture, Sym013, composed of six mAbs targeting EGFR, HER2, and HER3 (two mAbs directed against non-overlapping epitopes on each target), has also reached clinical development. The superior overall activity observed with mixtures against this receptor family may be explained by several factors, including more complete halting of this partially redundant signaling pathway, increased internalization and degradation via antibody-mediated receptor cross-linking, and increased Fc-mediated cell killing.

[0047] Another therapeutic area in which antibody mixtures have shown remarkable advantages is the treatment of infectious diseases (Oleksiewicz et al., Archives of Biochemistry and Biophysics 2012 526:124-131; Pohl et al., Infection and Immunity 2013 81(6):1880-1888). For example, multi-epitope targeting of soluble toxins such as botulinum toxin A not only neutralizes the toxin but also increases its clearance from the circulation. Overall, mAb approaches have not proven highly effective in treating infectious diseases, in contrast to other therapeutic areas. This may reflect the need for polyclonal antibody responses, similar to those of the innate immune system, for effective protection against complex organisms such as pathogens.

[0048] Roslolimus purpura (Roslolimus purpura) is a striking example of how complex antibody mixtures can be. This mixture of 25 mAbs against the rhesus D antigen was produced using a polyclonal cell bank approach, illustrated in Figure 1B, and developed for the treatment of immune thrombocytopenic purpura (ITP). The advantage of this complex mixture is that it provides effective coverage of the large number of rhesus D variants present in the population (Robak et al., Blood. 2012 120(18):3670-76).

[0049] These examples highlight the mechanistic advantages that cocktails can offer compared to single mAb therapy, including, but not limited to, increased receptor internalization and degradation, rapid and superior clearance of soluble targets, increased Fc-dependent effector function, synergistic targeting of multiple epitopes on a single target or multiple targets and pathways, better coverage of target variants, and prevention of escape mutations.

[0050] The present invention provides a means to generate defined antibody mixtures, either i) bispecific antibodies (BiAbs) only, or ii) monoclonal antibodies (mAbs) and BiAbs. The method relies on the coexpression of a single antibody heavy chain common to all Fv regions of the antibodies in the mixture with several light chains of either the kappa or lambda family. This coexpression and random incorporation into IgG molecules results in the secretion from a single cell of a mixture of monospecific mAbs or BiAbs, all containing the same heavy chain. The complete mixture can then be purified using, for example, an affinity reagent that binds to the Fc region of IgG, such as Protein A, as previously described in U.S. Patent Application Publication No. 20140179547. The present invention improves on previous methods by allowing for the selective purification of different subsets of the secreted mixture. In particular, it allows for the simple, cost-effective generation of mixtures of BiAbs. This opens the possibility of using mixtures of BiAbs for modes of action not possible with a single mAb or mAb mixture. The present invention combines the benefits of using a single production cell line with the ability to control the composition of the final purified mixture, maximizing the desired biological activity.

[0051] Depending on the number of light chains co-expressed with a common heavy chain, various types of antibody mixtures can be generated. This process can be generalized as follows: where n = the number of different light chains expressed with the common heavy chain; Number of different mAbs = n Number of different BiAbs = (n 2 -n) / 2 Total number of different antibodies = n + (n 2 -n) / 2

[0052] Table 2 lists these numbers for co-expression of 2 to 10 different light chains, with three examples detailed further below: When two antibody light chains are coexpressed with a single heavy chain, three different antibodies are secreted: two mAbs, each incorporating the same light chain in each Fab, and a single BiAb, incorporating a different light chain in each Fab (see Figure 2A). However, the abundance of the three forms depends on the relative expression and assembly of the two light chains. If expression and assembly are equivalent, the theoretical distribution is 25% for each mAb and 50% for the BiAb. When two or three antibody light chains are co-expressed with a single heavy chain, six different antibodies are secreted: three mAbs, each incorporating the same light chain in each Fab, and three BiAbs, each incorporating a different light chain in each Fab (see Figure 2B). If expression and assembly are comparable, the theoretical distribution is 11.1% for each mAb and 22.2% for each BiAb. When three or four antibody light chains are co-expressed with a single heavy chain, 10 different antibodies are secreted: four mAbs, each incorporating the same light chain in each Fab, and six BiAbs, each incorporating a different light chain in each Fab (see Figure 2C). If expression and assembly are comparable, the theoretical distribution is 6.25% for each mAb and 12.5% ​​for each BiAb. [Table 2]

[0053] The distribution of mAbs and BiAbs containing kappa or lambda light chains varies depending on whether the different co-expressed light chains are of the kappa or lambda type, further diversifying the types of mixtures that can be produced by this approach. For example, when two lambda light chains and one kappa light chain are co-expressed with a common heavy chain, two IgGλλ mAbs, one IgGκκ mAb, two IgGκλ BiAbs, and one IgGλλ BiAb are produced, as shown in Figure 3. When four light chains are co-expressed, the molecular distribution varies when two kappa and two lambda light chains are used (Figure 4) or when three lambda and one kappa light chain are used (Figure 5).

[0054] The resulting number of antibody forms generated in a given multispecific mixture can be generalized as follows: n = number of kappa light chains m = number of lambda light chains If Number of IgGκκ mAbs = n Number of IgGλλ mAbs = m Number of IgGκλ BiAbs = n × m Number of IgGκκ BiAbs = (n 2 -n) / 2 Number of IgGλ BiAb = (m 2 -m) / 2

[0055] If the expression and assembly of the different light chains were identical, the theoretical distribution of each resulting form can be generalized as follows: n = number of kappa light chains m = number of lambda light chains If Percentage of IgGκκ mAb = n / (n × m) Percentage of IgGλ mAb = m / (n × m) Percentage of IgGκλ BiAb = (n × m) × 2 / (n × m) Percentage of IgGκκ BiAb = (n 2 -n) / (n×m) Percentage of IgGλλ BiAb = (m 2 -m) / (n×m)

[0056] As described above, the present invention provides a means for purifying clearly defined subsets of either i) BiAbs only or ii) mAbs and BiAbs from the diverse mixtures that can be generated by co-expression of various numbers of kappa and / or lambda light chains. The present invention relies on multi-step affinity chromatography using a resin that specifically binds to the Fc portion and a resin that specifically binds to the kappa or lambda constant region. In this way, subsets of antibody forms comprising these complex mixtures can be easily isolated, and their composition can be adjusted depending on the type of resin used and the desired mode of action.

[0057] For example, coexpression of two lambda and one kappa light chain with a common heavy chain generates a mixture composed of two IgGλλ mAbs, one IgGκκ mAb, two IgGκλ BiAbs, and one IgGλλ BiAb (Figure 3). All of these antibody forms contain Fc portions and can be easily and efficiently purified using, for example, Protein A chromatography. From this mixture (Mixture 1 shown in Figure 3), distinct, defined subsets of antibodies can be easily isolated (see Submixtures 2 and 3 in Figure 3, respectively) using affinity chromatography media that specifically bind either kappa or lambda chains, thereby allowing capture of all antibody forms containing at least one kappa or lambda chain, respectively. Submixture 2 contains one IgGκκ mAb and two IgGκλ BiAbs. Submixture 3 contains two IgGλλ mAbs. By two successive affinity chromatography steps using media that specifically bind kappa and lambda chains, a submixture containing two IgGκλ BiAbs (but no mAb) can be isolated (submixture 4 in Figure 3).

[0058] When four different light chains are co-expressed, the resulting mixture is more complex and depends on the proportion of co-expressed kappa and lambda light chains. Two examples are shown in Figures 4 and 5, where two lambda and two kappa light chains or three lambda and one kappa light chain are co-expressed, respectively.

[0059] Co-expression of two lambda and two kappa light chains with a common heavy chain results in two IgGλλ mAb, two IgGκκ mAbs, four IgGκλ BiAbs, one IgGλλ A mixture (Mixture 1, Figure 4) composed of two IgGκκ mAbs, four IgGκλ BiAbs, and one IgGκκ BiAb is generated. All of these antibody forms contain Fc portions and can be effectively purified using, for example, Protein A chromatography. From this initial Mixture 1, distinct, defined subsets of antibodies can be readily isolated (Submixtures 2 and 3, Figure 4, respectively) using affinity chromatography media that specifically bind either kappa or lambda chains, thereby allowing capture of all antibody forms containing at least one kappa or lambda chain, as described above. Submixture 2 contains two IgGκκ mAbs, four IgGκλ BiAbs, and one IgGκκ BiAb. Submixture 3 contains two IgGλλ mAbs, four IgGκλ BiAbs, and one IgGλλ BiAb. Finally, by two successive affinity chromatography steps using media that specifically bind kappa and lambda chains, a mixture containing the four IgGκλ BiAbs (no mAbs, and no BiAbs containing only lambda or only kappa chains) can be isolated (submixture 4 in Figure 4).

[0060] In the situation where three lambda light chains and one kappa light chain are coexpressed with a common heavy chain, a mixture composed of three IgGλλ mAbs, one IgGκκ mAb, three IgGκλ BiAbs, three IgGλλ BiAbs, and one IgGκκ BiAb (Mixture 1, Figure 5) is generated. All of these antibody forms contain Fc portions and can be effectively purified using, for example, Protein A chromatography. As described above, different, defined subsets of antibodies can be easily isolated from this initial Mixture 1 (Figure 5) using affinity chromatography media (e.g., CaptureSelect Fab Kappa and CaptureSelect Fab Lambda Affinity Matrices (GE Healthcare)) that specifically bind to either the kappa or lambda light chain constant domains, thereby enabling capture of all antibody forms containing at least one kappa or lambda chain, respectively (Submixtures 2 and 3 in Figure 5, respectively). Submixture 2 contains one IgGκκ mAb and three IgGκλ BiAbs. Submixture 3 contains three IgGλλ mAbs, three IgGκλ BiAbs, and three IgGλλ BiAbs. Finally, by two successive affinity chromatography steps using media that specifically bind kappa and lambda chains, a submixture containing three IgGκλ BiAbs (no mAbs, and no BiAbs containing only lambda or kappa chains) can be isolated (Submixture 4 in Figure 5).

[0061] The above two situations highlight that although in each case four light chains are co-expressed, the nature of the chains expressed (i.e., two kappa and two lambda light chains or three lambda and one kappa light chain) leads to the generation and purification of very different mixture subsets that can be easily and effectively isolated by applying the methods of the present invention. These situations represent only selected examples and do not limit the application of the present invention to other situations.

[0062] It will be apparent that the present invention can generally be used to isolate subsets of mAbs and / or BiAbs from complex mixtures based on their respective content of kappa and lambda light chains. In either situation, affinity chromatography media that specifically bind the constant or variable domains of either kappa or lambda antibody light chains can be used to purify the three types of mixture subsets. 1. All antibody molecules containing at least one kappa light chain, including both mAbs and BiAbs 2. All antibody molecules containing at least one lambda light chain, including both mAbs and BiAbs 3. All BiAb molecules containing one kappa and one lambda light chain

[0063] The present invention can also be further applied to purify antibody subsets of antibody mixtures that contain hybrid light chains, as described in U.S. Patent Application Publication No. 20140179547. These hybrid chains are 1. A variable kappa domain fused to a constant lambda domain, or 2. Variable lambda domain fused to a constant kappa domain It consists of either:

[0064] Similar to full-length lambda or kappa light chains, these hybrid molecules Kappa constant domain (e.g., CaptureSelect™ LC-kappa (Hu) affinity matrix (Life Technologies, Zug, Switzerland) Some kappa variable domains (e.g., Protein L) Lambda constant domain (e.g., CaptureSelect™ LC-Lambda (Hu) Affinity Matrix (Life Technologies, Zug, Switzerland) By using a chromatographic medium that binds to either the variable or constant domains, they can be selectively separated according to their variable and constant domain composition.

[0065] A typical application of the present invention is illustrated in FIG.

[0066] The multispecific antibody mixtures of the present disclosure are useful in a variety of indications, and subsets of the antibody mixtures may have multiple applications for the development of therapeutic modalities that rely on modes of action not possible with mAbs, mAb mixtures, or BiAbs.

[0067] In particular, the present invention allows for the simple generation of fully human BiAb mixtures using a single cell line, which represents a major advantage. Indeed, as mentioned above, the combination of two individual mAbs leads to a significant increase in costs. This limitation is obviously even more important when two individual BiAbs must be combined, since the production of BiAbs is more complex and costly than standard mAbs. Examples of potentially interesting mixtures of BiAbs and how these mixtures can be generated using the present invention are described in more detail below.

[0068] Multi-epitope targeting of tumor-associated antigens (TAA) combined with CD47 blockade: CD47 is a ubiquitously expressed receptor that acts as a checkpoint in the innate immune system, suppressing phagocytosis through its interaction with SIRPα (e.g., Oldenborg, PA, CD47: A Cell Surface Glycoprotein (See, e.g., "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 multifaceted actor in the tumor microenvironment of potential therapeutic interest," Br J Pharmacol. 2012 Dec;167(7):1415-30). Blockade of CD47 with a BiAb approach avoids the toxicity observed with mAbs directed against CD47. CD47xTAA BiAbs allow for limited inhibition of CD47 only on TAA-expressing cells, thereby avoiding toxicity and poor pharmacokinetic properties. A mixture of two BiAbs targeting two epitopes on a single receptor, each with an anti-TAA arm and an anti-CD47 arm, should result in superior anti-CD47 blockade, increased Fc coverage, and ultimately better tumor cell killing (Figure 7A). Indeed, in this situation, for each TAA molecule, two CD47 receptors can be blocked, and two Fc receptors are present on the target cell surface, enhancing effector cell recruitment via Fc-gamma receptor interactions.

[0069] Co-expression of a common heavy chain with: A first lambda light chain that drives specificity for a first epitope on the TAA A second lambda light chain that promotes specificity for a second epitope on the TAA Kappa light chains drive specificity for CD47

[0070] From the resulting mixture, a subset containing two BiAbs (CD47xTAA epitope 1 and CD47xTAA epitope 2) could be purified by two-step affinity chromatography using kappa-specific affinity media followed by lambda-specific affinity media (or vice versa) (Figure 7B).

[0071] Multi-TAA targeting combined with CD47 blockade: The above example can be applied to two TAAs expressed by target cancer cells. A mixture of two BiAbs targeting two TAAs, each with both an anti-TAA arm and an anti-CD47 arm, should also result in superior anti-CD47 blockade, increased Fc coverage further combined with the effects associated with TAA blockade, and ultimately superior tumor cell killing (Figure 8A).

[0072] Co-expression of a common heavy chain with: A first lambda light chain that drives specificity for the first TAA A second lambda light chain to promote specificity for a second TAA Kappa light chains drive specificity for CD47

[0073] From the resulting mixture, a subset containing two BiAbs (CD47xTAA1 and CD47xTAA2) could be purified by two-step affinity chromatography using kappa-specific affinity media followed by lambda-specific affinity media (or vice versa) (Figure 8B).

[0074] Multi-TAA or epitope targeting combined with blockade of several checkpoint molecules: The approach used in the above example can be extended to target two TAAs (or two epitopes on the same TAA) expressed by the target cancer cells, combining arms that block two checkpoint receptors, e.g., CD47 and PD-L1. Such a mixture should result in CD47 blockade, PD-L1 blockade, increased Fc coverage, and ultimately superior tumor cell killing and a potentially much longer-lasting immune system response, while avoiding the toxicity associated with typical monospecific blockade of immune checkpoints (Figure 9A).

[0075] Co-expression of a common heavy chain with: A first lambda light chain that drives specificity for the first TAA A second lambda light chain to promote specificity for a second TAA Kappa light chains drive specificity for CD47 Kappa light chains drive specificity for PD-L1

[0076] From the resulting mixture, subsets containing four different BiAbs (CD47xTAA1; CD47xTAA2; PD-L1xTAA1; PD-L1xTAA2) could be purified by two-step affinity chromatography using kappa-specific affinity media followed by lambda-specific affinity media (or vice versa) (Figure 9B).

[0077] Multi-TAA Retargeting of T Cells: T cell retargeting is a clinically validated and widely pursued approach in oncology (Chames and Baty MAbs. 2009 1(6):539-47). A mixture of BiAbs would enable the retargeting of T cells to two different TAAs (or epitopes on the same TAA), potentially improving treatment efficacy (Figure 10A).

[0078] Co-expression of a common heavy chain with: A first lambda light chain that drives specificity for the first TAA A second lambda light chain to promote specificity for a second TAA Kappa light chains drive specificity for CD3

[0079] From the resulting mixture, subsets containing two different BiAbs (CD3xTAA1; CD3xTAA2) could be purified by two-step affinity chromatography using kappa-specific affinity media followed by lambda-specific affinity media (or vice versa) (Figure 10B).

[0080] The above examples are illustrative and do not limit the possible applications that can be pursued by applying the present invention. Furthermore, it is clear that any kappa chain provided in any example can be replaced with a lambda chain (and vice versa).

[0081] Another important type of application of the present invention is the generation of mAb and BiAb mixtures that exclude one or more forms of mAb. This feature is important for some targets, where monospecific bivalent engagement by mAbs can be detrimental, leading to toxicity and other undesirable effects. For example, anti-cMet antibodies are known to lead to undesirable agonist activity, which led to the development of monovalent antibodies. Using the present invention, generating mixtures containing anti-cMet antibodies but avoiding anti-c-Met mAbs is straightforward. As noted above, targeting CD47 by mAbs leads to significant toxicity in humans. Anti-CD47 mAbs can be removed from mixtures of mAbs targeting other receptors and BiAbs targeting CD47 in conjunction with another receptor. Similarly, monoclonal anti-CD3 also needs to be excluded from anti-CD3 mixtures containing CD3-binding components.

[0082] Combined monovalent and bivalent targeting of two tumor-associated antigens: for example, a mixture of mAbs targeting a first TAA combined with BiAbs targeting the first and second TAA that should avoid bivalent targeting. This strategy can be applied to TAAs such as cMet and EGFR (Figure 11A).

[0083] Co-expression of a common heavy chain with: Lambda light chains, which drive specificity for the first TAA Kappa light chains, which drive specificity for a second TAA

[0084] From the resulting mixture, a subset containing BiAb (TAA1xTAA2) and anti-TAA1 mAb could be purified by affinity chromatography using lambda-specific affinity media (FIG. 11B).

[0085] Again, other uses of subsets of antibody mixtures can be rationalized and the above examples do not limit the scope of the invention.

[0086] The same principles apply to any antibody isotype and antibody form non-human species, so long as affinity or other chromatographic reagents are available to separate different submixtures of antibodies.

[0087] Furthermore, the same principles of the present invention can be applied to F(ab')2 formats in which a single VHCH1 is co-expressed with two or more VκCκ or VλCλ, resulting in the secretion of a mixture of monospecific and bispecific F(ab')2 molecules. These can then be separated into well-defined submixtures using affinity chromatography media that bind portions of the kappa or lambda light chains, according to the methods of the present invention. Similarly, hybrid VκCλ and VλCκ can also be used when practicing the methods of the present invention. [Example]

[0088] Example 1: Selection of antibody candidates for bispecific antibody generation Four antibodies targeting different epitopes on hMSLN were selected for antibody mixture generation: an anti-hCD19 antibody containing a lambda light chain and an anti-hCD47 antibody containing a kappa light chain. All of these antibodies contain the same heavy chain as described in PCT Publication WO 2014 / 087248 and co-pending patent application U.S. Patent Application No. 62 / 511,669, entitled "Anti-CD47 x Anti-Mesothelin Antibodies and Methods of Use Thereof," filed May 26, 2017, and are suitable for generating bispecific antibodies based on the kappa body format described in U.S. Patent Application Publication No. 20140179547. The selected antibodies are listed in Table 3, and their sequences are shown below. [Table 3]

[0089] The anti-hMSLN, anti-hCD19 and anti-hCD47 antibodies in Table 3 each comprise a common heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1. [ka] [ka]

[0090] The anti-hMSLN, anti-hCD19 and anti-hCD47 antibodies in Table 3 each comprise a common variable heavy domain (SEQ ID NO:4) encoded by the nucleic acid sequence shown in SEQ ID NO:3. [ka] [ka]

[0091] The O25 antibody comprises a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence set forth in SEQ ID NO: 1, and a lambda light chain (SEQ ID NO: 6) encoded by the nucleic acid sequence set forth in SEQ ID NO: 5. In the amino acid sequence below, the variable region of the lambda light chain is in bold. [ka]

[0092] The O25 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a lambda variable light domain (SEQ ID NO: 8) encoded by the nucleic acid sequence set forth in SEQ ID NO: 7. [ka] [ka]

[0093] The O30 antibody comprises a common heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence set forth in SEQ ID NO:1, and a lambda light chain (SEQ ID NO:10) encoded by the nucleic acid sequence set forth in SEQ ID NO:9. In the amino acid sequence below, the variable region of the lambda light chain is in bold. [ka]

[0094] The O30 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a lambda variable light domain (SEQ ID NO: 12) encoded by the nucleic acid sequence set forth in SEQ ID NO: 11. [ka]

[0095] The O35 antibody comprises a common heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence set forth in SEQ ID NO:1, and a lambda light chain (SEQ ID NO:14) encoded by the nucleic acid sequence set forth in SEQ ID NO:13. In the amino acid sequence below, the variable region of the lambda light chain is in bold. [ka]

[0096] The O35 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a lambda variable light domain (SEQ ID NO: 16) encoded by the nucleic acid sequence set forth in SEQ ID NO: 15. [ka]

[0097] The O38 antibody comprises a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 and a lambda light chain (SEQ ID NO: 29) encoded by the nucleic acid sequence set forth in SEQ ID NO: 30. [ka] [ka]

[0098] The O38 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a lambda variable light domain (SEQ ID NO: 31) encoded by the nucleic acid sequence set forth in SEQ ID NO: 32. [ka]

[0099] The O41 antibody comprises a common heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence set forth in SEQ ID NO:1, and a lambda light chain (SEQ ID NO:18) encoded by the nucleic acid sequence set forth in SEQ ID NO:17. In the amino acid sequence below, the variable region of the lambda light chain is in bold. [ka] [ka]

[0100] The O41 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a lambda variable light domain (SEQ ID NO: 20) encoded by the nucleic acid sequence set forth in SEQ ID NO: 19. [ka]

[0101] The L7-2 antibody comprises a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 and a lambda light chain (SEQ ID NO: 22) encoded by the nucleic acid sequence set forth in SEQ ID NO: 21. [ka] [ka]

[0102] The C2 antibody comprises a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence set forth in SEQ ID NO: 113, and a lambda variable light domain (SEQ ID NO: 24) encoded by the nucleic acid sequence set forth in SEQ ID NO: 23. [ka]

[0103] The K2 antibody comprises a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 and a kappa light chain (SEQ ID NO: 26) encoded by the nucleic acid sequence set forth in SEQ ID NO: 25. [ka] [ka]

[0104] The K2 antibody comprises a common variable heavy domain (SEQ ID NO: 4) encoded by the nucleic acid sequence set forth in SEQ ID NO: 3, and a kappa variable light domain (SEQ ID NO: 28) encoded by the nucleic acid sequence set forth in SEQ ID NO: 27. [ka]

[0105] Example 2: Expression and purification of a bispecific antibody with lambda and kappa light chains. Co-expression of one heavy chain and two light chains in the same cell can lead to the assembly of three different antibodies. This can be achieved in different ways, such as by transfection of multiple vectors expressing one of the chains to be co-expressed, or by using multiple gene expression drive vectors. Co-expression can be achieved by using vectors similar to those described in U.S. Patent Application Publication No. 2012 / 0184716 and International Publication No. 2012 / 023053 (each of which is hereby incorporated by reference in its entirety). The vector pNoviκHλ was previously generated to allow co-expression of one heavy chain, one kappa light chain, and one lambda light chain. Expression of the three genes is driven by a human cytomegalovirus (hCMV) promoter, and the vector also contains a glutamine synthetase gene (GS), allowing for the selection and establishment of stable cell lines. For transient expression in mammalian cells, the VL genes of anti-hMSLN IgGλ, anti-hCD19 IgGλ, or anti-hCD47 IgGκ were cloned into the vector pNoviκHλ. Peak cells were amplified and cultured at 8 × 10 cells in 45 mL of culture medium containing fetal bovine serum. 6The cells were split into T175 flasks at a concentration of 1000 cells / flask. Cells were transfected with 30 μg of plasmid DNA using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. At several time points during production, antibody concentrations were measured in the serum-containing supernatants of transfected cells using biolayer interferometry (BLI) technology. An OctetRED96 instrument and Protein A biosensor were used for quantification (Pall, Basel, Switzerland). 200 μL of supernatant was used to determine IgG concentrations; the biosensor was primed and regenerated with 10 mM glycine, pH 1.7, and IgG calibrators diluted in conditioned PEAK cell medium were prepared for standard curve generation. Concentrations were determined using the dose-response 5PL-weighted Y standard curve equation and the initial slope binding rate equation. Supernatants were harvested 7–10 days after transfection according to antibody concentration and clarified by centrifugation at 1300 g for 10 minutes. The purification process consisted of three affinity steps. First, CaptureSelect™ IgG-CH1 affinity matrix (Thermo Fisher Scientific, Waltham, MA) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4°C, the supernatant was centrifuged at 1000g for 10 minutes, the flow-through was saved, and the resin was washed twice with PBS. The resin was then transferred to a spin column, and a solution containing 50 mM glycine at pH 3.0 was used for elution. Several elution fractions were generated, pooled, and desalted against 25 mM histidine / 125 mM NaCl pH 6.0 buffer using 50 kDa Amicon® ultracentrifugal filter units (Merck KGaA, Darmstadt, Germany). The final product containing total human IgG from the supernatant was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and incubated with an appropriate volume of CaptureSelect™ LC-kappa (Hu) affinity matrix (Thermo Fisher Scientific, Waltham, MA) for 30 minutes at room temperature and 20 rpm.The incubation, resin recovery, elution, and desalting steps were performed as described previously. The final affinity purification step was performed using a CaptureSelect™ LC-Lambda (Hu) affinity matrix (Thermo Fisher Scientific, Waltham, MA) using the same process as the two previous purifications. The final product was quantified using a Nanodrop. The purified bispecific antibody was analyzed by electrophoresis under denaturing and reducing conditions. An Agilent 2100 Bioanalyzer was used with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA). Four microliters of purified sample was mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, MO). The sample was heated at 95°C for 5 minutes and then loaded onto the chip. Aliquots from the first purification step (containing the bispecific antibody and both monospecific mAbs) and the final product were loaded onto an isoelectric focusing (IEF) gel to assess the purity of the final purified bispecific antibody (free of mAb contamination). Aggregation levels were determined by SEC-HPLC. Finally, binding of the bispecific antibody to both targets was assessed using OctetRED96. Briefly, biotinylated targets (hMSLN, hCD19, hCD47, and an unrelated target) were loaded onto a streptavidin biosensor. The biosensor was then immersed in a solution containing the bispecific antibody, and binding was monitored in real time. All samples were tested for endotoxin contamination using the Limulus Amebocyte Extract Test (LAL; Charles River Laboratories, Wilmington, MA). The following bispecific antibodies were expressed and purified: [Table 4]

[0106] Example 3: Activity of Individual Bispecific Antibodies Targeting hMSLN and hCD47 and Bispecific Antibody Combinations in Phagocytosis Assays Bispecific antibodies targeting proteins or antigens expressed on the surface of tumor cells that can co-engage CD47 on the same tumor cells can mediate increased phagocytic activity of macrophages by interfering with the inhibitory signal mediated by CD47 interaction with SIRPα. This principle is described in PCT Publication WO 2014 / 087248 and co-pending patent application U.S. Patent Application No. 62 / 511,669, filed May 26, 2017, entitled "Anti-CD47 x Anti-Mesothelin Antibodies and Methods of Use Thereof." Herein, we tested the hypothesis of multi-epitope targeting of a tumor-associated antigen (TAA) (in this case, hMSLN in combination with CD47 blockade), as shown in Figure 7. To evaluate the benefits of having a higher density of Fc per MSLN molecule and higher CD47 blockade, we compared the phagocytic activity mediated by either individual hCD47 / hMSLN bispecific antibodies or by pairwise combinations of bispecific antibodies. Because hCD19 was not present in the assay, the hCD47 / hCD19 bispecific antibody K2L7-2 was used as a monovalent anti-hCD47 control. Three tumor cell lines expressing different levels of hCD47 and hMSLN were tested: NCI-N87, HPAC, and Caov-3. The cell surface expression levels of CD47 and mesothelin for NCI-N87 cells were 43,000 and 27,000, respectively. The cell surface expression levels of CD47 and mesothelin for HPAC cells were 105,000 and 13,000, respectively. The levels of cell surface expression of CD47 and mesothelin in Caov-3 cells were 220,000 and 38,000, respectively.

[0107] Assays were performed using human macrophages differentiated from peripheral blood monocytes and NCI-N87, HPAC, or CaOV3 as target cells. Macrophages were co-incubated with calcein AM-labeled target cells (effector:target ratio 1:1) in the presence of increasing concentrations of bispecific or monovalent antibodies for 2.5 hours at 37°C. At the end of the incubation period, the supernatant was replaced with complete culture medium. Plates were imaged using a CX5 imaging platform, and 1,500 macrophages per condition were acquired and analyzed. Double-positive events confirmed phagocytosis, and the software calculated the phagocytic index.

[0108] These dose-response experiments show that in all three cell types, the phagocytic activity mediated by the combination of K2O30 + K2O41 was superior to that of either K2O30 or K2O41 (Figures 12A, 13A, and 14A). Similarly, in all three cell types, the phagocytic activity mediated by the combination of K2O35 + K2O41 was superior to that of either K2O35 or K2O41 (Figures 12B, 13B, and 14B). Additionally, in NCI-N87 and HPAC cells, the phagocytic activity mediated by the combination of K2O38 + K2O41 was superior to that of either K2O38 or K2O41 (Figures 12C and 14C). In NCI-N87 cells, the combination of K2O30 + K2O25 was superior to that of either K2O30 or K2O25 (Figure 15). In all cases, a negative control IgG (designated NI-0801, described in PCT Publication WO 2008 / 106200) or a monovalent anti-hCD47 control induced minimal or no phagocytic activity. In all these examples, the two bispecific antibodies were added in equal amounts (i.e., a 50:50 ratio). To assess whether slight variations in this equimolar ratio would affect activity, K2O30 and K2O25 were mixed and tested at ratios of 40:60 and 60:40. All ratios yielded similar results, indicating that slight variations in the ratio between the two bispecific antibodies do not significantly affect the increased phagocytic activity of the combination (Figure 15A).

[0109] Example 4: Expression of four antibody chains from a single vector The vector pNoviκHλ was modified to enable the expression of additional light chains. The new vector, pNoviH3L, contains four promoters driving the expression of one heavy chain and three light chains. The K2 anti-CD47 kappa light chain and two anti-hMSLN lambda light chains, O30 and O25, were cloned into this single vector. All coding sequences and cloning junctions were verified by sequencing. As described in Example 2, this vector was used in transient transfection to verify its functionality (i.e., that bispecific antibodies could be produced). The vector was then linearized for electroporation into Chinese hamster ovary (CHO) cells.

[0110] Example 5: Expression of antibody mixtures containing bispecific and monospecific antibodies. In the study presented herein, stable CHO lines were transfected and grown using a chemically defined, animal-component-free (CDACF) manufacturing process. After transfection by electroporation and selection with MSX, screening by FACS was performed. The highest-producing pool was selected for production under fed-batch conditions. Total IgG productivity was assessed for different pools using Octet technology. Affinity purification was performed as described in Example 2. After the Protein A chromatography step, the material, which contained all IgG forms, was analyzed for polypeptide content by isoelectric focusing gel (IEF) using an Agilent bioanalyzer, and aggregation levels were determined by SEC-HPLC. Finally, binding of the bispecific antibody to both targets was assessed using OctetRED96. Briefly, biotinylated targets (hMSLN, hCD47, and an unrelated target) were loaded onto a streptavidin biosensor. The biosensor was then immersed in a solution containing the bispecific antibody, and binding was monitored in real time. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate Test (LAL; Charles River Laboratories, Wilmington, MA).

[0111] Example 6: Purification of a submixture containing two bispecifics The mixture of all IgG forms (bispecific and monospecific IgG) expressed and purified in Example 5 was further subjected to two steps of chromatography using media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (GE Healthcare). As shown in Figures 6 and 7B, these two steps allowed for the recovery of all IgG forms containing both kappa and lambda light chains. The total IgG was then applied to a column containing CaptureSelect Fab Kappa affinity matrix (GE Healthcare) equilibrated with 10 volumes of PBS. The column was then washed with 5–10 column volumes of PBS. All immunoglobulin molecules bearing kappa light chains were eluted from the column by applying 5 column volumes of 0.1 M glycine pH 3.0, and fractions were collected. The antibody-containing fractions were pooled before buffer exchange on an Amicon buffer exchange column (Merck) equilibrated with PBS. The antibodies were then applied to a second column containing CaptureSelect Fab Lambda Affinity Matrix equilibrated with 10 volumes of PBS. The column was then washed with 5–10 column volumes of PBS. All immunoglobulin molecules bearing only kappa light chains bound to the column and were found in the flow-through. Antibodies bearing lambda light chains were eluted from the column by applying 5 column volumes of 0.1 M glycine pH 3.0, and fractions were collected. Fractions containing the bispecific antibodies were pooled before buffer exchange on an Amicon buffer exchange column (Merck) equilibrated with PBS. Analysis of the flow-through and elution fractions from each purification step by SDS-PAGE showed that antibodies bearing lambda light chains were found in the flow-through of the CaptureSelect Fab Kappa Affinity Matrix, and conversely, antibodies bearing kappa light chains were found in the flow-through of the CaptureSelect Fab Lambda Affinity Matrix.

[0112] The mixture of all IgG forms obtained after the Protein A step and the K2O25O30 submixture containing the two bispecific antibody forms purified as described above were analyzed by IEF (Figure 16A). As expected, six bands corresponding to the different mono- and bispecific antibody forms were visible for the mixture obtained after Protein A. After three affinity purification steps, two bands corresponding to the bispecific form containing kappa and lambda light chains were visible. The purified K2O25O30 submixture was further analyzed by hydrophobic interaction chromatography (HIC) to determine the relative content of the two bispecific antibody forms. Integration of the two distinct peaks revealed that K2O25O30 contained 69% K2O25 and 31% K2O30 (Figure 16B).

[0113] Example 7: Activity in phagocytosis assays of submixtures containing two bispecific antibodies targeting hMSLN and hCD47, as well as combinations of the same two bispecific antibodies The activity of the K2O25O30 submixture isolated in Example 6 was then compared to an equimolar combination of K2O25 and K2O30 in an in vitro phagocytosis assay using NCI-N87 cells as described in Example 3. The activity of K2O25O30 and the equimolar combination of K2O25 and K2O30 was identical (Figure 17), indicating that the submixture reproduces the increased activity of the combination compared to the activity of the individual bispecific antibodies as described in Example 3.

[0114] Example 8: In vivo antitumor activity of bispecific antibody combinations The antitumor activity of two CD47xMSLN κλ bodies (K2O38 and K2O41) and one combination of CD47xMSLN κλ bodies (K2O38 + K2O41) was evaluated in an MSLN-transfected HepG2 model of liver cancer. 6 HepG2-MSLN cells were subcutaneously implanted into NOD / SCID mice (following formula: (length x width) 2Tumor volumes were measured 2-3 times a week using a CT scan. 3 Treatment began once tumor volume reached 100%. Mice were randomized into eight groups (6–7 mice per group). In this experiment, the effects of the CD47xMSLN κλ body combination were compared with those of the CD47xMSLN κλ body alone and the MSLN MAb amatuximab. Antibodies were injected intravenously once weekly until the end of the experiment (d28). All antibodies were administered at 60 mg / kg / injection. Tumor volume measurements were used to calculate the area under the curve (AUC) for each individual mouse. For statistical analysis, one-way ANOVA was performed using GraphPad Prism, followed by a Tukey multiple comparison test. p<0.05 was considered statistically significant. The tumor growth inhibition (TGI) percentage was also determined based on tumor volume compared to the isotype control group using the formula: %TGI={1-[(Tt-T0) / (Vt-V0)]}×100; Tt=median treated tumor volume at time t; T0=median treated tumor volume at time 0; Vt=median control tumor volume at time t and V0=median control tumor volume at time 0.

[0115] As shown in Figure 18, treatment with two CD47xMSLN κλ-bodies significantly reduced tumor growth compared to the hIgG1 control, with TGI of 21% for K2O41 and 46% for K2O38. The combination showed superior efficacy against tumor growth compared to the single approaches, with TGI of 80% for K2O41 + K2O38. Finally, both the single CD47xMSLN κλ-bodies and their combination showed superior antitumor efficacy compared to amatuximab (a neutralizing mAb that recognizes MSLN), which is currently in clinical investigation. Other embodiments

[0116] While the present invention has been described in conjunction with its detailed description, the foregoing description is illustrative and not limiting of 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. The present invention provides, for example, the following items. (Item 1) A subset of antibodies isolated from an antibody mixture composed of two or more monospecific antibodies and one or more bispecific antibodies, wherein all antibodies in said subset comprise a common heavy chain. (Item 2) The subset of item 1, all consisting of antibodies that comprise at least a portion of a kappa light chain. (Item 3) 2. The subset of item 1, all consisting of antibodies that comprise at least a portion of a lambda light chain. (Item 4) The subset of item 1, all consisting of antibodies that comprise at least a portion of a kappa light chain and at least a portion of a lambda light chain. (Item 5) 3. A method for isolating the subset according to item 2, comprising purifying the subset from an antibody mixture using at least one affinity chromatography step. (Item 6) 6. The method according to item 5, wherein the purification step is carried out using a kappa constant or variable domain-specific affinity chromatography medium. (Item 7) 4. A method for isolating the subset according to item 3, comprising purifying the subset from an antibody mixture using at least one affinity chromatography step. (Item 8) 8. The method according to item 7, wherein the purification step is carried out using a lambda constant specific affinity chromatography medium. (Item 9) 5. A method for isolating the subset according to item 4, comprising purifying the subset from an antibody mixture using at least a first affinity chromatography step and a second affinity chromatography step. (Item 10) 10. The method of claim 9, wherein the first purification step is carried out using a kappa constant or variable domain-specific affinity chromatography medium and the second purification step is carried out using a lambda constant or variable domain-specific affinity chromatography medium. (Item 11) 10. The method of claim 9, wherein the first purification step is carried out using a lambda constant domain-specific affinity chromatography medium and the second purification step is carried out using a kappa constant or variable domain-specific affinity chromatography medium. (Item 12) 1. A method for isolating a subset of antibodies isolated from an antibody mixture composed of three or more antibodies, wherein at least two antibodies in the mixture are non-identical antibodies; The method comprises: i) producing a mixture of antibodies by expressing in a single recombinant host cell one or more nucleic acid sequences encoding a common immunoglobulin heavy chain and at least a first immunoglobulin light chain and a second immunoglobulin light chain, wherein said first and second immunoglobulin light chains are non-identical and each of said first and second immunoglobulin light chains is capable of pairing with said common immunoglobulin heavy chain to form a functional antigen-binding domain, thereby producing three or more antibodies comprising said common heavy chain; and ii) isolating a subset of antibodies using at least one affinity chromatography step. A method comprising: (Item 13) 13. The method of claim 12, wherein the first immunoglobulin light chain comprises at least a portion of a kappa light chain. (Item 14) 13. The subset of item 12, wherein the second immunoglobulin light chain comprises at least a portion of a lambda light chain. (Item 15) 13. The subset of item 12, wherein the first immunoglobulin light chain comprises at least a portion of a kappa light chain and the second immunoglobulin light chain comprises at least a portion of a lambda light chain. (Item 16) 14. The method of any one of items 12 or 13, wherein the isolating step is carried out using a kappa stationary specific affinity chromatography medium. (Item 17) 15. The method of any one of items 12 or 14, wherein the isolating step is carried out using a lambda constant-specific affinity chromatography medium. (Item 18) Item 16. The method according to item 15, wherein the isolation step comprises at least a first affinity chromatography step and a second affinity chromatography step. (Item 19) The first purification step is carried out using a kappa constant or variable domain specific affinity chromatography medium, and the second purification step is carried out using a lambda constant or variable domain specific affinity chromatography medium. Item 19. The method according to item 18, wherein the second purification step is carried out using a medium. (Item 20) 20. The method of claim 18, wherein the first purification step is carried out using a lambda constant domain-specific affinity chromatography medium and the second purification step is carried out using a kappa constant or variable domain-specific affinity chromatography medium. (Item 21) 13. The method of claim 12, further comprising recovering the three or more non-identical antibodies from the recombinant host cells or from a culture of the host cells. (Item 22) 13. The method of claim 12, wherein at least two of the three or more antibodies target different epitopes of the same target antigen. (Item 22) 13. The method of claim 12, wherein at least two of the three or more antibodies target different antigens. (Item 23) 13. The method of claim 12, wherein the three or more antibodies comprise monospecific antibodies and bispecific antibodies.

Claims

[Claim 1] The invention as described in the drawings.

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