METHODS FOR GENERATING AND PREPARING A SPECIFIC ANTIBODY, AS WELL AS FOR GENERATING AN ANTIBODY MIXTURE

Description

1 / 61 Descriptive Report of the Invention Patent for METHODS FOR GENERATING AND PREPARING A BIESPECTIVE ANTIBODY, AS WELL AS FOR GENERATING A MIXTURE OF ANTIBODIES. Related Application

[0001] This application claims the benefit of U.S. Provisional Application No. 61 / 374,159, filed August 16, 2010, U.S. Provisional Application No. 61 / 443,008, filed February 15, 2011, U.S. Provisional Application No. 61 / 509,260, filed July 19, 2011, the contents of each of which are incorporated herein by reference in their entirety. Field of Invention

[0002] The present invention relates to the generation of novel bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule. The antibodies of the invention are composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other a Lambda constant domain. This invention in particular relates to the isolation of antibodies of different specificities but sharing a common heavy chain. The invention also relates to the controlled co-expression of two light chains and a single heavy chain leading to the assembly of monospecific and bispecific antibodies. The invention provides a means of producing a completely human bispecific and bivalent antibody that is sequence-unaltered and does not involve the use of non-human ligands or other sequences, as well as antibody mixtures of two monospecific antibodies and one bispecific antibody.The invention similarly provides the means to effectively purify the bispecific antibody. Background of the Invention Petition 870220016476, dated 24 / 02 / 2022, p. 15 / 98 2 / 61

[0003] An antibody is composed of four polypeptides: two heavy chains and two light chains. The antigen-binding portion of an antibody is formed by the variable light chain (VL) domain and the variable heavy chain (VH) domain. At one end of these domains, six loops form the antigen-binding site and are also referred to as the complementarity-determining regions (CDRs). Three CDRs are located in the VH domain (H1, H2, and H3), and the other three are in the VL domain (L1, L2, and L3). During B cell development, a single immunoglobulin region is formed by somatic recombination known as V(D)J recombination. The variable region of the immunoglobulin heavy or light chain is encoded by different gene segments. The heavy chain is encoded by the three segments called variable (V), diversity (D), and binding (J) segments, while the variable light chain is formed by the recombination of only two segments, V and J.A large number of antibody paratopes can be generated by recombination between one of the multiple copies of the V, D, and J segments present in the genome. The V segment encodes CDR1 and CDR2, while CDR3 is generated by recombination events. During the course of the immune response, further diversity is introduced at the antigen-binding site by a process called somatic hypermutation (SHM). During this process, point mutations are introduced into the variable genes of the heavy and light chains, and in particular in the regions encoding CDRs. This additional variability allows the selection and expansion of B cells expressing antibody variants with improved affinity for their cognate.

[0004] The vast majority of immunoglobulins are bivalent and monospecific molecules carrying the same specificity in both arms as they are composed of two identical heavy chain polypeptides and two identical light chain polypeptides. Petition 870220016476, dated 24 / 02 / 2022, p. 16 / 98 3 / 61 However, it was recognized very early during the development of hybridoma technology that hybrid hybridomas can be created by a fusion event between two hybridomas (Suresh MR et al., Methods Enzymol 1986; 121: 210-228). These 'frameworks' express two different heavy chains and two different light chains and therefore produce a variety of different antibody species resulting from the random pairing of heavy and light chains. Among these different species, bispecific antibodies (bsAbs) are generated, carrying a different specificity in each arm. Another naturally occurring exception is the IgG4 isotype immunoglobulin which will be able to undergo heavy chain switching due to a less stable dimerization mediated by the binding region of that isotype (van der Neut Kolfschoten M et al., Science. 2007 317(5844): 1554-7). Although this switching appears to occur in vivo, its biological significance remains obscure.

[0005] Monoclonal antibodies have emerged as a successful and attractive class of molecules for therapeutic intervention in various areas of human disease. However, targeting or neutralizing a single protein is not always sufficient to achieve efficacy in certain diseases, which limits the therapeutic use of monoclonal antibodies. It is increasingly clear that in several indications, neutralizing one component of a biological system is not sufficient to achieve efficacy. One solution to this problem is the co-administration of multiple monoclonal antibodies. This approach is, however, complicated by regulatory aspects if the antibodies to be combined have not been previously approved individually. Furthermore, combination approaches are also expensive from an industrial perspective. Thus, there is a need for antibodies and therapeutics that allow targeting of multiple antigens with a single molecule. Petition 870220016476, dated 24 / 02 / 2022, page 17 / 98 4 / 61 Summary of the Invention

[0006] The invention allows the identification, production, and purification of bispecific antibodies that are indistinguishable in sequence from standard antibodies. The invention similarly allows the production and purification of a single antibody mixture of three or more antibodies all bearing the same heavy chain. The unaltered nature of the antibodies of the invention gives them favorable industrial characteristics similar to standard monoclonal antibodies.

[0007] The bispecific antibodies of the invention are generated using the following steps: Two antibodies with different specificities and sharing the same variable heavy chain domain, but with different variable chain domains isolated, are used. This step is facilitated by the use of antibody libraries having a fixed heavy chain or transgenic animals containing a single VH gene. - The variable heavy chain domain is fused to the constant region of a heavy chain, one variable light chain domain is fused to a Kappa constant domain, and the other variable light chain domain is fused to a Lambda constant domain. Preferably, the variable light chain domain fused to the Kappa constant domain is of the Kappa type, and the variable light chain domain fused to the Lambda constant domain is of the Lambda type. However, the invention also allows the generation of hybrid light chains such that two variable light chain domains of the same type can be used to generate bispecific antibodies of the invention. The three chains are co-expressed in mammalian cells, leading to the assembly and secretion in the supernatant of a mixture of three antibodies: two monospecific antibodies and one bispecific antibody carrying two different light chains. The relationship of Petition 870220016476, dated 24 / 02 / 2022, page 18 / 98 5 / 61 different antibodies depend on the relative expression of the chains and their assembly into an IgG. The invention provides methods for fine-tuning these ratios and maximizing the production of bispecific antibodies. The antibody mixture is purified using standard chromatographic techniques used for antibody purification. The antibody mixture can be characterized and used as a multi-targeting agent. The bispecific antibody is purified using affinity chromatography in a manner that consecutively binds specifically to human Kappa and human Lambda constant regions. This purification process is independent of the sequence of variable light chain domains and is therefore generic for all bispecific antibodies of the invention. The isolated bispecific antibody bearing a light chain containing a Kappa constant domain and a light chain containing a Lambda constant domain is characterized using different biochemical and immunological methods. The bispecific antibody of the invention can be used for therapeutic intervention or as a research or diagnostic reagent.

[0008] The invention provides monoclonal antibodies carrying a different specificity at 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.

[0009] In some antibodies, at least one first portion of the first light chain is of the Kappa type and at least one portion of the second light chain is of the Lambda type. In some antibodies, the first light chain includes at least one constant Kappa region. In some antibodies, the first light chain also includes a variable region. Petition 870220016476, dated 24 / 02 / 2022, page 19 / 98 6 / 61 Kappa. In some antibodies, the first light chain also includes a variable Lambda region. In some antibodies, the second light chain includes at least one constant Lambda region. In some antibodies, the second light chain also includes a variable Lambda region. In some antibodies, the second light chain also includes a variable Kappa region. In some antibodies, the first light chain includes a constant Kappa region and a variable Kappa region, and the second light chain includes a constant Lambda region and a variable Lambda region.

[00010] In some modalities, the sequences of constant and variable structure regions are human.

[00011] The invention likewise provides methods for producing and generating a bispecific antibody by a) Isolate an antibody or antibody fragment region having a specificity determined by a variable heavy chain domain combined with a first variable light chain domain; b) Isolate an antibody or antibody fragment region having a different specificity determined by the same variable heavy chain domain as the antibody in step a) combined with a second variable light chain domain; c) Co-express in a cell: (i) the common variable heavy chain domain fused to an immunoglobulin heavy chain constant region; (ii) the first variable light chain domain either fused to a kappa-type constant light chain domain or fused to a lambda-type constant light chain domain; and (iii) the second variable light chain domain fused to a constant light chain domain of a different type than the first variable constant domain.

[00012] Some methods similarly include the additional step of d) isolating the bispecific antibodies produced from the monospecific antibodies produced. For example, in some Petition 870220016476, dated 24 / 02 / 2022, page 20 / 98 In 7 / 61 methods, isolation is performed using an affinity chromatography purification step. In some methods, the purification step is performed using a specific Kappa constant domain, a specific Lambda constant domain, or both affinity chromatography means.

[00013] In some methods, a variable Kappa light chain domain is merged into a constant Kappa-type region. In some methods, a variable Kappa light chain domain is merged into a constant Lambda-type region. In some methods, a variable Lambda light chain domain is merged into a constant Kappa-type region. In some methods, a variable Lambda light chain domain is merged into a constant Lambda-type region.

[00014] In some methods, steps a) and b) are facilitated by the use of antibody libraries having a common heavy chain and limited diversity to the variable light chain domain. The variable heavy chain domain that is forced into one of these libraries may be based on different variable germline genes and have different sequences in both the CDR and Structure regions. In some methods, such libraries have been designed using different types of variable heavy chain domains and could be used to generate antibodies of the invention.

[00015] In some methods, the antibody library is required in filamentous bacteriophage, on the surface of yeast, bacteria or mammalian cells or used for ribosome or other type of in vitro display.

[00016] The invention similarly provides methods of preparing a bispecific antibody that specifically binds to a first antigen and a second antigen, wherein the first and second antigens are different, by a) providing a first nucleic acid molecule encoding a first polypeptide comprising Petition 870220016476, dated 24 / 02 / 2022, page 21 / 98 8 / 61 a variable heavy chain region of an immunoglobulin polypeptide or fragment thereof that binds the first antigen coupled to an immunoglobulin constant region; b) providing a second nucleic acid molecule encoding a second polypeptide comprising a variable light chain region of the immunoglobulin polypeptide or fragment thereof that binds the first antigen coupled to a first constant light chain region of the kappa or lambda type; c) providing a third nucleic acid molecule encoding a third polypeptide comprising a variable light chain region of an immunoglobulin polypeptide or fragment thereof that binds the second antigen coupled to a second constant light chain region of the kappa or lambda type, wherein the first and second constant light chain domains are of different types;ed) to cultivate a host cell comprising the first, second, and third nucleic acid molecules under conditions that allow expression of the first, second, and third polypeptides.

[00017] Some methods similarly include the other step of e) recovering the bispecific antibody. For example, in some methods, the bispecific antibody is recovered in step e) using an affinity chromatography purification step. In some methods, the purification step is performed using specific Kappa constant domain, Lambda constant domain, or both means of specific Kappa constant domain and Lambda constant domain affinity chromatography.

[00018] In some methods, the second nucleic acid encodes a variable Kappa light chain domain. In some methods, the second nucleic acid encodes a Kappa-type constant region. In some methods, the second nucleic acid encodes a Lambda-type constant region. In some methods, the second nucleic acid encodes a Petition 870220016476, dated 24 / 02 / 2022, page 22 / 98 9 / 61 variable domain of Lambda-type light chain. In some methods, the second nucleic acid encodes a constant region of Kappa-type. In some methods, the second nucleic acid encodes a constant region of Lambda-type. In some methods, the third nucleic acid encodes a variable domain of Kappa-type light chain. In some methods, the third nucleic acid encodes a constant region of Kappa-type. In some methods, the third nucleic acid encodes a constant region of Lambda-type. In some methods, the third nucleic acid encodes a variable domain of Lambda-type light chain. In some methods, the third nucleic acid encodes a constant region of Kappa-type. In some methods, the third nucleic acid encodes a constant region of Lambda-type.

[00019] The invention similarly provides an antibody mixture that includes two monospecific antibodies and one bispecific antibody, all having a common heavy chain. For example, the bispecific antibody is any of the bispecific antibodies described herein or made using methods described herein.The invention similarly provides methods of generating such an antibody mixture by a) isolating an antibody or antibody structure region having a specificity determined by a variable heavy chain domain combined with a first variable light chain domain; b) isolating an antibody or antibody structure region having a different specificity determined by the same variable heavy chain domain as the antibody of step a) combined with a second variable light chain domain; c) co-expressing in a cell: (i) the common variable heavy chain domain fused to an immunoglobulin constant heavy chain region; (ii) the first variable light chain domain either fused to a kappa-type constant light chain domain or fused to a lambda-type constant light chain domain; and (iii) the second variable heavy chain domain. Petition 870220016476, dated 24 / 02 / 2022, page 23 / 98 10 / 61 light or fused into a constant domain of Kappa-type light chain or fused into a constant domain of Lambda-type light chain. Some methods similarly include the additional step of d) isolating the antibody mixture produced in step c) from cell culture supernatant.

[00020] The invention similarly provides methods for producing two or more, for example, three or more non-identical antibodies in a single recombinant host cell by a) expressing in the single recombinant host cell one or more nucleic acid sequences encoding a common immunoglobulin heavy chain and at least two, for example, at least three, different immunoglobulin light chains that are capable of pairing with the common immunoglobulin heavy chain to form functional antigen-binding domains to produce two or more, for example, three or more, non-identical antibodies comprising the common heavy chain. Some methods similarly include the step of harvesting or otherwise purifying the two or more, for example, three or more, non-identical antibodies from the recombinant host cell or from a culture of the host cell. The host cell is, for example, a mammalian cell.In some methods, non-identical antibodies include both monospecific and bispecific antibodies.

[00021] In some methods, non-identical antibodies target different epitopes of the same target antigen. In some methods, non-identical antibodies have discrepant affinities for the same target epitope. In some methods, non-identical antibodies bind to different antigens.

[00022] In some methods, two or more, for example, three or more, non-identical antibodies are selected independently from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Petition 870220016476, dated 24 / 02 / 2022, p. 24 / 98 11 / 61

[00023] In some methods, two or more, for example, three or more, non-identical antibodies contain a modified Fc region that modifies the effector functions of the antibodies such as Antigen-Dependent Cell-Mediated Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antigen-Dependent Cellular Phagocytosis (ADCP) or their pharmacokinetic properties by altering their binding to the neonatal Fc receptor.

[00024] In some methods, two or more, for example three or more different immunoglobulins are in the F(ab')2 format.

[00025] In some methods, one or more nucleic acid sequences are stably expressed in the host cell.

[00026] In some methods, two or more, for example three or more, non-identical antibodies are produced by the host cell in vitro.

[00027] Some methods similarly include the additional steps of selecting a host cell by analyzing at least two or more, for example, three or more, non-identical antibodies produced by the recombinant host cell for their ability to bind a target antigen; culturing the recombinant host cell; and isolating the three or more non-identical antibodies. Antibodies may be isolated using any of the techniques described herein or any other method recognized in the appropriate art.

[00028] In some methods, different immunoglobulin light chains have identical constant regions. In some methods, different immunoglobulin light chains have different constant regions. Brief Description of the Drawings

[00029] Figure 1 is a schematic representation of different bispecific antibody formats. A. Formats based on antibody fragments: X-binding Fab, Fab fragments Petition 870220016476, dated 24 / 02 / 2022, page 25 / 98 A. Formats based on crosslinks: 12 / 61; tascFv / BiTE, tandem T-cell engager; scFv / Bispecific; Db, diantibody; taDb, tandem diantibody. B. Formats based on Fc fusions: Db-Fc, diantibody-Fc fusion; taDb-Fc fusion, tandem diantibody-Fc fusion; taDb-CH3, tandem diantibody-CFB fusion; scFv>Fc, tetra scFv-Fc fusion; DVD-Ig, dual variable domain immunoglobulin. C. IgG formats: buttonhole and SEED, domain created by filament exchange; CrossMab, buttonhole combined with heavy and light chain domain exchange; bsAb, bispecific quadroma-derived antibody; sdAb, single domain-based antibody.

[00030] Figure 2 is a schematic representation of possible modes of action permitted by bispecific antibodies. 2A. Targeting of two antigens. 2B. Redirecting a toxic portion or activity onto a target cell. 2C. Increased selectivity mediated by avidity.

[00031] Figures 3A-3C are a schematic representation of the structure of different bispecific antibodies of the invention composed of two copies of a single heavy chain polypeptide and two different light chain polypeptides. The locations and / or arrangements of the Kappa light chain and Lambda light chain (or portions thereof) shown in these figures are not intended to be limiting. Those of ordinary skill in the art will appreciate that the Kappa light chain and the Lambda light chain (or portions thereof) can similarly be arranged to produce the mirror image of the bispecific antibodies shown in Figures 3A-3C. Those of ordinary skill in the art will likewise appreciate that the bispecific antibodies that are represented in a total IgG format in Figures 3A-3C can similarly be generated using other immunoglobulin isotypes or in other immunoglobulin formats such as F(ab')2. 3A. Variable Kappa domain fused into a domain Petition 870220016476, dated 24 / 02 / 2022, page 26 / 98 13 / 61 Kappa constant and Lambda variable domain merged into a Lambda constant domain. 3B. Kappa variable domains merged into a Kappa constant domain and a Lambda constant domain. 3C. Lambda variable domains merged into a Kappa constant domain and a Lambda constant domain.

[00032] Figure 4 is an illustration of an ELISA assay testing specific clones for hCXCL10-NusA or hIL6RC and bearing the same variable heavy chain domain. Each clone was tested against both targets to demonstrate specificity.

[00033] Figures 5A-C are a series of illustrations describing the three types of libraries used in the Examples; for each library type, Vk and VX libraries were kept separate. Figures 5A and C: Two groups of libraries containing a fixed VH3-23 variable domain that differ only by their H3 CDR sequence, which is indicated below the H3 (definition of CDR according to IMGT). The light chain repertoire was diversified by inserting random sequences into CDRL3 from selected light chain variable genes (Figures 5A and 5C) or by capturing naturally rearranged light chain variable domains isolated from human donors that may include all human variable genes and contain diversity in all 3 CDRs (Figure 5B). The different diversification strategies are illustrated by horizontal lines below the diversified region of the light chain repertoires.

[00034] Figures 6A-6B are graphs depicting the results of ELISA using selected monospecific lgGX and IgGK against hlFNy and IL6RC, respectively, and supporting a common heavy chain. The ELISA formats are schematically represented next to each graph. In Figure 6A, INFy was immobilized on the plate, incubated with either anti-INFy lgGX or anti-IL6RC (i.e., IL-6R receptor / soluble IL-6 complex) IgGK, and both were detected with anti-Ck antibodies. Petition 870220016476, dated 24 / 02 / 2022, page 27 / 98 14 / 61 human or CX anti-human coupled to horseradish peroxidase. The signal was revealed by colorimetry and quantified using a microtiter plate reader.

[00035] Figure 7 is a schematic representation of vectors used for the co-expression of one heavy chain and two light chains in mammalian cells. Both vectors contain three promoters to direct gene expression, a glutamine synthetase gene for stable cell line selection. In the second vector, pNovI kHXk, the expression of an additional kappa light chain is directed by an internal ribosome entry site (IRES). The different genes and genetic control elements are indicated. hCMV, human cytomegalovirus promoter; SV40, V40 promoter; pA ​​polyadenylation signal; VH, heavy chain variable domain; VK, kappa light chain variable domain; CK, kappa light chain constant domain; VL, lambda light chain variable domain; CL2, lambda light chain constant domain; GS cDNA, glutamine synthetase cDNA; AmpR; selectable marker for ampicillin resistance. A selected number of restriction sites are indicated.

[00036] Figure 8A is a schematic representation of the purification process for bispecific antibodies of the invention. Figure 8B is an illustration describing co-expression, purification, and SDSPAGE analysis of bispecific antibodies of the invention. The gel was stained using simply blue. PA: Protein A; K: Kappa select; λ: Lambda select; FT: total flow column; E: elution fraction.

[00037] Figure 9 is an illustration of an SDS-PAGE analysis of total IgG purified from mammalian cells transfected with vectors allowing different levels of Kappa light chain expression using different IRES elements within the pNovI kHXk vector (lanes 1-5) and compared to the pNovI kHX vector. The relative intensities of the Kappa and Lambda light chains indicate that the expression levels may Petition 870220016476, dated 24 / 02 / 2022, page 28 / 98 15 / 61 to be modulated.

[00038] Figure 10A is an illustration depicting IEF gel analysis of purified monospecific IgG (kk and / / ) and bispecific IgG (κλ). Figure 10B is an illustration of IEX-HPLC analysis of monospecific and bispecific antibodies. The three antibodies were injected independently and their elution profile is plotted on the graph. The gradient used in the experiment is shown.

[00039] Figure 11 describes the ELISA assays used to determine the ability of the bispecific antibody to equally bind the target and the presence of a Kappa and a Lambda light chain in the molecule. Figure 11A is a schematic representation of the ELISA format. Figure 11B is a graph depicting the results of the ELISA with INFy immobilized on the plate. Figure 11C is a graph depicting the results of the ELISA with IL-6RC immobilized on the plate. IgGK, monospecific anti-IL6RC antibody; IgG / , monospecific anti-INFy antibody; IgGK / , bispecific anti-IL6RC / anti-INFy antibody. Secondary detection antibodies anti-human Lambda HRP and anti-human Kappa HRP are indicated.

[00040] Figure 12 is an illustration of SPR analysis of bispecific IgGK / antibodies. In Figure 12A, INFy was immobilized on the surface of the Biacore fragment and monospecific anti-IL6RC antibody (IgGK), monospecific anti-INFy antibody (IgG / ), and bispecific anti-IL6RC / anti-INFy antibody (IgGK / ) were injected onto the surface followed by IL6RC injection. In Figure 12B, bispecific anti-IL6RC / anti-INFy antibody (IgGK / ) was immobilized on the fragment surface and anti-human Kappa and anti-human Lambda antibodies were injected at the same concentration. The experiment was repeated by reversing the antibody injection order with identical results.

[00041] Figure 13 is a schematic representation of an evaluation of a method for generating bispecific antibodies and Petition 870220016476, dated 24 / 02 / 2022, page 29 / 98 16 / 61 multispecific cells described here in CHO cells.

[00042] Figure 14A is a graph depicting the growth and antibody production profiles of CHO cell sets at a small-scale production level in an Erlenmeyer flask. Antibody production levels were determined by Protein A-HPLC analysis. VCC represents viable cell concentration and Ab represents antibody. Figure 14B is a graph depicting the growth and antibody production profile comparison between small-scale and medium-scale fermentation. Antibody production levels were determined by Protein A-HPLC analysis. VCC represents viable cell concentration and Ab represents antibody.

[00043] Figures 15A and 15B are a series of graphs depicting antibody productivity in a 96-well plate (96wpl) of mono Kappa (KK), mono Lambda (LL), and bispecific Kappa Lambda (KL) antibodies expressing cell lines five weeks post-transfection in two independent experiments. Antibody production levels were determined by ELISA. mAb represents monoclonal antibody. Figures 15C and 15D are a series of graphs depicting antibody productivity in a 24-well plate (24wpl) huge batch culture of mono Kappa (KK), mono Lambda (LL), and bispecific Kappa antibodies.

[00044] Figure 16A is an illustration depicting the results of reduced SDS-PAGE analysis of monospecific κ IgG molecules (i.e., monospecific molecules having Kappa light chains, similarly referred to here as mono κ molecules), monospecific λ IgG molecules (i.e., monospecific molecules having Lambda light chains, similarly referred to here as mono λ molecules), and κλ antibodies (i.e., antibodies having both Kappa and Lambda light chains) through the purification steps described above. Figure 16B is an illustration depicting the results of SDS-PAGE analysis. Petition 870220016476, dated 24 / 02 / 2022, p. 30 / 98 17 / 61 reduced mono κ, mono λ and κλ antibodies obtained following the elution steps described above. In Figures 16A and 16B, the gel was stained using simply blue, and E represents elution fraction, FT represents total column flow and MM represents molecular weight marker. Figure 16C is an illustration describing isoelectric focusing (IEF) gel analysis of purified monospecific IgG molecules (κκ and λλ) and the bispecific IgG molecule (κλ).

[00045] Figure 17A-D is a series of graphs and illustrations describing that the methods of generating bispecific antibodies of the invention produce antibodies that equally include a Kappa light chain and a Lambda light chain and that the purified antibodies exhibit bispecificity. The graphs depict the results of ELISA using purified κλ bodies against hlFNy and IL6RC. The ELISA was performed using anti-Kappa or anti-Lambda detection antibodies as indicated. Figures 17A-D illustrate that the Lambda light chain binds to hlFNy, while the Kappa light chain binds to IL6RC. NI-0501 is a control anti-hlFNy Lambda light chain antibody, and NI-1201 is a control anti-IL6RC Kappa light chain antibody.

[00046] Figure 18 is an illustration of an IEF gel of different monospecific and bispecific antibodies, indicating that the difference in pI can vary depending on the slightly variable antibody sequence. Lane 1, IgGi < anti-NusA; Lane 2, IgGi <?v anti-NusA / anti-INFy; Pista 3, ^Θλ anti-INFy; Pista 4, ^Θκ anti-IL6RC; Pista 5, ^Θκλ anti-IL6RC / antiIL6RC; Pista 6, ^Θλ anti-IL6RC.

[00047] Figure 19 is a schematic representation of three different hybrid proteins obtained by combining a variable Lambda gene and a constant Kappa gene. The fusion points differ between the different hybrids: in 19A, VLambda fused to CKappa; in 19B, VLambda up to CDR3 fused to VKappa FR4 and CKappa; and in 19C, VLambda and the first four amino acids of Petition 870220016476, dated 24 / 02 / 2022, page 31 / 98 18 / 61 CLambda and CKappa excluding the first four amino acids. CDR, Complementary Determination Region; FR, Structure Region.

[00048] Figure 20 is an illustration of the analysis of two hybrid light chain constructs in a Bionalyzer 2100 system using a Protein 80 fragment (Agilent Technologies). The electropherogram corresponding to the gel image is indicated.

[00049] Figure 21 is a series of graphs depicting dose-response ELISA results using scFv specific for INFy (A) or IL6RC (B) where the VH domain is the originally selected common VH (top curves) or other VH domains that allow scFv expression and purification (bottom curves).

[00050] Figure 22 is a graph describing the results obtained for quantification of bispecific IgGKα antibody using a sandwich ELISA format. The dose response was performed using purified bispecific antibody alone or mixed with monospecific Kappa or Lambda antibodies in different ratios as indicated, to assess the interference of these molecules in the assay. Detailed Description

[00051] To overcome the limitations of monoclonal and monovalent antibody therapeutics that can only target a single antigen, or to overcome the limitations of combinations of monovalent antibody therapeutics, intense efforts have been directed towards targeting multiple antigens using bispecific antibody formats. Such antibodies carrying more than one specificity are of interest in biotechnology and have great potential as therapeutic agents, allowing for new therapeutic approaches (Fischer and Léger, Pathobiology 2007; 74:3-14; Morrison SL Nature Biotechnol 2007; 25: 1233-1234). Bispecific antibodies are advantageous when they allow for multiple targeting, they increase therapeutic potential, and they address redundancy. Petition 870220016476, dated 24 / 02 / 2022, page 32 / 98 19 / 61 biological systems, and they provide new mechanisms of action through abilities such as targeting and / or increased specificity. As validated single therapeutic targets become more exhausted, combinations enabled by bispecific antibodies provide a new and expansive universe of targets for therapeutic agents and applications.

[00052] Several strategies have been used to generate such bispecific molecules, such as chemical crosslinking of antibody fragments, forced heterodimerization, quadroma technology, fusion of antibody fragments using polypeptide linkers, and the use of single domain antibodies. The availability of recombinant DNA technologies leads to the generation of a multitude of bispecific antibody formats (see, for example, Ridgway JB et al. (1996) Protein Eng 9: 617-621). Linkers and mutations have often been introduced into different regions of the antibody to force heterodimer formation or to connect different binding portions into a single molecule. However, these created molecules often have poor industrial characteristics, as well as an increased risk of immunogenicity, which limits or prevents their progression to the clinic.Furthermore, previous attempts to develop bispecific formats have been limited due to factors such as poor stability and / or expression. These approaches are also described below, and the formats discussed are illustrated in Figure 1.

[00053] Chemical crosslinking. The use of chemical crosslinking reagents to covalently link two antibodies is a conceptually straightforward approach. Antibody fragments generated from their respective antibody origins by enzymatic digestion or generated through recombinant technologies are conjugated using bifunctional reagents (Glennie MJ et al., J Exp Med 1992; 175:217225). Product homogeneity is the main limitation of this approach. Petition 870220016476, dated 24 / 02 / 2022, page 33 / 98 The 20 / 61 approach, as bispecific species have to be purified from homodimers, and the modification steps can alter the integrity and stability of the proteins. The multiple steps involved make this approach challenging in terms of manufacturing and product homogeneity.

[00054] Quadromas. Quadromas and triomas can be generated by fusing two hybridomas or a hybridoma with a B lymphocyte, respectively (Suresh MR et al., Methods Enzymol 1986; 121: 210228). In this case, the simultaneous expression of two heavy and two light chains leads to the random assembly of 10 antibody combinations, and the desired bsAb represents only a small fraction of the secreted antibodies. The bsAb has to be purified using a combination of chromatographic techniques, dramatically reducing production yields. A major limitation is that quadromas produce rodent-derived bsAb, which limits their therapeutic potential due to immunogenicity issues.

[00055] Recombinant bispecific antibodies. Most bispecific antibody formats have been generated by genetic engineering techniques using antibody fragments such as scFv or Fab fragments as building blocks connected via polypeptide linkers. Formats based on linked antibody fragments include tandem scFv (BiTE), diabodies, and tandem diabodies (Kipriyanov SM. Methods Mol Biol 2003; 207: 323-333; Korn T et al., Int J Cancer 2002; 100: 690-697). These building blocks can also be linked to a specific Fc region of immunoglobulin elevation for 'IgG-like' molecules. These formats include Fc-diantibody, Fc-diantibody tandem, CH3-diantibody tandem, (scFv)4-Fc, and DVD-Ig (Lu D et al., J Immunol Methods 2003; 279: 219-232; Lu D et al., J Biol Chem 2005; 280: 19665-19672; Lu D et al., J Biol Chem 2004; 279: 2856-2865; Wu C et al., Nat Petition 870220016476, dated 24 / 02 / 2022, p. 34 / 98 21 / 61 (Biotechnol 2007 25: 1290-7). A potential limitation of using ligands is that the flexible nature of these peptides makes them more prone to proteolytic cleavage, potentially leading to poor antibody stability, aggregation, and increased immunogenicity. Furthermore, these foreign peptides could elicit an immune response against the binding site between the protein and the ligand or the ligand itself. In general, bsAbs based on linked building blocks are challenging molecules to manufacture, which limits their therapeutic potential.

[00056] An ideal bispecific molecule for human therapy should be indistinguishable from a normal IgG. Strategies based on forcing heterodimerization of two heavy chains have been explored. One early approach created 'buttonhole' points by forcing the pairing of two different IgG heavy chains by introducing mutations in the CH3 domains to modify the contact interface (Ridgway JB et al., Protein Eng 1996; 9: 617-621). Amino acids from one chain with large side chains were introduced to create a 'button.' Conversely, bulky amino acids were replaced by amino acids with short side chains to create a 'hole' in the other CH3 domain. By co-expressing these two heavy chains, more than 90% heterodimer formation was observed ('buttonhole') versus homodimer formation ('hole-hole' or 'button-button').A similar concept was developed using filament exchange domain-created (SEED) human CH3 domains based on human IgG and human IgA sequences (Davis JH et al., 2010, PEDS 23: 195-202). These created domains lead to the formation of heterodimeric molecules that can carry two different specificities. These two approaches are attractive when they favor the production of the heterodimer of interest (up to 95%), but do not completely prevent homodimer formation. Therefore, downstream purification procedures capable of removing homodimers are necessary. Petition 870220016476, dated 24 / 02 / 2022, page 35 / 98 22 / 61 The homodimers of the heterodimers are still required. Another potential issue with these approaches is that the mutated domains are not completely human and may lead to immunogenicity and could similarly affect domain stability and molecule aggregation tendency. As these strategies allow forced pairing of heavy chains, different light chains may randomly pair with either of the two heavy chains and lead to the generation of different antibodies that need to be purified from each other. Recently, an improvement on the 'button-in-hole' approach has been described to address the light chain pairing issue (WO 2009 / 080253 Al). This method involves exchanging some of the light chain and heavy chain domains in addition to the 'button-in-hole' mutations.The main advantage of this method is that a bispecific bivalent antibody having two different variable heavy chain domains and two different variable light chain domains can be generated, as was created with CrossMab. However, the sequences of this bispecific antibody are not completely human as they contain both mutations in the Fc to force heterodimerization and unnatural junction points between the different immunoglobulin domains. Furthermore, these modifications lead to reduced expression levels of the bispecific format compared to a standard monoclonal antibody (Schaefer et al., PNAS 2011; 108: 1187-1192).

[00057] Single-domain based antibodies. The immune systems of camelids (llamas and camels) and cartilaginous fish (nurse sharks) use single V domains fused into an Fc cluster, demonstrating that a single domain can confer high antigen-binding affinity. Camelid, shark, and even human V domains represent alternatives to antibodies, but they will also be used to generate βsAbs. They can be reformatted into an IgG. Petition 870220016476, dated 24 / 02 / 2022, page 36 / 98 The classic 23 / 61 design, in which each arm has the potential to bind two targets through its VH or VL domain, is a unique IgG domain. This single IgG domain would have biochemical properties similar to an IgG and could potentially solve problems encountered with other bsAb formats in terms of production and heterogeneity. However, steric hindrance is likely to frequently prevent simultaneous binding of both antigens to both antibody arms.

[00058] A representation of bispecific antibody formats described above is shown in Figure 1. Some of these format representations are derived from Fischer and Léger, Pathobiology 2007; 74:3-14; and Morrison SL Nature Biotechnol 2007; 25: 1233-1234.

[00059] In contrast to these previous formats, the bispecific antibodies, multispecific antibodies, compositions, and methods provided here overcome such developmental obstacles. The bispecific antibodies provided here have a common heavy chain, two light chains—one Kappa (κ), one Lambda (λ)—each with a different specificity (i.e., two light chains, two specificities). Preferably, the bispecific antibodies do not contain any ligands or other modifications, including amino acid mutations. The methods provided here produce molecules having specific binding where diversity is restricted to the VL region. These methods produce the bispecific antibodies through controlled co-expression of the three chains (one VH chain, two VL chains), and purification of the bispecific antibody.The bispecific and / or multispecific antibodies described here exhibit similar affinities for a given target when compared to the affinities of monospecific antibodies for that same target. Preferably, the bispecific and / or multispecific antibodies described here are virtually indistinguishable from standard IgG molecules.

[00060] The methods provided here also provide the Petition 870220016476, dated 24 / 02 / 2022, page 37 / 98 24 / 61 means of generating simple antibody mixtures of two monospecific antibodies and one bispecific antibody that are useful, for example, for multiple targeting without purification of the bispecific antibody from the mixture. Possible modes of action of bispecific antibodies

[00061] Simultaneous inhibition of two targets. By definition, bispecific antibodies carry two specificities and can therefore inhibit more than one target. These targets can be soluble factors or located on the surface of a cell. A targeting format of multiple cytokines has been successfully generated (Wu C et al., Nat Biotechnol 2007 25: 1290-7).

[00062] Redirection. As a majority of bispecific antibody formats are capable of binding two molecules simultaneously, they can therefore be used as binding molecules in cytotoxic effector cells or cytotoxic agents to cells involved in a disease process. This application has been explored in cancerology. In some examples, an antibody specificity has been directed against tumor cell markers such as CD19, CD20, HER2, carcinoembryonic antigen (CEA). The second arm of the bispecific antibody carries in close proximity a toxic or active moiety such as drugs, toxins, cytokines, or an effector cell of the immune system (T cells, NK cells, monocytes, and neutrophils, targeting CD3, CD16, CD64, and CD89, respectively) (Thielemans K, Blood 1996; 87: 4390-398; Goldstein J et al., J Immunol 1997; 158: 872-879).

[00063] Enhanced specificity through avidity. In a classic IgG format, antibody binding is driven equally by the affinity of each binding site for its antigen and by the avidity effects provided by bivalent binding. The avidity effect dramatically increases the apparent affinity of the antibody for markers of Petition 870220016476, dated 24 / 02 / 2022, page 38 / 98 25 / 61 cell surface as two dissociation events have to occur for the antibody to be released. Some of the bispecific formats described above are bivalent (i.e., one binding site for each target) whereas others are tetravalent. The latter has four or more binding sites and has the potential to bind to each target in a bivalent manner. Bispecific bivalent therapeutic agents selectively target cell populations that express a combination of cell surface markers. This single mode of action is in principle restricted to molecules that can benefit from an avidity component to discriminate between cells expressing antigens and those expressing only one marker.

[00064] A representation of possible modes of action mediated by bispecific antibodies is shown in Figure 2. This representation is derived from Fischer and Léger, Pathobiology 2007; 74:3-14. Characteristics and limitations of bispecific antibody formats

[00065] The main characteristics of current bispecific antibody formats are summarized in Table I. All formats except those based on human domains contain sequences that are not of human origin or contain non-human protein sequences generated by the fusion of different protein domains. Most formats using ligands lead to potential manufacturing issues due to domain aggregation. The presence of foreign sequences and unfavorable stability characteristics can potentially significantly increase the risk of immunogenicity. A key difference between formats is the valence of their binding sites, which is directly linked to their ability to mediate redirection or avidity-mediated selective binding. Thus, not all formats may allow all modes of action.In particular, the only format that is indistinguishable from a completely human immunoglobulin cannot be mediated. Petition 870220016476, dated 24 / 02 / 2022, page 39 / 98 26 / 61 Redirecting or Enhanced Selectivity activities. There is, therefore, a need to generate new bispecific antibodies with properties favorable for the development of therapeutics, that is, being indistinguishable from a completely human immunoglobulin molecule, having good manufacturability properties and allowing the full spectrum of possible modes of action. Petition 870220016476, dated 24 / 02 / 2022, page 40 / 98 Table I. Characteristics of different bispecific antibody formats. Crosslinked Fragments Quadromes Recombinant Formats - Linked Antibody Fragments Recombinant Formats - Forced Heterodimers Recombinant Formats - Based on Single Domains Binding Mode Bivalent Bivalent Tetravalent (or more) Bivalent Tetravalent Mode of Action D1, R, IS D1, R, IS D1, IS D1, R, IS D1 Manufacturing Complex, Multi-step Process Purification from a complex antibody mixture Can be challenging due to antibody fragment instability and aggregation Simple mixture (main part of the product is bispecific) Simple Sequence Origin Human modified sites by crosslinking process Rodent sequences Human, presence of non-human protein ligands and junctions Human, presence of mutations to force heterodimerization Human Modes of Action: DI, Dual Inhibition; R, Redirection; IS, Enhanced Selectivity. 27 / 61 Petition 870220016476, dated 24 / 02 / 2022, page 41 / 98 28 / 61 Improved methods for generating bispecific and bivalent antibodies.

[00066] The present invention provides methods for generating bispecific antibodies that are identical in structure to a human immunoglobulin. This type of molecule is composed of two copies of a single heavy chain polypeptide, a first variable light chain region fused into a constant Kappa domain and a second variable light chain region fused into a constant Lambda domain. Each combining site exhibits a different antigen specificity to which both heavy and light chains contribute. The variable light chain regions can be of the Lambda or Kappa family and are preferably fused into constant Lambda and Kappa domains, respectively. This is preferred to avoid the generation of unnatural polypeptide couplings.However, it is similarly possible to obtain bispecific antibodies of the invention by fusing a variable Kappa light chain domain into a constant Lambda domain for a first specificity and by fusing a variable Lambda light chain domain into a constant Kappa domain for a second specificity (Figure 3). The bispecific antibodies described here are similarly called IçGkà antibodies or κλ bodies, a novel fully human bispecific IgG format. This κλ body format allows for affinity purification of a bispecific antibody that is indistinguishable from a standard IgG molecule with characteristics that are indistinguishable from a standard monoclonal antibody and is therefore advantageous when compared to previous formats.

[00067] An essential step of the method is the identification of two Fv antibody regions (each composed of a variable light chain and a variable heavy chain domain) having different antigen specificities that share the same variable heavy chain domain. Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof (see, for example, Petition 870220016476, dated 24 / 02 / 2022, page 42 / 98 29 / 61 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 CDRs 1 and 2, arise from human genes. The CDR3 region may be of human origin or designed by synthetic means. Such antibodies are called human antibodies, or fully human antibodies herein. Human monoclonal antibodies can be prepared using the triome 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 can be used and can be produced 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).

[00068] Monoclonal antibodies are generated, for example, 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 encoding the 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 xenogeneic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which are incorporated herein by reference in their entirety.

[00069] Alternatively, antibodies are obtained by evaluating Petition 870220016476, dated 24 / 02 / 2022, page 43 / 98 30 / 61 a library containing antibody or antigen-binding domain sequences to bind to the target antigen. This library is prepared, for example, in bacteriophage as protein or peptide fusions into a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the coding DNA sequences contained within the phage particles (i.e., phage-displayed library).

[00070] Hybridomas resulting from myeloma / B-cell fusions are then evaluated 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 suitable host animal is typically immunized with an immunization agent to extract lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunization agent. Alternatively, lymphocytes can be immunized in vitro.

[00071] Although not strictly impossible, the accidental identification of different antibodies having the same variable heavy chain domain but directed against different antigens is highly improbable. Indeed, in most cases the heavy chain contributes largely to the antigen-binding surface and is likewise the most variable in sequence. In particular, CDR3 in 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 variable heavy chain domains.

[00072] The method of the invention overcomes this limitation and greatly facilitates the isolation of antibodies having the same variable heavy chain domain by using antibody libraries in which the domain Petition 870220016476, dated 24 / 02 / 2022, page 44 / 98 The variable heavy chain domain (31 / 61) is the same for all library members, and thus diversity is limited to the variable light chain domain. Such libraries are described, for example, in copending application PCT / US2010 / 035619, filed on May 20, 2010, and published on November 25, 2010, as PCT Publication No. WO 2010 / 135558, and copending application PCT / US2010 / 057780, filed on November 23, 2010, each of which is incorporated herein by reference in its entirety. However, since the variable light chain domain is expressed together with the variable heavy chain domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing the same variable heavy chain domain and / or a diversity of variable Lambda light chains or variable Kappa light chains can be used in parallel for in vitro screening of antibodies against different antigens.This approach allows the identification of two antibodies having a common heavy chain, one carrying a variable Lambda light chain domain and the other a variable Kappa light chain domain, which can be used as building blocks for the generation of a bispecific antibody in the form of the total immunoglobulin of the invention. The bispecific antibodies of the invention can be of different isotypes and their Fc portion can be modified to alter the binding properties to different Fc receptors and in this way modify the effector functions of the antibody as well as pharmacokinetic properties. Numerous methods for modifying 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; US ​​Pat No. 6,528,624; PCT / US2009 / 0191199 filed January 9, 2009).The methods of the invention can likewise be used to generate bispecific antibodies and antibody mixtures in one. Petition 870220016476, dated 24 / 02 / 2022, p. 45 / 98 32 / 61 format of F(ab')2 which is missing the Fc portion.

[00073] Another fundamental step of the invention is the optimization of co-expression of the common heavy chain and two different light chains in a single cell to allow the assembly of a bispecific antibody of the invention. If all polypeptides are expressed at the same level and are assembled equally well to form an immunoglobulin molecule, then the ratio of monospecific (same light chains) to bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and / or not assembled with the same efficiency. Therefore, the methods of the invention similarly provide means to modulate the relative expression of different polypeptides to compensate for their intrinsic expression characteristics or different tendencies to assemble with the common heavy chain.This modulation can be achieved through promoter resistance, 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 resistance could include CMV (Cytomegalovirus Immediate-Early Virus promoter); EF1-^ (Human Elongation Factor ^-Subunit promoter); Ubc (Human Ubiquitin C promoter); SV40 (Simian Virus 40 promoter). Different IRES have similarly been described from mammalian and viral origins (see, for example, Hellen CU and Sarnow P. Genes Dev 2001 15: 1593-612). These IRES can greatly differ in their length and ribosome recruitment efficiency. Furthermore, it is also possible to adjust the activity by introducing multiple copies of IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541).Modulation of expression can similarly be achieved through transfections. Petition 870220016476, dated 24 / 02 / 2022, page 46 / 98 33 / 61 multiple sequential cell patterns to increase the copy number of individual genes expressing one or the other light chain and thereby modify their relative expressions. The examples provided here demonstrate that controlling the relative expression of the different chains is critical to maximizing the assembly and overall yield of the bispecific antibody.

[00074] The co-expression of the heavy chain and two light chains generates a mixture of three different antibodies in 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 here greatly facilitates this purification procedure by using affinity chromatography media that specifically interact with the constant domains of the Kappa or Lambda light chain, 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 stark contrast to specific purification methods that have to be developed and refined for each bispecific antibody 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 techniques such as ion-exchange chromatography may be challenging or not possible at all.

[00075] The invention similarly provides new means of producing simple antibody mixtures of two or more monospecific antibodies and one or more bispecific antibodies that share the same heavy chain and can be purified using standard chromatography techniques used for antibody purification. Petition 870220016476, dated 24 / 02 / 2022, page 47 / 98 34 / 61 monoclonal. (see for example, Lowy, I and others N Engl J Med 2010; 362: 197-205; Goudsmit, J., and others J Infect Dis. 2006. 193, 796-801). Such simple mixtures can be used as multi-targeting agents for therapeutic use.

[00076] Successful co-expression, purification and characterization of the heavy chain and two light chains and purification of bispecific antibodies are shown in the Examples. The genes encoding the common heavy chain and the two light chains were cloned into a vector containing three promoters. After transient transfection, the PEAK cell supernatant was collected.

[00077] The co-expression of the three chains led to the assembly of three different antibodies: two monospecific antibodies and one bispecific antibody. Their theoretical relative ratios should be 1:1:2 provided the expression levels and assembly rates are similar for both light chains. The bispecific antibodies were purified using a three-step affinity chromatography procedure: (1) Protein A: capture IgG (mono- and bi-), (2) Kappa selection: capture IgG containing a Kappa light chain, and (3) Lambda selection: capture IgG containing a Lambda light chain. Kappaselect and Lambdaselect are affinity chromatography media developed by BAC, BV, and GE Healthcare.

[00078] The purified bispecific antibodies were characterized as follows. The total flow and elution of each affinity purification step was analyzed by SDS-PAGE. The results indicate that, at each step, bispecific antibodies are enriched (Figure 8). The κλ body contained equivalent amounts of Kappa and Lambda light chains. The κλ body exhibited an intermediate migration pattern on an isoelectric focusing gel and ion-exchange chromatography compared to the two monospecific antibodies (Figure 10). The specificity and affinity of κλ bodies were determined by ELISA. Petition 870220016476, dated 24 / 02 / 2022, page 48 / 98 35 / 61 and surface plasmon resonance. The methods of the invention allow the identification of antibodies with affinities in the subnanomolar to nanomolar range without optimization. This is not obvious as the diversity in antibody libraries described here is restricted to the light chain that contributes less to the binding energy in standard antibodies.

[00079] To avoid the requirement of having access to two antibodies having variable light chain domains of the Kappa and Lambda type being perceived as a limitation to the present invention, the methods described herein allow the generation of a hybrid light chain in which a variable Lambda domain can be fused into a constant Kappa domain and conversely a variable Kappa domain can be fused into a constant Lambda domain as described in Figure 3. This broadens the applications of the invention to antibody pairs that share a light chain of the same type. As described in the Examples provided herein, the fusion point between the variable and constant domains is important and can affect the purification process of bispecific antibodies.

[00080] An evaluation of a method for producing the bispecific and / or multispecific antibodies of the invention is shown in Figure 13. In some embodiments, the methods for generating bispecific and / or multispecific antibodies use a chemically defined, serum-free process. These methods incorporate the most widely used mammalian cell line in the pharmaceutical industry, the Chinese Hamster Ovary (CHO) cell line. The methods described herein are used to generate both semi-stable and stable cell lines. The methods can be used to manufacture the bispecific and / or multispecific antibodies of the invention on a small scale (e.g., in an Erlenmeyer flask) or on a medium scale (e.g., in a 25L Wave bag). The methods are of the same Petition 870220016476, dated 24 / 02 / 2022, page 49 / 98 36 / 61 forms are easily adaptable for larger-scale production of the bispecific and / or multispecific antibodies, as well as antibody mixtures of the invention.

[00081] The methods for generating the bispecific and / or multispecific antibodies of the invention are advantageous because they employ a generic purification process as shown in Figure 8A. Figure 16 demonstrates purification and product integrity testing of bispecific antibodies purified from a semi-stable cell line. The bispecific antibodies were purified using the following three-step affinity chromatography procedure: (i) Protein A purification to capture IgG molecules, including both monospecific and bispecific; (ii) KappaSelect purification to capture IgG containing Kappa light chain(s); (iii) LambdaSelect purification to capture IgG containing Lambda light chain. The total flow and elution of each affinity purification step were analyzed by SDS-PAGE.The results demonstrated the removal of each monospecific form (i.e., monospecific IgG molecules having Kappa light chains and monospecific IgG molecules having Lambda light chains) during the purification process (Figure 16A). The purified κλ-containing antibodies (i.e., antibodies having Kappa and Lambda light chains) contained equivalent amounts of Kappa and Lambda light chains (Figure 16B). The purified κλ-containing antibodies showed an intermediate migration pattern on an isoelectric focusing gel when compared to the two monospecific antibodies (Figure 16C).

[00082] The chemically defined processes for manufacturing the bispecific and / or multispecific antibodies of the invention can be used with CHO cell sets or with established cell lines. The results obtained with the chemically defined process using established cell sets or cell lines Petition 870220016476, dated 24 / 02 / 2022, page 50 / 98 37 / 61 demonstrate comparable productivity and growth characteristics to those expressing the corresponding monospecific Kappa or Lambda antibodies. Thus, the κλ body retains the structure and industrial characteristics of a classic human IgG.

[00083] Previous approaches to producing bispecific antibody formats aimed at forcing the production of a homogeneous bispecific molecule using the different antibody creation approaches described above were made at the cost of productivity, scalability, and product stability. The present invention is a different approach that allows the production of a simple antibody mixture that has the standard productivity and scalability characteristics of monoclonal antibodies and provides efficient and generic means to purify the bispecific antibody from the mixture or to purify the antibody mixture. EXAMPLES EXAMPLE 1: Generation of antibody libraries having fixed heavy chains.

[00084] Antibody libraries in which the variable heavy chain domain is identical for all library members were generated as follows. First, the VH3-23 variable heavy chain domain containing a defined CDR3 AKSYGAFDY (SEQ ID NO: 1) (CDR nomenclature according to IMGT) and a defined FR4 sequence was cloned into the pNDS vector using the Sfil and Xhol restriction sites to obtain the pNDS_Vhfixed phagemid vector. The amino acid sequence of VK FR4 corresponds to the FR4 region encoded by the germline genes JK1. The amino acid sequence of Vλ FR4 corresponds to the FR4 region encoded by the germline genes JL2 and JL3. Two variants of the Vk FR4 sequence were generated with a single amino acid substitution at position 106 (Arginine or Glycine). A total of 6 Kappa (VK1-33, VK1-39, VK3-11, Petition 870220016476, dated 24 / 02 / 2022, page 51 / 98 38 / 61 VK3-15, VK3-20, VK4-1) and 5 Lambda variable domain genes (VX-44, VX1-51, VX6-57, VX2-14, VX1-40) containing a wrinkling fragment instead of a CDR3 coding sequence were cloned into pNDS_VHfixed to generate 17 acceptor vectors in which diversity of synthetic or natural origin can be cloned and high diversity libraries can be generated according to the methods described in copending application PCT / US2010 / 035619, filed May 20, 2010, published as WO2010 / 135558, and the methods described in Ravn et al. (2010) Nucl Acid Res 38(21):e193, each of which is incorporated herein by reference in its entirety. This process resulted in the generation of 17 libraries, all having a common VH3-23 domain and VKappa or diversified VLambda domains in the variable light chain complementarity determination region 3 (CDRL3 region), containing a total of 6.9x109 variants (Figure 5A).Sequencing of 180 randomly selected transformants indicated that >90% of the clones had integrated a CDRL3 sequence that was structurally sound and therefore potentially functional. EXAMPLE 2: Rescuing Phages from Libraries

[00085] Each library was independently rescued according to standard phage display procedures briefly summarized below. A cell volume from frozen library aliquots sufficient to coat 10 times the theoretical diversity of the library was added to 500 mL of 2xTYAG (100 pg / mL ampicillin; 2% glucose) and cultured at 37°C with shaking (240 rpm) until an OD600 of 0.3 to 0.5 was reached. The culture was then superinfected with helper phage MK13K07 and incubated for one hour at 37°C (150 rpm). The medium was then changed by centrifuging the cells at 2000 rpm for 10 minutes, removing the medium and resuspending the pellet in 500 mL of 2xTY-AK (100 pg / mL Petition 870220016476, dated 24 / 02 / 2022, p. 52 / 98 39 / 61 ampicillin; 50 pg / mL kanamycin). The culture was then grown overnight at 30°C (240 rpm). The culture was centrifuged at 4,000 rpm for 20 minutes to pellet the cells. The supernatant was collected and 30% (v / v) PEG 8000 (20%) / 2.5M NaCl was added to precipitate the phage particles, incubating the mixture for 1 hour on ice. The phage particles were collected by centrifugation at 10,000 rpm for 30 minutes and resuspended in 10 mL of TE buffer (10 mM Tris-HCl pH 8.0; 1 mM EDTA). The resuspended solution was centrifuged at 10,000 rpm to clear bacterial residues and the precipitation procedure was repeated. After final resuspension, the phage was titrated by E. coli infection and absorption at 260 nm. The level of scFv display on the phage surface was similarly assessed by Western blot analysis using an anti-c-myc monoclonal antibody.Phage purified from different libraries was stored frozen at -80°C after the addition of glycerol at a final concentration of 15% (w / v). EXAMPLE 3: Phage display selections using Fixed Heavy Chain Libraries

[00086] Liquid-phase selections against biotinylated hCXCL10-NusA fusion protein (hCXCL10-NusA) and biotinylated hIL6 receptor complex (hIL6RC): Aliquots of VH-Vk and VH-VX phage libraries (1011-1012Pfu) were kept separate and blocked with PBS containing 3% (w / v) skim milk for one hour at room temperature in a rotary mixer. Blocked phage was then deselected onto streptavidin magnetic beads (Dynal M-280) for one hour at room temperature in a rotary mixer. Deselectioned phage was then incubated with biotinylated hCXCL10-NusA in vivo or hIL6RC (100 nM) for two hours at room temperature in a rotary mixer. Beads were added to the target and captured using a magnetic filament. Petition 870220016476, dated 24 / 02 / 2022, page 53 / 98 40 / 61 followed by four washes with PBS / 0.1% Tween 20 and 3 washes with PBS. Beads were then added directly to 10 mL of exponentially cultured TGI cells and incubated for one hour at 37°C with slow shaking (100 rpm). An aliquot of the infected TGI was serially diluted to titrate the selection yield. The remaining infected TGI was spun at 3000 rpm for 15 minutes and resuspended in 0.5 mL of 2xTYAG (2xTY medium containing 100 pg / mL ampicillin and 2% glucose) and spread on 2xTYAG agar bioassay plates. After overnight incubation at 30°C, 10 mL of 2xTYAG was added to the plates and the cells were scraped from the surface and transferred to a 50 mL polypropylene tube. 2xTYAG containing 50% glycerol was added to the cell suspension to obtain a final glycerol concentration of 17%. Aliquots from the selection circle were kept at -80°C.

[00087] Phage rescue: 100 pg of cell suspension obtained from previous selection circles was added to 20 mL of 2xTYAG and cultured at 37°C with shaking (240 rpm) until an OD600 of 0.3 to 0.5 was reached. The culture was then superinfected with 3.3 x 1010 of helper phage MK13KO7 and incubated for one hour at 37°C (150 rpm). The medium was then changed by centrifuging the cells at 3800 rpm for 10 minutes, removing the medium and resuspending the pellet in 20 mL of 2xTY-AK (100 pg / mL ampicillin; 50 pg / mL kanamycin). The culture was then cultured overnight at 30°C (240 rpm). The following day, an aliquot of the centrifuged supernatant was used as a contribution to the next round of selection.

[00088] Monoclonal phage rescue for ELISA: Single clones were selected in a microtiter plate containing 150 µl of 2xTYAG medium (2% glucose) per well and cultured at 37°C (100-120 rpm) for 5-6 hours. M13K07 helper phage was added to each well to obtain a multiplicity of infection (MOI) of 10 (i.e., Petition 870220016476, dated 24 / 02 / 2022, page 54 / 98 41 / 61 phages per cell in the culture) and incubated at 37°C (100 rpm) for 1 hour. Following growth, plates were centrifuged at 3,200 rpm for 10 minutes. Supernatant was carefully removed, cells resuspended in 150 µl of 2xTYAK medium and cultured overnight at 30°C (120 rpm). For ELISA, phages are blocked by adding 150 µl of 2x concentration PBS containing 5% skimmed milk powder followed by one hour of incubation at room temperature. Plates were then centrifuged for 10 minutes at 3000 rpm and the phage-containing supernatant used for ELISA.

[00089] Phage ELISA: ELISA plates (Maxisorp, NUNC) were coated overnight with 2 pg / mL hCXCLlO-NusA in PBS or 2 pg / mL ML6RC in PBS. Plates were then blocked with 3% skim milk / PBS at room temperature for 1 hour. Plates were washed 3 times with 0.05% Tween 20 PBS before transferring the pre-blocked phage supernatants and incubating for one hour at room temperature. Each clone was tested against both targets to test its specificity. Plates were then washed 3 times with 0.05% Tween 20 PBS. 50 µl of 3% skim milk / PBS containing anti-M13 antibody conjugated by (HRP) (Amersham, diluted 1:10,000) was added to each well. Following incubation at room temperature for 1 hour, the plates were washed 5 times with 0.05% PBS Tween 20. The ELISA was then developed by adding 50 µl of TMB (Sigma) and 50 µl of 2N H2SO4 to stop the reaction. Absorption intensity was read at 450 nm.Specific clones for hCXCL10-NusA or hIL6RC bearing the same variable heavy chain domain could be identified. (Figure 4).

[00090] Phage clone sequencing: Single clones were cultured in 5 mL of 2xTYAG medium (2% glucose) per well and cultured at 37°C (120 rpm) overnight. The next day DNA. Petition 870220016476, dated 24 / 02 / 2022, page 55 / 98 42 / 61 phagemid was purified and used for DNA sequencing using a specific primer for pNDS1: mycseq, 5'CTCTTCTGAGATGAGTTTTTG. (SEQ ID NO: 2).

[00091] Large-scale scFv purification: A 1 mL primer culture of 2xTYAG was inoculated onto a single colony from a freshly streaked 2xTYAG agar plate and incubated with shaking (240 rpm) at 37°C for 5 hours. 0.9 mL of this culture was used to inoculate a 400 mL culture of the same media and was cultured overnight at 30°C with vigorous shaking (300 rpm). The following day, the culture was induced by adding 400 µl of 1M IPTG and incubation was continued for an additional 3 hours. Cells were collected by centrifugation at 5,000 rpm for 10 minutes at 4°C. Pelletized cells were resuspended in 10 mL of TES ice-cold buffer supplemented with protease inhibitors as described above. Osmotic shock was achieved by adding 15 mL of 1:5 diluted TES buffer and incubating for 1 hour on ice. Cells were centrifuged at 10,000 rpm for 20 minutes at 4°C to pelletize cell debris.The supernatant was carefully transferred to a fresh tube. Imidazole was added to the supernatant at a final concentration of 10 mM. 1 mL of Ni-NTA resin (Qiagen), equilibrated in PBS, was added to each tube and incubated in a rotary mixer at 4°C (20 rpm) for 1 hour. The tubes were centrifuged at 2,000 rpm for 5 minutes and the supernatant carefully removed. The pelleted resin was resuspended in 10 mL of chilled Wash Buffer (4°C) 1 (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The suspension was added to a polyprep column (Biorad). 8 mL of chilled Wash Buffer 2 (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) were used to wash the column by gravity flow. The scFv were eluted from the column with 2 mL of buffer. Petition 870220016476, dated 24 / 02 / 2022, page 56 / 98 43 / 61 elution (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Fractions were analyzed by absorption at 280 nm, and protein-containing fractions were pooled before buffer change on a NAP5 desalting column (Amersham) equilibrated with PBS. The scFv in PBS were analyzed by SDS-PAGE and quantified by absorption at 280 nm. The purified scFv were aliquoted and stored at -20°C and 4°C. The purified scFv were used in ELISA to confirm specific binding to the target against which they were selected. EXAMPLE 4: Additional selections using different fixed VH libraries

[00092] Antibody libraries were similarly generated by capturing naturally rearranged light chain repertoires and cloning them in the context of a single VH domain described in Example 1. In this case, the total light chain variable gene region was enlarged from human cDNA using primers corresponding to the 5' and 3' regions of human rearranged variable regions and cloned into the pNDS_VHfixed vector described in Example 1. Another set of libraries was generated as described in Example 1 but using a fixed VH3-23 domain containing a different CDRH3 sequence ARGDDVS (SEQ ID NO: 3). The libraries described above are schematically represented in Figure 5. These fixed VH libraries were used against a panel of target proteins using the selection and evaluation methodology described in Examples 2 and 3. Selections were performed using the following targets: hCXCLlO-NusA, IL-6RC, CD47, CD16, CD8, and hlFNy.Candidates that have been identified and shown to be specific for their targets are listed in Table II. These results demonstrate that different antibody-binding targets having a common heavy chain can be generated and that light chain-restricted diversity is sufficient to confer antigen specificity. Candidates could be generated from libraries containing one. Petition 870220016476, dated 24 / 02 / 2022, page 57 / 98 44 / 61 VH3-23 domain with different CDRH3 sequences or having diversified VL repertoires using different strategies. Therefore, the results demonstrate that the approach is not restricted to a particular VH sequence or a particular variable light chain domain diversification strategy. Table II. Number of independent clones identified against a panel of targets using libraries with a fixed VH sequence containing two different CDRH3 sequences. NA: selection not performed. Targets: Total Fixed CDRI-H SEQ ID:1 Fixed CDRH3 SEQ IDS IC λ K λ K λ hCD16 5 1 4 - - 1 hcos 5 12 2 10 3 2 Í1CD47 21 9 16 4 5 5 IL6RC 17 14 8 7 9 7 fFNy - 5 5 - NusA-CXCLIO 2 4 2 4 NA NA EXAMPLE 5: Fixed VH candidates reformatting into IgG and transient expression in mammalian cells.

[00093] After evaluation, scFv candidates were reformatted into IgG and expressed by transient transfection into PEAK cells. Several IgGα (n=5) and IβκGk (n=9) having a common heavy chain specific for IFNγ and IL6RC, respectively, and having different light chain sequences were generated as follows. The selected scFv VH and VL sequences were amplified with specific oligonucleotides and cloned into an expression vector containing the constant regions of heavy and light chains, and the constructs were verified by sequencing. The expression vectors were transfected into mammalian cells using Fugene 6 Transfection Reagent (Roche, Basel, Switzerland). Briefly, Pico cells were cultured in 6-well plates. Petition 870220016476, dated 24 / 02 / 2022, pp. 58 / 98 45 / 61 cells were transfused at a cell concentration of 6 x 10⁵ per well in 2 mL of culture medium containing fetal bovine serum. Expression vectors encoding candidate VH and VL sequences were co-transfected into the cells using Fugene Transfection Reagent 6 according to the manufacturer's instructions. One day following transfection, the culture media were aspirated, and 3 mL of fresh serum-free media were added to the cells and cultured for three days at 37°C. Following the three-day culture period, the supernatant was harvested for purified IgG on G-Sepharose 4B protein-fast flow columns (Sigma, St. Louis, MO) according to the manufacturer's instructions. Briefly, transfected cell supernatants were incubated overnight at 4°C with ImmunoPure IgG-binding buffer (G) (Pierce, Rockford IL). Samples were then passed over fast-flow columns of G-Sepharose 4B protein and the IgG was subsequently purified using elution buffer.The eluted IgG fraction was then dialyzed against PBS and the IgG content quantified by absorption at 280 nm. IgG purity and integrity were verified by SDS-PAGE. IgG was then tested for IFNγ binding and the IL-6 / IL-6R receptor complex, referred to here as IL6RC, by ELISA. Biotinylated IFNγ and IL6RC were immobilized in streptavidin-coated microplates (Streptawell, Roche) and the plates were then blocked with PBS supplemented with 2% BSA at room temperature for 1 hour. Plates were washed 3 times with 0.05% Tween 20 PBS before adding purified anti-INFγ IgGX or anti-IL6RC IgGK into wells coated with either target. After an hour incubation at room temperature and washing, the antibodies bound to the plates were detected with anti-human Ck or anti-human CX antibodies coupled to horseradish peroxidase.The ELISA was then revealed by adding 50 μl of TMB (Sigma) and 50 μl of 2N H2SO4 to stop the reaction. Petition 870220016476, dated 24 / 02 / 2022, p. 59 / 98 46 / 61 Absorption intensity was read at 450 nm. The results indicate that the binding specificity of scFv isolated from fixed HV libraries was maintained in the IgG format and demonstrated that two IgGs having the same heavy chain but different light chains can be specific for distinct targets (Figure 6).

[00094] The heavy and light chain amino acid sequences of Anti-INFy IgGA or anti-IL6RC IgGK are indicated below: VKappa Light Chain Anti-IL6RC EIVLTQSPATLSLSPGERATLSCRASQSVS3YLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQWLPTTPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLK3 GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGL3SPVTK3FNRGEC (SEQ ID NO: 4) VLambda Anti-INFy Light Chain NFMLTQPHSVSESPGKTVTI3CTR3SGSIASNYVQWYQQRPG3SPTTVIYEDNQRPSGVPDRFS G3ID333N3A3LTI3GLKTEDEADYYCQ3Q3WDGNHIVFGGGTKLTVLGQPKAAP3VTLFPP33 EELQANKATLVCLI3DFYPGAVTVAWKADSSPVKAGVETTTP3KQSNNKYAAS3YLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS{SEQ ID NO: 5) Common heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTTkALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYFSDIAVEWESNG QPENNYKTTFPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG {SEQ ID NO: 6). EXAMPLE 6: Co-expression of a single heavy chain and two light chains in mammalian cells

[00095] The simultaneous expression of one heavy chain and two light chains in the same cell can lead to the assembly of three different antibodies (Figure 8A). Simultaneous expression can be achieved in different ways, such as by transfecting multiple vectors expressing one of the chains to be co-expressed or by using vectors that direct multiple gene expression. A pNovi κHλ vector was designed to allow the co-expression of one heavy chain, one Kappa light chain, and one Lambda light chain (Figure 7A). The expression Petition 870220016476, dated 24 / 02 / 2022, p. 60 / 98 47 / 61 of the three genes are driven by human cytomegalovirus (hCMV) promoters, and the vector similarly contains a glutamine synthetase (GS) gene that allows for the selection and establishment of stable cell lines. Another vector, pNovi kHà, was similarly constructed, in which a second copy of a kappa light chain was inserted after the lambda light chain, and its expression was driven by an internal ribosome entry site (IRES). Using this bicistonic design, the relative expression of the kappa light chain can be increased. The two vectors are schematically represented in Figure 7. EXAMPLE 7: Transient coexpression of a single heavy chain and two light chains in mammalian cells and purification of total IgG.

[00096] The VH and VL genes of IgGX anti-INFy or IgGK anti-IL6RC were cloned into the pNovi kHà vector described in Example 5, for transient expression in mammalian cells. Spike cells were cultured in 6-well plates at a concentration of 6 x 10⁵ cells per well in 2 mL of culture medium containing fetal bovine serum. 2 µg of plasmid DNA was transfected into the cells using TransIT-LTl transfection reagent (Mirus) according to the manufacturer's instructions. One day following transfection, the culture media were aspirated, and 3 mL of fresh serum-free medium was added to cells and cultured for five days at 37°C. Following a five-day culture period, the supernatant was harvested, centrifuged at 4000 rpm, and applied to MabSelect Sure PA resin (GE Healthcare) according to the manufacturer's instructions.Total IgGs were eluted under acidic conditions followed by neutralization and buffer exchange in PBS. IgG content was quantified by absorption at 280 nm and analyzed by SDS-PAGE. The presence of two bands corresponding to the two light chains and one band corresponding to the heavy chain indicated that all three chains could be expressed simultaneously in the same cell (Figure 8B). Petition 870220016476, dated 24 / 02 / 2022, pp. 61 / 98 48 / 61 EXAMPLE 8: Purification of bispecific antibodies carrying a Lambda and a Kappa light chain.

[00097] The co-expression of one heavy chain and two light chains in the same cells can lead to the assembly of three different antibodies: two monospecific antibodies bearing the same light chain in both arms and one bispecific antibody bearing a different light chain associated with the heavy chain in each arm. The latter can be isolated from the monospecific antibodies by chromatographic techniques that take advantage of the different biochemical properties of monospecific and bispecific antibodies. To develop a generic purification approach that is applicable to the entire generation of bispecific antibodies, we used an affinity chromatography approach schematically described in Figure 8A. Supernatants from transfected cell cultures were applied to MabSelect Sure PA resin (GE Healthcare) column and purified total IgG as described in Example 7.Total IgG was then applied to a column containing CaptureSelect Fab Kappa affinity matrix (BAC BV, Netherlands) equilibrated with ten volumes of PBS. The column was then washed with 5-10 column volumes of PBS. Immunoglobulin molecules bearing a kappa light chain were eluted from the column by applying 5 column volumes of 0.1 M Glycine pH 2.0, and fractions were collected and neutralized. The antibody-bearing fractions were pooled before buffer exchange on a PD10 desalting column (Amersham) equilibrated with PBS. The antibody was then applied to a second column containing CaptureSelect Fab Lambda affinity matrix equilibrated with ten volumes of PBS. The column was then washed with 5-10 column volumes of PBS. Immunoglobulin molecules bearing only a kappa light chain bound to the column and were found in the continuous stream. Antibodies carrying a... Petition 870220016476, dated 24 / 02 / 2022, page 62 / 98 49 / 61 Lambda light chain molecules were eluted from the column by applying 5 volumes of 0.1 M Glycine pH 3.0 column, and fractions were collected. Fractions containing the bispecific antibody were pooled before buffer change on a PBS-equilibrated PD10 desalting column (Amersham). Immunoglobulin molecules bearing a Kappa light chain were eluted from the column by applying 5 volumes of 0.1 M Glycine pH 2.0 column, and fractions were collected and neutralized. Fractions containing the antibody were pooled before buffer change on a PBS-equilibrated PD10 desalting column (Amersham). The antibody was then applied to a second column containing CaptureSelect Fab Lambda affinity matrix equilibrated with ten volumes of PBS. The column was then washed with 5-10 volumes of PBS column. Immunoglobulin molecules bearing only the Kappa light chain bind to the column and are found in the continuous flow.Antibodies carrying a Lambda light chain were eluted from the column by applying 5 volumes of 0.1 M Glycine pH 3.0 column, and a fraction was collected. The fractions containing the bispecific antibody were pooled before buffer exchange on a PD10 desalting column (Amersham) equilibrated with PBS. The continuous flow and elution fraction of each purification step were analyzed by SDS-PAGE and indicated that antibodies supporting Lambda light chains were found in the continuous flow of the CaptureSelect Fab Kappa affinity matrix and, conversely, that antibodies supporting Kappa light chains were found in the continuous flow of the CaptureSelect Fab Lambda affinity matrix (Figure 8B). As expected, in the final elution fraction, the intensity of the bands corresponding to the two light chains is equivalent. Thus, these three-step approaches allow the purification of bispecific antibodies supporting a Kappa and a Lambda light chain.The final recovery of bispecific antibody supporting a. Petition 870220016476, dated 24 / 02 / 2022, pp. 63 / 98 50 / 61 Kappa and Lambda light chain was approximately 10-21% in different experiments. The Protein A elution fraction similarly indicated that more Lambda light chain than Kappa light chain is assembled in the purified IgG, suggesting that increased Kappa light chain expression could enhance the assembly and recovery of bispecific antibodies. EXAMPLE 9: Modulation of light chain expression to enhance bispecific antibody assembly

[00098] To increase kappa light chain expression, the common VH gene, the VLambda gene from the anti-INFy antibody, and two copies of VKappa from the anti-IL6RC were cloned into the pNovi kHàk vector described in Example 6. The expression of the second copy of the VKappa light chain is driven by IRES. Different IRES elements were tested to obtain different levels of VKappa light chain expression. This resulted in the construction of five independent vectors: pNovi kHàk 1 to 5. These vectors were used for transient transfections in mammalian cells as described in Example 7, and total IgG was purified to form the supernatant using Protein A affinity purification. SDS-PAGE analysis indicates that the different pNovi kHàk vectors increased kappa light chain expression and assembly in IgG compared to expression using the pNovi kHàk vector (Figure 9).The total IgG fractions obtained after purification of Protein A for each of these constructs were also purified in two consecutive steps using CaptureSelect Fab Kappa and CaptureSelect Fab Lambda as described in Example 8. The yield of bispecific antibodies was 20% for pNovi kHà and ranged between 33 and 41% for pNovi kHàk constructs, indicating that increased Kappa light chain expression led to increased bispecific antibody assembly. To further confirm these results, cotransfections were performed using pNovi kHà and the vector expressing anti-IL6RC IgGK. Petition 870220016476, dated 24 / 02 / 2022, pp. 64 / 98 51 / 61 monospecific assays were performed. In this way, the relative levels of kappa light chain as well as the yields of bispecific antibody were similarly increased, indicating that several approaches can be employed to adjust the relative light chain expression. EXAMPLE 10: Characterization of the purified bispecific antibody

[00099] The purified bispecific antibodies isolated as described in the Examples above were characterized using different techniques. [000100] Isoelectric focusing gel (IEF). The purified bispecific antibodies isolated as described in Example 9 were analyzed using IsoGel Agarose IEF plates with a pH range of 3–10 (Lonza) and compared to the monospecific anti-INFy IgGX and anti-IL6RC IgGK antibodies. After focusing, the gel was placed in fixative solution for 30 minutes, washed with water for 5 minutes, and then dried at room temperature. The gel was stained with Coomassie stain for 15 minutes, briefly rinsed with water, and then with decolorizing solution for 2 x 15 minutes. Finally, the gel was dried at room temperature before imaging. The results shown in Figure 10A indicate that the bispecific IgGKα antibody has a different and intermediate pI compared to the pI of the two monospecific antibodies, as predicted from the antibody shape and theoretical calculations.The staining similarly demonstrated that the bispecific antibody is highly pure after the three-step purification processes described in Example 8. [000101] Ion-exchange chromatography (IEX). 50 µg of purified monospecific and bispecific antibodies were analyzed by Ion-Exchange Liquid Chromatography-High Performance (IEX-HPLC) (Waters e2695 HPLC / detector 2489) using an Agilent Bio Mab column. Petition 870220016476, dated 24 / 02 / 2022, pp. 65 / 98 52 / 61 NP5, (Agilent): the mobile phases were: A: 10mM Na2HPO4 / NaH2PO4, pH 6.5; B: 10 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 6.5; using a flow rate of 0.8 mL / min and a 20%-60% gradient for 133 minutes. The three antibodies have different retention times, with the peak corresponding to the bispecific antibody having an intermediate profile compared to the monospecific antibodies (Figure 10B). [000102] ELISA. Monospecific anti-INFγ IgGX, anti-IL6RC IgGK, and bispecific IgGKα antibodies were tested by ELISA for binding to INFγ and IL6RC. The results shown in Figure 11 indicate that the bispecific IgGKα is capable of binding to both targets and can be detected equally with anti-human Ck and secondary CX antibodies. [000103] Surface Plasmon Resonance (SPR). The affinity and binding kinetics of monospecific anti-INFy IgGX, anti-IL6RC IgGK, and bispecific IgGKα antibodies were characterized using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). 200RU of a goat anti-human polyclonal IgI (ahlgG; Biacore) were immobilized by EDC / NHS chemistry on a Biacore CM5 fragment. This surface was used to capture monospecific or bispecific human IgG. The surface was regenerated after each cycle by injection of 10mM glycine pH=1.5 at 30 µl / min for 30s followed by a 1 min stabilization time in HBSEP buffer. The data were fitted according to a 1:1 Langmuir model and the Kon, Koff, and Kd values ​​were determined (Table III). Similar affinity values ​​were obtained for the monospecific antibodies and the bispecific IgGKα antibodies.The data indicate that the two antibody combining sites bind to hlFNγ and IL-6RC similarly in a monospecific and bispecific manner. [000104] The ability of bispecific IgGKα to interact with both targets simultaneously was tested by SPR. Biotinylated INFγ was Petition 870220016476, dated 24 / 02 / 2022, p. 66 / 98 53 / 61 immobilized on a CM5 Biacore fragment coated with streptavidin. Monospecific and bispecific antibodies were injected onto this surface followed by IL6RC injection. The sensorgram shown in Figure 11A shows that the bispecific IgGKα antibody was able to bind to immobilized INFγ and was able to capture IL6RC simultaneously. SPR was similarly used to assess the relative amounts of Kappa and Lambda light chains in the purified bispecific antibody. IgGKα was directly immobilized via amine coupling on the surface of a CM5 Biacore fragment, and an anti-human CKappa antibody was injected followed by an anti-human CLambda antibody at the same concentration. Equivalent responses were obtained with both anti-light chain antibodies, indicating that, as predicted by the shape, equivalent amounts of Kappa and Lambda light chains are present in the bispecific IgGKα antibody (Figure 12B). Table III. Binding kinetics analysis for monospecific antibodies and bispecific IgGKα antibodies for IFNγ and IL6RC measured on a Biacore 2000 system. Analyzed Ligand KD (M) kon (1 / Ms) koff (1 / s) IFNy IgGKÀ 1.84E-10 9.19E+05 1.69E-04 IgGÀÀ 1.96E-10 6.08E+05 1.19E-04 IL6RC IgGKÀ 2.72E-07 7.44E+03 2.02E-03 IgGKK 2.66E-07 8.08E+03 2.15E-03 EXAMPLE 11: Manufacturing of Bispecific IgGKα Antibodies [000105] The expression of bispecific IgGKα antibody was similarly performed in Chinese Hamster Ovary (CHO) cells, which are widely used for the manufacture of monoclonal antibodies. In the example presented here, both semi-stable sets of transfected CHO cells as well as a stable CHO cell line were generated for the production of IgGKα antibodies. Petition 870220016476, dated 24 / 02 / 2022, pp. 67 / 98 54 / 61 bispecific. In the studies presented here, stable CHO lines were generated and cultivated using a chemically defined, animal component-free manufacturing process (CDACF). The entire process is described in Figure 13. [000106] CHO Sets. CHO cells were electroporated with the linearized pNovi kHàk vector encoding the bispecific IgGKÀ anti-INFy / anti-IL6RC antibody described in the Examples above, as well as with plasmids directing the expression of the monospecific IgGKÀ anti-INFy and IgGK anti-IL6RC. After electroporation, transfected cell sets were cultured under unfed day 10 huge conditions. Cells transfected with the bispecific construct showed similar growth profiles when compared to cells transfected with monospecific expression vectors. Furthermore, productivities were similarly comparable and reached a typical antibody productivity range: between 100-200 mg / L for unfed huge set cultures (Figure 14A). Graduation between small-scale production in an Erlenmeyer flask (100 mL) and medium-scale production in a 25 L Wave Bag was successfully achieved (Figure 14B).These results indicate that similar growth curves and productivities are obtained during expression of bispecific IgGKα antibody in CHO cell lines and the corresponding monospecific antibodies. [000107] Stable CHO cell lines. Recombinant cell lines producing bispecific antibody were generated by electroporation of CHO cells with the pNovi kHàk vector. Post-transfection, recombinant cell lines were selected by diluting the cell culture in the presence of a final concentration of 50 pM of methionine sulfoximine (MSX). After 6 weeks of incubation, colonies of recombinant cell lines were Petition 870220016476, dated 24 / 02 / 2022, pp. 68 / 98 55 / 61 cells were evaluated for total IgG productivity by FastELISA® (R&D Biotech) (Figure 15A-B). Selected cell lines were expanded in cell culture medium containing 25 pm MSX, transferred to 24-well microtiter plates, and evaluated for productivity and growth characteristics in suspension culture (Figure 15C-D). The results revealed that the bispecific antibody IgGKα can be produced under huge batch conditions at a level comparable to cell lines expressing standard monospecific antibodies. Superior production cell lines were selected and operated in huge 50 mL batch cultures in shake flasks for a maximum of 10 days. Protein A HPLC was used for quantification of total IgG in the supernatant.Total IgG from the supernatants of the top 10 production cell lines was purified by MabSelect SuRE chromatography using 1 mL of pre-packed HiTrap columns (GE Healthcare). The relative amounts of monospecific and bispecific antibodies in the purified total IgG were evaluated by IEX-HPLC as described in Example 10. For most cell lines, the bispecific IgGKα antibody fraction ranged from 37-42% of the total IgG, and two cell lines expressed lower amounts of IgGKα (22 and 25%). The results for the CHO 10 cell lines are summarized in Table IV. Table IV. Total antibody titer (mg / mL) Total antibody post-purification MabSelect SuRr (mg) Relative amount of bispecific antibody from IEX-HPLC (%) Total amount of bispecific antibody (mg) cell line 1 0.35 10.73 37 3.97 cell line 0.32 9.54 25 2.37 Petition 870220016476, dated 24 / 02 / 2022, pp. 69 / 98 56 / 61 Total antibody titer (mg / mL) Total antibody post-purification MabSelect SuRr (mg) Relative amount of bispecific antibody from IEX-HPLC (%) Total amount of bispecific antibody (mg) Cell 2 Cell line 3 0.31 10.02 37 3.72 Cell line 4 0.42 10.01 40 4.00 Cell line 5 0.38 11.59 38 4.39 Cell line 6 0.43 11.10 43 4.75 Cell line 7 0.49 12.67 42 5.32 Cell line 8 0.33 8.96 22 2.01 Cell line 9 0.38 9.94 42 4.18 Cell line 10 0.39 10.98 42 4.61 [000108] Purification and characterization of bispecific IgGα antibody expressed in CHO. The supernatant from CHO cell assemblies transfected with bispecific and monospecific constructs was used for purification. The monospecific anti-INFγ IgGX and anti-IL6RC IgGK were purified using Protein A affinity chromatography and desalted in PBS, while the bispecific IgGKα antibodies were purified using the three-step affinity chromatography process described in Example 8. The elution and total flow fractions from the different steps, as well as the final purified samples, were analyzed by SDS-PAGE and IEF (Figure 16). The specificity of the IgGKα was monitored after each purification step by ELISA (Figure Petition 870220016476, dated 24 / 02 / 2022, pp. 70 / 98 57 / 61 17). The results demonstrate that the purification process was robust and compatible with expression in CHO and bispecific IgGKα antibody of highly pure yield. EXAMPLE 12: Additional examples of bispecific IgGKα antibodies [000109] Other examples of bispecific IgGKα antibodies were isolated, expressed, and purified as described in the Examples above. These included a bispecific IgGKα antibody anti-NusA / anti-INFy and a bispecific IgGKα antibody anti-IL6RC / anti-IL6Rc in which both combining sites bind to IL6RC but carry a Kappa and a Lambda light chain. These purified bispecific antibodies were analyzed by IEF together with their respective monospecific counterparts (Figure 18). The results show that the pl of the bispecific antibody is always intermediate between the pl of the monospecific antibodies, but that the differences can vary significantly depending on the sequence of the variable light chain domain.This illustrates that purification of the bispecific antibody based on cost differences could be difficult if the two light chains have similar biochemical properties, and highlights the advantage of the affinity purification approach of the invention. EXAMPLE 13: Generation of bispecific IgGKα antibodies using two variable Kappa or two Lambda domains [000110] Bispecific antibodies can similarly be generated using two variable domains of the same type (Lambda or Kappa). When affinity purification steps require the presence of constant Kappa and constant Lambda domains of the light chains, any variable light chain domain can in principle be fused to these constant domains to generate hybrid light chains as illustrated in Figure 3. This was demonstrated using two antibodies directed against INFγ and IL6RC isolated from fixed VH antibody libraries described in Example 1. In this case, both Petition 870220016476, dated 24 / 02 / 2022, p. 71 / 98 58 / 61 antibodies share the same VH and have a variable Lambda light chain domain. The VLambda domain of the anti-INFy antibody was combined with the constant Lambda domain, while the VLambda domain of the anti-IL6RC antibody was combined with the constant Kappa domain. For the latter, three different constructs were generated including different fusion points between the VLambda domains and the CKappa domain. In one construct (Hybrid 1), the fusion point was at the end of the structure 4 (FR4) region of the VLambda domain and included the entire constant Kappa domain (Figure 19A). In another construct (Hybrid 2), the FR4 Lambda region was replaced by an FR4 Kappa region (Figure 19B). In the third construct (Hybrid 3), the first 4 amino acids of the constant Kappa domain were replaced by the 4 amino acids of the constant Lambda domain (Figure 19C).These different hybrid Lambda-Kappa light chains were cloned along with the common heavy chain and Lambda light chain in the pNovi kHà vector. Mammalian cell transfection, protein expression, and three-step affinity purification of IgGKÀ were performed as described in the Examples above. Analysis of the elution fractions indicated that Hybrid 3 light chain did not bind to the Kappaselect resin and therefore did not allow efficient bispecific antibody purification. Hybrid 1 and Hybrid 2 allowed efficient bispecific IgGKÀ purification. This purified IgGKÀ supporting a hybrid light chain was analyzed on an Agilent Bionalyzer 2100 using a Protein 80 fragment (Agilent Technologies), and the peaks in the electropherogram corresponding to the light chains were shown to be equivalent (Figure 20).The results show that bispecific antibodies of the invention can be generated using two antibodies having a common heavy chain and two light chains having variable domains of the same type (Kappa or Lambda). EXAMPLE 14: Similarly, VH and VL of the antibodies described are Petition 870220016476, dated 24 / 02 / 2022, pp. 72 / 98 59 / 61 involved in antigen binding [000111] To test the contribution of common VH to antigen binding, different scFv light chains supporting a common VH selected in Examples 1 and 4 were combined with two different VHs that are different from the original common VH. The different scFv could be expressed and purified as described in Example 3 and tested for binding against the target they were selected for. Examples shown in Figure 21 show that an INFγ-specific scFv and an IL6RC-specific scFv can only bind their respective targets when combined with the VH domain with which they were originally selected, and no binding is observed when combined with two other VH domains that allow their expression and purification. The results indicate that despite the fact that diversity is restricted to the VL domain, both common VH and VL equally contribute to antigen binding. EXAMPLE 15: Development of an ELISA for quantification of bispecific IgGKα antibody. [000112] A sandwich ELISA for quantifying bispecific IgGKα antibodies was developed. 96-well Maxisorp plates (Nunc) were coated with 10 µg / mL of mouse Lambda anti-human antibody (Southern Biotech) and incubated at 4°C overnight. After washing (0.05% PBS Tween 20, 3 washes), the plate was blocked with 3% PBS-BSA (Sigma) for 2 hours at room temperature. Purified IgGKα standard was serially diluted in 1% PBS-BSA between 500 ng / mL and 1 ng / mL to obtain a good linearity range for sample quantification. Depending on their origin, the samples were diluted to fall within the quantification range as follows: Raw CHO supernatant 1 / 1500; Purified Protein A 1 / 15000. The samples were then serially diluted 1:2. After washing the plates, 50 µL of each dilution Petition 870220016476, dated 24 / 02 / 2022, pp. 73 / 98 A 60 / 61 prepared plate was added in duplicate and incubated for 1 hour at room temperature. Plates were washed again and incubated for 1 hour at room temperature with 50 μL of 1:2000 anti-human Kappa antibody (Southern Biotech). After the last wash (0.05% PBST, 5 washes), the reaction was revealed with 50 μL of TMB substrate (Sigma) and stopped after 15 minutes by adding 50 μL of H2SO4 (2N sodium hydroxide). Absorbance at 450 nm was recorded using a precision microplate reader (Epoch, Witec). Because monospecific antibodies could affect the assay by binding to coated capture antibodies, booster experiments were performed by adding increasing amounts of monospecific IgGK and monospecific lgGX antibodies to the bispecific IgGKα standard. Different ratios were tested: (50% of IgGKα bispecific, 25% • monospecific IgGK, 25% monospecific IgGX); (67% bispecific IgGK; 33% monospecific IgGX); (50% bispecific IgGK, 50% monospecific IgGX); (25% bispecific IgGK, 75% monospecific IgGX); (50% bispecific IgGK, 50% IgGK monospecific). [000113] The results shown in Figure 22 indicate that the assay is not significantly affected by monospecific antibodies and therefore can be used for quantification of bispecific IgGKα antibody in complex samples such as cell culture supernatants. ELISA was used to quantify bispecific IgGKα antibody in the supernatant of stable CHO cell lines and then total IgG purified from the same supernatants by Protein A affinity chromatography. The ELISA quantification results were compared to the total IgG content determined by Protein A HPLC or by absorption at 280 nm and are summarized in Table V. The concentrations obtained by ELISA corresponded to 30-40% IgG. Petition 870220016476, dated 24 / 02 / 2022, pp. 74 / 98 61 / 61 total, a bispecific ratio that is expected. The data show that this assay can be used to determine the amount of bispecific IgGKα antibody in a cell culture supernatant and facilitates the evaluation of stable cell lines for their productivity in bispecific IgGKα antibody. Table V. Quantification of bispecific IgGKα antibody for different stable CHO cell lines using crude supernatants or total IgG by ELISA and compared to HPLC of Protein A and A280 quantification results of total IgG. Cell line CHO supernatants - Total IgG purified by Protein A Prot A HPLC ELISA A280nm ELISA #11 0.3 mg / mL 0.18 mg / mL 4.38 mg / mL 2.3 mg / mL #14 0.42 mg / mL 0.234 mg / mL 4.13 mg / mL 3.3 mg / mL #20 0.38 mg / mL 0.18 mg / mL 4.48 mg / mL 1.7 mg / mL 10E4 0.39 mg / mL 0.19 mg / mL 4.81 mg / mL 3 mg / mL Other Modalities [000114] While the invention has been described together with a 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. Petition 870220016476, dated 24 / 02 / 2022, p. 75 / 98

Claims

1 / 7 CLAIMS 1. A method for generating a bispecific antibody, characterized in that it carries a different specificity at each combining site and consists of 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, and wherein the sequences of the constant and variable structure region are human, comprising: (a) isolating an antibody or antibody fragment region having a specificity determined by a variable heavy chain domain combined with a first variable light chain domain; (b) isolating an antibody or antibody fragment region having a different specificity determined by the same variable heavy chain domain, such as the antibody of step (a) combined with a second variable light chain domain; (c) co-expressing in a cell: i.i. the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region; ii. the first light chain variable domain fused to a kappa-type light chain constant domain or fused to a lambda-type light chain constant domain; and iii. the second light chain variable domain fused to a light chain constant domain of a different type than the constant domain of the first light chain.

2. Method according to claim 1, characterized in that a Kappa light chain variable domain is fused to a Kappa-type constant region, or a Kappa light chain variable domain is fused to a Lambda-type constant region, or a Lambda light chain variable domain is fused to a Kappa-type constant region, or a Lambda light chain variable domain is fused to a Lambda-type constant region.

3. Method according to claim 1 or 2, characterized in that it further comprises step (d) of purifying the bispecific antibodies produced in step (c) from the monospecific antibodies produced in step (c), wherein step (d) is optionally an affinity chromatography purification step.

4. Method according to claim 3, characterized in that the purification step is carried out using specific Kappa constant domain affinity chromatography media, specific Lambda constant domain affinity chromatography media, or both specific Kappa constant domain and specific Lambda constant domain affinity chromatography media.

5. Method according to claim 1 or 2, characterized in that steps (a) and (b) are facilitated by the use of antibody libraries having a common heavy chain and diversity limited to the variable light chain domain, wherein the antibody library is optionally displayed on filamentous bacteriophage, on the surface of yeast, bacteria or mammalian cells or used for ribosome or other type of in vitro display.

6. A method according to any one of claims 1 to 5, characterized in that the generated bispecific antibody is determined by the first light chain comprising at least one constant Kappa region, or at least one constant Kappa region and one variable Kappa region, or at least one constant Kappa region and one variable Lambda region.

7. Method, according to any one of claims 1 to 6, characterized in that the generated bispecific antibody is determined by the second light chain comprising at least a constant Lambda region, or at least a constant Lambda region and a variable Lambda region, or at least a constant Lambda region and a variable Kappa region.

8. A method, according to any one of claims 1 to 7, characterized in that the generated bispecific antibody is determined by the first light chain comprising a constant Kappa region and a variable Kappa region, and in that the second light chain comprises a constant Lambda region and a variable Lambda region.

9. A method, according to any one of claims 1 to 8, characterized in that the bispecific antibody generated is determined by the sequences of the constant and variable structural regions being human.

10. A method for preparing a bispecific antibody that binds specifically to a first antigen and a second antigen, wherein the first and second antigens are different, and wherein the sequences of the constant and variable structure regions are human, characterized in that it comprises: (a) providing a first nucleic acid molecule encoding a first polypeptide comprising a variable heavy chain region of an immunoglobulin polypeptide or a fragment thereof that binds to the first antigen, coupled to a constant immunoglobulin region; (b) providing a second nucleic acid molecule encoding a second polypeptide comprising a variable light chain region of an immunoglobulin polypeptide or a fragment thereof that binds to the first antigen coupled to a first constant light chain region of the Kappa or Lambda type;(c) provide a third nucleic acid molecule encoding a third polypeptide comprising a variable light chain region of an immunoglobulin polypeptide or fragment thereof that shares the same variable heavy chain region of the immunoglobulin polypeptide or fragment thereof from step (a) and binds to the second antigen, coupled to a second constant light chain region of the Kappa or Lambda type, wherein the first and second constant light chain domains are of different types; and (d) grow a host cell comprising the first, second and third nucleic acid molecules under conditions permitting expression of the first, second and third polypeptides.

11. Method according to claim 10, characterized in that it further comprises step (e) of recovering the bispecific antibody.

12. Method according to claim 10, characterized in that the second nucleic acid encodes a variable kappa-type light chain domain or a variable kappa-type light chain domain and a constant kappa-type region, or a variable kappa-type light chain domain and a constant lambda-type region.

13. Method according to claim 10, characterized in that the second nucleic acid encodes a Lambda-type variable light chain domain, or a Lambda-type variable light chain domain and a Kappa-type constant region, or a Lambda-type variable light chain domain and a Lambda-type constant region.

14. Method according to claim 10, characterized in that the third nucleic acid encodes a variable kappa-type light chain domain, or a variable kappa-type light chain domain and a constant kappa-type region, or a variable kappa-type light chain domain and a constant lambda-type region.

15. Method according to claim 10, characterized in that the third nucleic acid encodes a Lambda-type variable light chain domain, or a Lambda-type variable light chain domain and a Kappa-type constant region, or a Lambda-type variable light chain domain and a Lambda-type constant region.

16. Method according to any one of claims 10 to 15, characterized in that the bispecific antibody is recovered in a step (and) using an affinity chromatography purification step, wherein the purification step is optionally performed using specific affinity chromatography media of the specific Kappa constant domain, specific Lambda constant domain or both specific affinity chromatography media of the specific Kappa constant domain and specific Lambda constant domain.

17. Method for generating an antibody mixture, characterized in that it comprises two monospecific antibodies and one bispecific antibody, all having a common heavy chain, comprising: (a) isolating an antibody or antibody fragment region having a specificity determined by a variable heavy chain domain combined with a first variable light chain domain; (b) isolating an antibody or antibody fragment region having a different specificity determined by the same variable heavy chain domain, such as the antibody of step (a) combined with a second variable heavy chain domain; (c) co-expressing in a cell: Petition 870220016476, dated 24 / 02 / 2022, page 80 / 98 6 / 7 i. the common variable heavy chain domain fused to a constant heavy chain region of the immunoglobulin; ii.iii. the first variable light chain domain fused to a constant light chain domain of the Kappa type or fused to a constant light chain domain of the Lambda type; and iii. the second variable light chain domain fused to a constant light chain domain of the Kappa type or fused to a constant light chain domain of the Lambda type, and wherein the sequences of the constant and variable structure region of the common heavy chain, the first light chain and the second light chain are human.

18. Method according to claim 17, characterized in that it further comprises step (d) of purifying the antibody mixture produced in step (c) from cell culture supernatant.

19. Method for generating an antibody mixture, characterized in that it comprises three or more monospecific antibodies and three or more bispecific antibodies, comprising: (a) isolating an antibody or antibody fragment region having a specificity determined by a variable heavy chain domain combined with a first variable light chain domain; (b) isolating several antibodies or antibody fragment regions having a different specificity determined by the same variable heavy chain domain, such as the antibody of step (a) combined with different variable light chain domains; (c) co-expressing in a cell: i. the common variable heavy chain domain fused to a constant immunoglobulin heavy chain region; Petition 870220016476, dated 24 / 02 / 2022, page 81 / 98 7 / 7 ii.all antibody light chains isolated in steps (a) and (b) fused to a constant kappa-type light chain domain or fused to a constant lambda-type light chain domain, wherein the constant and variable structure region sequences are human.

20. Method according to claim 19, characterized in that it further comprises step (d) of purifying the antibody mixture produced in step (c) from cell culture supernatant. Petition 870220016476, dated 24 / 02 / 2022, pp. 82 / 98

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