Antigen-binding polypeptide constructs comprising kappa and lambda light chains and uses thereof

Abstract

To provide multispecific antigen-binding polypeptides comprising immunoglobulin lambda light chains and immunoglobulin kappa light chains.SOLUTION: Provided is an antigen-binding polypeptide construct comprising a first heterodimer and a second heterodimer. The first heterodimer comprises a first immunoglobulin G heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) forming a first Fab region that specifically binds to a first antigen. The second heterodimer comprises a second immunoglobulin G heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) forming a second Fab region that specifically binds to a second antigen. H1 is different from H2, and each of H1 and H2 comprises a heavy chain variable domain and a heavy chain constant domain 1. L1 comprises a lambda light chain variable domain and a lambda light chain constant domain, and L2 comprises a kappa light chain variable domain and a kappa light chain constant domain.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on December 1, 2015, is labeled 0966216 and is 20.0 bytes in size. [Background technology]

[0002] Bispecific antibodies can bind to two different epitopes and are often prepared based on the immunoglobulin heavy and light chains of two different monospecific parent antibodies. Their ability to bind to two different epitopes or antigens makes them attractive tools for therapeutic applications where targeting more than one antigen or epitope is beneficial in the treatment of disease. However, because antibody heavy chains have evolved to bind to antibody light chains in a relatively promiscuous manner, it can be difficult to efficiently produce bispecific antibodies in a form similar to naturally occurring antibodies. As a result of this promiscuous pairing, co-expression of two different heavy chains and two different light chains in a bispecific antibody naturally leads to scrambling of heavy-light chain pairing. This scrambling is a major challenge in creating bispecific therapeutics, where homogeneous pairing is a prerequisite for good manufacturability and biological efficacy.

[0003] Several approaches to preparing bispecific antibodies in a form similar to naturally occurring antibodies have been described, however, these approaches have been developed and exemplified for the case where both parent antibodies used to prepare the bispecific antibody have light chains of the kappa gene family.

[0004] The majority of known therapeutic antibodies (and therefore potential parent antibodies) have kappa light chains, although some also have lambda light chains. Kappa and lambda light chains differ from each other in both structure and sequence.

[0005] A review of various approaches for producing bispecific antibodies from two parent antibodies in a format similar to naturally occurring antibodies can be found in Klein et al., (2012) mAbs 4:6, 1-11. International Patent Application No. PCT / EP2011 / 056388 (WO2011 / 131746) describes an in vitro method for producing heterodimeric proteins in which asymmetric mutations are introduced into the CH3 regions of two monospecific starting proteins to promote directional exchange of "Fab arms" or "half molecules" between two monospecific IgG4 or IgG4-like antibodies upon incubation under reducing conditions.

[0006] U.S. Patent Publication No. 2009 / 0182127 (Novo Nordisk, Inc.) describes the generation of bispecific antibodies by modifying amino acid residues at the Fc connecting portion and the CH1:CL connecting portion of light-heavy chain pairs to reduce the ability of one light chain to interact with the other heavy chain. International Patent Publications WO2014 / 081955 (Amgen) and WO2014 / 150973 (Eli Lilly) describe amino acid residues in lambda light chains that may be modified to confer desired pairing specificity. Neither of these publications describes complementary amino acid modifications that can be used to prepare bispecific antibodies from one parent antibody having a kappa light chain and another parent antibody having a lambda light chain. International Patent Publication No. WO2012 / 131555 (Glenmark) describes replacing the connecting portion between the heavy chain and lambda light chain of an antibody with that of a TCR (T cell receptor) domain connecting portion. Summary of the Invention

[0007] The present disclosure provides a multispecific antigen-binding polypeptide comprising an immunoglobulin lambda light chain and an immunoglobulin kappa light chain. In one aspect, the antigen-binding polypeptide is a construct comprising a first heterodimer and a second heterodimer. In one embodiment, the first heterodimer (H1L1) comprises a first immunoglobulin heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) that form a first Fab region that specifically binds to a first antigen; the second heterodimer (H2L2) comprises a second immunoglobulin heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) that form a second Fab region that specifically binds to a second antigen. In some embodiments, H1 is different from H2. In some embodiments, H1 and H2 comprise a heavy chain variable domain (VH domain) and a heavy chain constant domain 1 (CH1 domain). In one embodiment, L1 comprises a lambda light chain variable (VL-lambda) domain and a lambda light chain constant (CL-lambda) domain. In one embodiment, L2 comprises a kappa light chain variable (VL-kappa) domain and a kappa light chain constant (CL-kappa) domain. In some embodiments, one or more of H1, H2, L1, and L2 comprise amino acid modifications compared to the corresponding wild-type H1, H2, L1, and L2 polypeptide sequences, wherein the amino acid modifications promote preferential pairing of L1 to H1 relative to L2 and / or promote preferential pairing of L2 to H2 relative to L1. In some embodiments, the amino acid modifications do not introduce new cysteine ​​residues. In one embodiment, the amino acid modifications do not remove naturally occurring cysteine ​​residues.

[0008] In some embodiments, the construct comprises amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 and / or promote preferential pairing of L2 to H2 relative to L1 when H1, H2, L1, and L2 are co-expressed in a cell or mammalian cell, or when H1, H2, L1, and L2 are co-expressed in a cell-free expression system, or when H1 and L1 are produced in a (first) cell and H2 and L2 are produced in a second (e.g., different) cell and the products of the two cells are mixed via a redox-generating method, or when H1 and L1 are produced in a first cell-free expression system and H2 and L2 are produced in a second (e.g., different) cell-free expression system and the products of the two cell-free expression systems are mixed.

[0009] In some embodiments of the construct, each heterodimer comprises a single Fab.

[0010] In some embodiments of the antigen-binding polypeptide constructs described herein, a. H2 comprises an amino acid substitution at position 143; L2 comprises an amino acid substitution at position 124; and i. H1 comprises an amino acid substitution at position 186 or 179, and L1 comprises an amino acid substitution at position 180; ii. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; iii. H1 comprises an amino acid substitution at position 143; L1 comprises an amino acid substitution at position 133; or iv. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 178; b. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; i. H2 contains amino acid substitutions at positions 186 or 124 and 186, and L2 contains amino acid substitutions at positions 133 or 133 and 160 or 124 and 133 or 176 and 180; or ii. H2 contains an amino acid substitution at position 188; L2 contains an amino acid substitution at position 131; iii. H2 contains an amino acid substitution at position 143; L2 contains amino acid substitutions at positions 124 and 133 or 124, 133 and 180; c. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; i. H2 comprises amino acid substitutions at positions 124 and 186 or 124 and 179 or 188, and L2 comprises amino acid substitutions at positions 176 and 178 or 176 and 180 or 131; or ii. H2 contains amino acid substitutions at positions 143 and 188 or 143 or 124 and 143; L2 contains amino acid substitutions at positions 124, 176 and 178 or 124 and 178 or 124 and 180 or 124, 176 and 180, or 124, or 124 and 176; d. H1 contains an amino acid substitution at positions 179, 186, 143, and / or 188; L1 contains an amino acid substitution at positions 180, 133, and / or 176 and 178; H2 contains an amino acid substitution at position 143, and L2 contains an amino acid substitution at positions 131 and / or 124; e. H1 contains an amino acid substitution at position 39 or does not contain an amino acid substitution that promotes preferential pairing; L1 contains an amino acid substitution at position 38 or does not contain an amino acid substitution that promotes preferential pairing; H2 contains an amino acid substitution at position 39 and L2 contains an amino acid substitution at position 38; f. H1 comprises an amino acid substitution at position 143; L1 comprises an amino acid substitution at position 131; i. H2 comprises amino acid substitutions at positions 188 or 124 and 186, and L2 comprises amino acid substitutions at positions 176 and 178 or 176 and 180 or 131; or ii. H2 contains an amino acid substitution at position 143 or 186; L2 contains amino acid substitutions at positions 124 and 133 or 124, 133 and 180; g. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at positions 176 and 178 or 178; and i. H2 comprises amino acid substitutions at positions 177 and 188, and L2 comprises amino acid substitutions at positions 176 and 178; or ii. H2 contains amino acid substitutions at positions 186 or 124 or 124 and 179; L2 contains amino acid substitutions at positions 176 or 131 and 176; h. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; i. H2 comprises an amino acid substitution at position 188 and L2 comprises an amino acid substitution at position 131; or ii. H2 contains amino acid substitutions at positions 177 and 188; L2 contains amino acid substitutions at positions 176 and 178; or H1 contains amino acid substitutions at positions 124 and 190; L1 contains amino acid substitutions at position 135; H2 contains amino acid substitutions at positions 124 or 188, and L2 contains amino acid substitutions at positions 176 or 176 and 178; i. H1 contains amino acid substitutions at positions 177 and 188; L1 contains amino acid substitutions at positions 176 and 178; and a. H2 comprises an amino acid substitution at position 188 and L2 comprises an amino acid substitution at positions 176 and 178 or 131; b. H2 contains an amino acid substitution at position 186; L2 contains amino acid substitutions at positions 133 or 124 and 160 and 180; c. H2 contains amino acid substitutions at positions 124 or 124 and 179 or 124 and 186; L2 contains amino acid substitutions at positions 176 or 176 and 178 or 176 and 180; or d. H2 contains an amino acid substitution at position 143; L2 contains amino acid substitutions at positions 133 or 124 and 133; j. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 178; H2 contains an amino acid substitution at positions 124 or 188; L2 contains an amino acid substitution at positions 176 and 178 or 176 and 180 or 176; k. H1 comprises an amino acid substitution at positions 145 and 188; L1 comprises an amino acid substitution at position 178; H2 comprises an amino acid substitution at positions 124 and / or 188; L2 comprises an amino acid substitution at one or more of 124, 133, and 178; l. H1 contains an amino acid substitution at position 174, 179 or 186; L1 contains an amino acid substitution at position 176 or 180; H2 contains an amino acid substitution at position 143 or 190, and L2 contains an amino acid substitution at position 131, 135 or 124; or m. H1 contains an amino acid substitution at position 174; L1 contains an amino acid substitution at position 176; H2 contains an amino acid substitution at position 190; L2 contains no amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 135; n. H1 contains amino acid substitutions at positions 143 and 190; L1 contains amino acid substitutions at position 133; H2 contains amino acid substitutions at position 124; L2 contains amino acid substitutions at positions 131 and 135; o. H1 contains an amino acid substitution at position 143 and / or 186; L1 contains an amino acid substitution at position 133; H2 contains an amino acid substitution at position 124; L2 contains an amino acid substitution at position 131; p.H1 contains amino acid substitutions at positions 143 and 179; L1 contains amino acid substitutions at positions 124 and 178; H2 contains an amino acid substitution at position 186, and L2 contains amino acid substitutions at positions 178 and 180 or 160 and 180; q. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 124; H2 contains an amino acid substitution at position 179 or 186, and L2 contains amino acid substitutions at positions 124, 160, and 180; r. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at positions 180 or 178 and 180; H2 contains an amino acid substitution at positions 143 and / or 179, and L2 contains an amino acid substitution at positions 124 and 178 or 131; s. H1 contains an amino acid substitution at position 179; L1 contains an amino acid substitution at position 180; H2 contains an amino acid substitution at position 143, and L2 contains an amino acid substitution at position 124; t. H1 contains an amino acid substitution at position 143 or 186; L1 contains an amino acid substitution at position 180 or no amino acid substitution that promotes preferential pairing; H2 contains amino acid substitutions at positions 143 and 145, and L2 contains an amino acid substitution at position 124; u. H1 does not contain an amino acid substitution that promotes preferential pairing; L1 contains an amino acid substitution at position 135; H2 contains an amino acid substitution at position 139, and L2 contains an amino acid substitution at position 116; v. H1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 45; L1 does not contain an amino acid substitution that promotes preferential pairing; H2 contains an amino acid substitution at position 45, and L2 contains an amino acid substitution at position 44; w. H1 comprises an amino acid substitution at position 139; L1 comprises an amino acid substitution at position 116; H2 does not comprise an amino acid substitution that promotes preferential pairing, and L2 comprises an amino acid substitution at position 135; or x. H1 comprises an amino acid substitution at position 124; L1 comprises an amino acid substitution at position 176; H2 comprises an amino acid substitution at position 124; L2 comprises an amino acid substitution at position 176.

[0011] In some embodiments, the affinity of the first Fab region for a first antigen is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, or 100-fold of the affinity of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences for the first antigen, and / or the affinity of the second Fab region for a second antigen is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, or 100-fold of the affinity of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences for the second antigen.

[0012] In some embodiments, the melting temperature (Tm) of the first Fab region is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Tm of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences for the first antigen, and / or the melting temperature (Tm) of the second Fab region is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Tm of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences for the second antigen.

[0013] In some embodiments of the antigen-binding polypeptide construct, a. H1 and L1 are wild-type polypeptide sequences, and H2 and L2 each comprise at least one amino acid modification; b. one or more of H1, L1, and H2 comprises at least one amino acid modification, and L2 is a wild-type polypeptide sequence; c. one or more of H1, L1, and L2 comprises at least one amino acid modification, and H2 is a wild-type polypeptide sequence; d. one or more of H1, H2, and L2 comprises at least one amino acid modification, and L1 is a wild-type polypeptide sequence; e. one or more of L1, H2, and L2 comprises at least one amino acid modification, and H1 is a wild-type polypeptide sequence; or f. H1, L1, H2, and L2 each comprise at least one amino acid modification.

[0014] In some embodiments, the amino acid modification is a. CH1 domains of H1 and H2, CL-lambda domain of L1, and CL-kappa domain of L2; or b. In the CH1 and VH domains of H1 and H2, the CL-lambda and VL-lambda domains of L1, and the CL-kappa and VL-kappa domains of L2.

[0015] In some embodiments, the amino acid modification is a. at least two of the CH1 domain of H1, the CH1 domain of H2, the CL-lambda domain of L1, and the CL-kappa domain of L2; b. at least two of the CH1 and VH domains of H1 and H2, the CL-lambda and VL-lambda domains of L1, and the CL-kappa and VL-kappa domains of L2; or c. In at least two of the VH domain of H1, the VH domain of H2, the VL-lambda domain of L1, and the VL-kappa domain of L2.

[0016] In some embodiments, H1, L1, H2, and / or L2 comprise at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid mutations in the Fab region. In some embodiments, at least one of H1, H2, L1, and L2 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications in at least one constant domain and / or at least one variable domain.

[0017] In one embodiment, one or more of H1, H2, L1, and L2 contain amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 to form H1L1, or that promote preferential pairing of L2 to H2 relative to L1 to form H2L2, such that the relative pairing of at least one of H1L1 or H2L2 is at least about 10% greater than wild type and the relative pairing of the other is within about 10% of wild type or at least about 10% greater than wild type.

[0018] In some embodiments, the amino acid modifications promote preferential pairing of L1 and H1 relative to L2 to form H1L1, or promote preferential pairing of L2 and H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least 40:60 and the ratio of H2L2:H2L1 is at least 60:40; or the amino acid modifications promote preferential pairing of L1 and H1 relative to L2 to form H1L1, or promote preferential pairing of L2 and H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least 40:60 and the ratio of H1L1:H1L2 is at least 60:40.

[0019] In some embodiments, the thermal stability of the first Fab region is within about 0, 1, 2, or 3°C of the Tm of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences. In some embodiments, the thermal stability of the second Fab region is within about 0, 1, 2, or 3°C of the Tm of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences.

[0020] In some embodiments, the amino acid modification is selected from the group consisting of the unique identifier Mab design sets shown in Table 4A or 4B. In some embodiments, the amino acid modification is selected from the group consisting of the unique identifier Mab design sets shown in one or more of Tables 10-A1 through 10-A12. In some embodiments, the amino acid modification is selected from the group consisting of the unique identifier Mab design sets shown in any one of Tables 10-B1 through 10-B10.

[0021] In some embodiments, the construct further comprises a dimeric Fc having two Fc polypeptides, each comprising a CH3 domain sequence, linked, with or without a linker, to one of the first and second Fab regions. In some embodiments, the Fc is a human Fc, human IgG1 Fc, human IgA Fc, human IgG Fc, human IgD Fc, human IgE Fc, human IgM Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc comprises one or more modifications compared to wild-type in at least one of the CH3 domain sequences that promote the formation of a heterodimeric Fc.

[0022] In some embodiments, Fc is i) a heterodimeric IgG1 Fc with the modifications L351Y_F405A_Y407V in the first Fc polypeptide and T366L_K392M_T394W in the second Fc polypeptide; ii) a heterodimeric IgG1 Fc with the modifications L351Y_F405A_Y407V in the first Fc polypeptide and T366L_K392L_T394W in the second Fc polypeptide; iii) a heterodimeric IgG1 Fc with the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide and T350V_T366L_K392L_T394W in the second Fc polypeptide; iv) a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide and T350V_T366L_K392M_T394W in the second Fc polypeptide; or v) A heterodimeric IgG1 Fc having the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide.

[0023] In some embodiments, the Fc further comprises at least one CH2 domain sequence. In one embodiment, the Fc comprises one or more modifications to promote selective binding of an Fc-γ receptor, reduce or eliminate binding to an Fc-γ receptor, or promote binding to FcRn.

[0024] In some embodiments, when H1, L1, H2, and L2 are co-expressed, the change in the total amount of correct pairing as measured by the sum of H1L1 and H2L2 pairing is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains without amino acid substitutions in the Fab region that promote preferential pairing, or the change in the total amount of correct pairing as measured by the amount of bispecific antibody produced as a percentage of species other than half antibodies produced that promote preferential pairing. greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the ab region, or the change in the total amount of correct pairing as measured by the amount of bispecific antibody produced as a percentage of all species produced is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab region that promote preferential pairing.

[0025] In some embodiments, the linker comprises one or more polypeptide linkers, one or more antibody hinge regions, or one or more IgG1 hinge regions. In one embodiment, the one or more polypeptide linkers comprise one or more modifications compared to a wild-type polypeptide linker.

[0026] In some embodiments, the amino acid modification comprises an amino acid substitution.

[0027] In some embodiments, one or more of the sequences H1, H2, L1, and L2 are derived from human or humanized sequences.

[0028] In some embodiments, the constructs described herein are conjugated to a therapeutic agent or drug.

[0029] In another aspect, the present disclosure provides an isolated recombinant polynucleotide or a set of isolated recombinant polynucleotides encoding the constructs described herein. In some embodiments, a vector or a set of vectors is provided that includes one or more of the polynucleotides or sets of polynucleotides described herein. In some embodiments, the vector or at least one vector of the set of vectors is polycistronic.

[0030] In another aspect, the present disclosure provides an isolated cell comprising a polynucleotide or set of polynucleotides, or a vector or set of vectors described herein. In some embodiments, the cell is a yeast cell, a bacterial cell, an insect cell, or a mammalian cell. In some embodiments, the isolated cell is stably or transiently transfected with a vector or set of vectors described herein.

[0031] In another aspect, there is provided a pharmaceutical composition comprising the antigen-binding polypeptide construct described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises one or more substances selected from the group consisting of a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, and an excipient.

[0032] In another aspect, methods for preparing the constructs described herein are described. In some embodiments, the methods include: (a) obtaining a host cell containing a polynucleotide or set of polynucleotides encoding an antigen-binding polypeptide construct; (b) culturing the host cells in the host cell culture under conditions that allow expression of the antigen-binding polypeptide construct; and (c) harvesting the antigen-binding polypeptide construct from the host cell culture.

[0033] In some embodiments, host cells are transiently or stably transfected with a polynucleotide or set of polynucleotides described herein.

[0034] In another aspect, a computer-readable storage medium is provided. In some embodiments, the computer-readable storage medium stores a dataset comprising data representing complementary amino acid modifications in a first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin lambda light chain polypeptide sequence (L1); and a second heterodimer comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin kappa light chain polypeptide sequence (L2). In some embodiments, the H1 and H2 polypeptide sequences stored in the dataset comprise at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain) and are different from each other. In some embodiments, the L1 and L2 polypeptide sequences stored in the dataset comprise at least a light chain variable domain (VL domain) and a light chain constant domain (CL domain). In some embodiments, the complementary amino acid modifications stored in the dataset promote preferential pairing of L1 to H1 relative to L2, and promote preferential pairing of L2 to H2 relative to L1. In some embodiments, the dataset includes data representing those modifications or a subset of those modifications listed in Table 4A or Table 4B. In some embodiments, the dataset includes data representing those modifications or a subset of those modifications listed in one or more of Tables 10-A1 through 10-A12 or Tables 10-B1 through 10-B10.

[0035] In another aspect, methods for producing a bispecific antigen-binding polypeptide construct are described. In some embodiments, the bispecific antigen-binding polypeptide construct produced by the method comprises: a. a first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin lambda light chain polypeptide sequence (L1); and b. A second heterodimer comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin kappa light chain polypeptide sequence (L2).

[0036] In some embodiments, the H1 and H2 polypeptide sequences produced by the method comprise at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain) and are distinct from one another. In some embodiments, the L1 and L2 polypeptide sequences produced by the method comprise a light chain variable domain (VL domain) and a light chain constant domain (CL domain). In some embodiments, one or more of the H1, L1, H2, and L2 polypeptide sequences produced by the method comprise amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 and that promote preferential pairing of L2 to H2 relative to L1.

[0037] In some embodiments, the method for producing a bispecific antigen-binding polypeptide construct comprises: a. introducing one or more complementary amino acid modifications from the datasets described herein into H1, L1, H2, and / or L2; and b. Co-expressing H1, L1, H2, and L2 in a host cell to produce an expression product comprising the bispecific antigen-binding polypeptide construct.

[0038] In some embodiments, the method further comprises determining the amount of the bispecific antigen-binding polypeptide construct in the expression product relative to other polypeptide products and selecting a preferred subset of complementary amino acid modifications that result in an increased amount of the bispecific antigen-binding polypeptide construct compared to the amount of the bispecific antigen-binding polypeptide construct in the expression product obtained from co-expression of wild-type H1, L1, H2, and L2. In some embodiments, the bispecific antigen-binding polypeptide construct is produced with a purity of 70% or greater relative to the other polypeptide products. In some embodiments, the construct produced by the method comprises an Fc comprising at least two CH3 domain sequences, wherein the Fc is linked to the first heterodimer and the second heterodimer with or without one or more linkers. In some embodiments, the Fc is a heterodimeric Fc comprising one or more amino acid modifications that promote the formation of a heterodimeric Fc over a homodimeric Fc.

[0039] In some embodiments of the methods of generating a bispecific antigen-binding polypeptide construct, when H1, L1, H2, and L2 are co-expressed, the change in the total amount of correct pairing as measured by the sum of %H1L1 and %H2L2 produced compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%; or the change in the total amount of correct pairing as measured by the amount of bispecific antibody produced as a percentage of species other than half antibodies produced. the change is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing; or the change in the total amount of correct pairing as measured by the amount of bispecific antibody produced as a percentage of all species produced is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1 shows the D3H44, pertuzumab, and CAT-2200 heavy and light chain amino acid sequences aligned to human germline sequences of the variable and constant domains. The translated protein sequences for each domain, germline, and allele were obtained by direct query to IMGT / GENE-DB (http: / / www.imgt.org / genedb / query). IMGT / DomainGapAlign (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) was used to determine the closest gene / allele. Consensus sequences were identified by BoxShade (http: / / www.ch.embnet.org / software / BOX_form.html) with a 0.8 cutoff. Black-shaded amino acid residues represent amino acid sequence identity, while gray-shaded residues represent amino acid sequence similarity. The assignment of amino acids to each domain in Figure 1 was made according to the IMGT definition described in Lefranc M.-P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains," Dev. Comp. Immunol., 2005, 29, 185-203, and Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, G., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27, 55-77 (2003). FIG. 1A shows the pertuzumab and D3H44 variable heavy (VH) domains aligned to human IGHV and IGHJ germline subgroups (one representative sequence is shown per gene and allele).The gene and allele sequences closest to the pertuzumab IGHV and IGHJ are X92218|IGHV3-66*01 and J00256|IGHJ4*01, respectively. The gene and allele sequences closest to the D3H44 IGHV and IGHJ are X92218|IGHV3-66*01 and J00256|IGHJ4*01, respectively. Figure 1B shows the pertuzumab and D3H44 variable light chain (VL) domains aligned to the human kappa IGKV and IGKJ germline subgroups (one representative sequence is shown for each gene and allele). The gene and allele sequences closest to the pertuzumab IGKV and IGKJ are Y14865|IGKV1-NL1*01 and J00242|IGKJ2*01, respectively. The closest gene and allelic sequences to D3H44 IGKV and IGKJ are X59315|IGKV1-39*01 and J00242|IGKJ1*01, respectively. Figure 1C shows the pertuzumab and D3H44 constant heavy chain 1 (CH1) domains aligned to the human CH1 IGHG germline subgroup. The closest gene and allelic sequences to pertuzumab and D3H44 IGHG are J00228|IGHG1*01. Figure 1D shows the pertuzumab and D3H44 constant light chain (CL) domains aligned to the human kappa IGKC germline subgroup. The closest gene and allelic sequences to pertuzumab and D3H44 IGKC are J00241|IGKC*01. Figure 1E shows the CAT-2200 VH domain aligned to human IGHV and IGHJ germline subgroups (one representative sequence is shown for each gene and allele). The closest gene and allele sequences to CAT-2200 IGHV and IGHJ are M99660|IGHV3-23*01 and J00256|IGHJ4*01, respectively. Figure 1F shows the CAT-2200 VL domain aligned to human lambda IGLV and IGLJ germline subgroups (one representative sequence is shown for each gene and allele). The closest gene and allele sequences to CAT-2200 IGLV and IGLJ are Z73673|IGLV6-57*01 and M15641|IGLJ2*01, respectively.Figure 1G shows the CAT-2200 CH1 domain aligned to the human CH1 IGHG germline subgroup. The closest gene and allelic sequence to CAT-2200 IGHG is J00228|IGHG1*01. Figure 1H shows the CAT-2200 CL domain aligned to the human lambda IGLC germline subgroup. The closest gene and allelic sequence to CAT-2200 IGLC is J00253|IGLC2*01. [Figure 2]

[0033] Figure 1 shows a flowchart for computational modeling of interface residues and design with preferred heavy-light chain pairings. [Figure 3] Figure 3 shows a 3D structural alignment between the constant domains of D3H44 (PDB ID 1JPT) and CAT-2200 (PDB-ID 2VXS). Figure 3A illustrates the general conformational differences observed between kappa and lambda light chains when aligned on their respective heavy chains. Figure 3B provides a diagram of the light chain junction (with the heavy chain removed) of the model presented in Figure 3A to further illustrate the conformational differences. The dotted arrow indicates the conformational rearrangement of secondary structure elements at the junction between the heavy and light chains. [Figure 4]Figure 1 shows a high-level schematic of the engineering requirements for forming bispecific antibodies and the assay requirements necessary to quantify heavy-chain / light-chain (HL) pairing. The design goal of engineering bispecific antibodies with high purity (i.e., little to no mismatched HL association) can be achieved by rational engineering of the preferential pairing of two unique heavy chains to unique cognate light chains (through the introduction of specific amino acid mutations). This process is shown schematically; here, H1 has been engineered to preferentially pair with L1 (indicated by a check mark) rather than L2 (indicated by an "X"). Similarly, H2 has been engineered to preferentially pair with L2 rather than L1. Arrows on the H1L1 and H2L2 heterodimers represent the affinity for pairing between these HL pairs, while arrows on the H1L2 and H2L1 heterodimers represent disruption of pairing between the latter HL pair. Experimental screening of designs to promote preferential pairing requires an assay capable of simultaneously quantifying H1L1:H1L2 and H2L2:H2L1. These assay requirements can be simplified by assuming that each bispecific Fab arm can be independently engineered. In this case, the assay need only quantify H1L1:H1L2 or H2L2:H2L1, and not both simultaneously. [Figure 5] A schematic diagram is provided showing how heavy and light chains can be tagged and how preferential pairing is determined. In this schematic, the circular boundary represents cells transfected with three constructs (one heavy chain and two unique light chains). The expression products are secreted from the cells, and the supernatant (SPNT) is passed over a detection device, in this case, an SPR chip. Based on the detection of two different tags fused to the two light chains that compete for heavy chain pairing, a quantitative estimate of the preferential pairing of the heavy chain to the two light chains can be obtained. [Figure 6]Performance filtering criteria based on two LCCA results for each design are shown. These performance filtering criteria were used to identify the KL design library and the KK-derived KL design library. To be included, a design should have a positive LCCA result above the neutral zone (the neutral zone is defined as the region between a match:mismatch ratio of 40:60 and 60:40), while the other LCCA must be above the lower limit of the neutral zone (a match:mismatch ratio of 40:60). Scenarios A and B represent designs that pass the filtering criteria. Note that scenario B is included because the H2L2:H2L1 LCCA is above the neutral zone and the H1L1:H1L2 LCCA is within (but not below) the neutral zone. Scenario C represents a design that is excluded if both LCCA results are positive but neither are above the neutral zone. Scenarios D and E represent designs that are excluded because at least one of the LCCA results is below the neutral zone, even if the other LCCA is above the neutral zone (see Scenario D). Designs with the H1L1 and H2L2 designations reversed are also included. This diagram description assumes a 50:50 wild-type pairing ratio. [Figure 7] Figure 1 shows the performance of selected KL designs and KK-derived KL designs (based on the LCCA data for the Mab design set in Tables 4A and 4B), defined by design strength = ΔH1:L1:L2_scalar + ΔH2:L2:L1_scalar. This metric is an indicator of overall pairing success at the design level. [Figure 8] Shown are the possible heavy chain associated products expected when two different light chains are co-expressed with two different heavy chains in a cell. [Figure 9] FIG. 1 shows a general method for preparing bispecific antigen-binding polypeptide constructs using the Mab design set libraries provided herein. [Figure 10]Figure 10 shows min-max boxplots summarizing the performance of all KL designs tested in SMCA in the three bispecific systems by design cluster. Figure 10A shows performance as measured by total bispecific calculation (Δbispecific%). Figure 10B shows performance as measured by total association calculation (Δassociation%). [Figure 11] Min-max box-and-whisker plots summarizing the performance of KL designs and KK-derived KL designs per bispecific system by transferability group are shown; "kl 3 / 3" indicates KL designs transferable in a 3 / 3 bispecific system, "kl 3 / 3 + 2 / 3" indicates KL designs transferable in at least two bispecific systems, and "kl all" indicates all KL designs tested. Similarly, "kk 3 / 3" indicates KK-derived KL designs transferable in a 3 / 3 bispecific system, "kk 3 / 3 + 2 / 3" indicates KK-derived KL designs transferable in at least two bispecific systems, and "kk all" indicates all KK-derived KL designs tested; results are presented based on the total bispecific calculation (Δbispecificity%). [Figure 12] DSC sensorgrams of bispecific antibodies produced using Mab design set 3972 (SMCA design ID) (in each of the three bispecific systems) and the wild-type parent antibody per system are shown. Figure 12A shows wild-type CAT-2200 mAb (dark gray), wild-type Pertuzumab mAb (medium gray), and design 3972 CAT-2200 / Pertuzumab SMCA (light gray); Figure 12B shows wild-type CAT-2200 mAb (dark gray), wild-type SGN-CD19a mAb (medium gray), and design 3972 CAT-2200 / SGN-CD19a SMCA (light gray); Figure 12C shows wild-type SGN-CD19a mAb (dark gray), wild-type CR8071 mAb (medium gray), and design 3972 CR8071 / SGN-CD19a SMCA (light gray). [Figure 13] A min-max boxplot summarizing the effect of amino acid substitutions in Mab designs on the Tm of the Fabs tested is shown. Results are reported as change in Fab Tm relative to wild type, and are shown for all designs where Tm was measured ("All") and separated by paratope. [Figure 14]A min-max boxplot summarizing the effect of amino acid substitutions in Mab designs on the affinity of the Fabs tested for that antigen is shown. Results are reported as the difference in log(KD) of the appropriate Fab of the bispecific from wild type (-(log(KD_mutant)-log(KD_wt)). Results are shown for all designs where affinity was measured ("All") and separated by paratope. [Figure 15] Figure 15 shows UPLC-SEC profiles of Protein A and preparative SEC-purified bispecific and parental antibodies. Figure 15A shows the wild-type parental CAT-2200 mAb; Figure 15B shows the wild-type parental CR8071 mAb; Figure 15C shows the wild-type parental SGN-CD19a mAb; Figure 15D shows the wild-type parental Pertuzumab mAb; Figure 15E shows the bispecific antibody produced using Design 3972 CAT-2200 / Pertuzumab SMCA; Figure 15F shows the bispecific antibody produced using Design 3972 CAT-2200 / SGN-CD19a SMCA; and Figure 15G shows the bispecific antibody produced using Design 3972 CR8071 / SGN-CD19a SMCA. [Figure 16]

[0111] Figure 16A shows the process for selecting wild-type reference values ​​to calculate the "change in total association relative to wild-type" and the "change in total bispecificity relative to wild-type" for designs tested in SMCA when the corresponding wild-type bispecific constructs were not evaluated by SMCA. Figure 16A shows the process for selecting wild-type reference values ​​for "total association" ("% H1L1 and % H2L2 association") for each of the three bispecific systems; Figure 16B shows the process for selecting wild-type reference values ​​for "total bispecificity" ("H1L1_H2L2 and H1L2_H2L1**") for each of the three bispecific systems. DETAILED DESCRIPTION OF THE INVENTION

[0041] Provided herein are engineered antibodies (also referred to herein as multispecific antigen-binding polypeptide constructs) that can include a first heterodimer (H1L1) having a first immunoglobulin heavy chain (H1) and an immunoglobulin lambda light chain (L1) paired to form a first Fab region, and a second heterodimer (H2L2) having an immunoglobulin heavy chain (H2) and an immunoglobulin kappa light chain (L2) paired to form a second Fab region. The first Fab region typically binds a first antigen, and the second Fab region typically binds a second antigen. In some embodiments, the first and second antigens are different from each other. H1 is different from H2. One or more of the immunoglobulin heavy and light chains are engineered to contain amino acid modifications that promote preferential pairing of the correctly paired heavy and light chains (H1L1 or H2L2) when co-expressed or co-produced. More specifically, the amino acid modifications promote preferential pairing between each heavy chain and the correct light chain, such that the heavy chain of the first heterodimer (H1) preferentially pairs with L1 over L2, and the heavy chain of the second heterodimer (H2) preferentially pairs with L2 over L1. As a result, coexpression of the H1, L1, H2, and L2 polypeptides enables the production of correctly paired bispecific antibodies with reduced or limited mispairing, potentially reducing the number and amount of mispaired species produced and improving manufacturability. In one embodiment, amino acid modifications in the Fab region pair with amino acid modifications in the Fc region to promote the formation of the heterodimeric Fc region and further reduce the amount of mispaired heavy chains. The amino acid modifications do not significantly affect the thermal stability of the correctly paired heterodimer or the binding affinity of the correctly paired heterodimer for antigen, compared to heterodimers formed from wild-type H1 and L1 or H2 and L2 polypeptides, respectively.

[0042] Also provided herein are methods of making the multispecific antigen-binding polypeptide constructs described above.

[0043] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In the event that there are multiple definitions for terms herein, those in this section prevail. When citation is made by a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may change, but that similar information may be found by searching the Internet. Additionally, reference evidences the availability and public dissemination of such information.

[0044] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the subject matter of any claimed invention. As used herein, the use of the singular includes the plural unless specifically stated otherwise.

[0045] In describing the present invention, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer, etc.), unless otherwise indicated. As used herein, "about" means ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the recited range, value, sequence, or structure, unless otherwise indicated. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components, unless otherwise indicated or dictated by context. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably. Additionally, it should be understood that the individual single chain polypeptides or immunoglobulin constructs derived from various combinations of the structures and substituents described herein are disclosed by this application to the same extent as if each single chain polypeptide or heterodimer were described individually. Thus, the selection of the particular components to form the individual single chain polypeptides or heterodimers is within the scope of this disclosure.

[0046] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, manuals, and treatises, are expressly incorporated herein by reference in their entirety for any purpose.

[0047] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0048] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of illustration and disclosure, such as the constructs and methodology described in the publications, which may be used in connection with the methods, compositions, and compounds described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0049] In this application, amino acid names and atom names (e.g., N, O, C, etc.) are used as defined by the Protein Data Bank (PDB) (www.pdb.org) based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984), as modified from Eur. J. Biochem., 152, 1 (1985). The term "amino acid residue" primarily refers to the 20 naturally occurring amino acids, namely, alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Glycine or Glycine), and ribozyme (Ribozyme). (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

[0050] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. These terms apply not only to naturally occurring amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0051] The term "nucleotide sequence" or "nucleic acid sequence" is intended to indicate a contiguous stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combination thereof.

[0052] "Cells," "host cells," "cell lines," and "cell cultures" are used interchangeably herein and it should be understood that all such terms include progeny resulting from the propagation and culturing of a cell. "Transformation" and "transfection" are used interchangeably to refer to the process of introducing a nucleic acid sequence into a cell.

[0053] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., compounds in which a carbon is linked to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Reference to amino acids includes, for example, naturally occurring proteinogenic L-amino acids; D-amino acids, chemically modified amino acids (such as amino acid variants and derivatives); naturally occurring non-proteinogenic amino acids such as alanine and ornithine; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, N-methylamino acids (e.g., methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, hydroxy-histidine, homohistidine), amino acids with an extra methylene in the side chain ("homo" amino acids), and amino acids in which a carboxylic acid functionality in the side chain has been replaced with a sulfonic acid group (e.g., cysteic acid). Incorporation of non-natural amino acids, including synthetic, non-native amino acids, substituted amino acids, or one or more D-amino acids, into the proteins of the antigen-binding polypeptide constructs described herein can be advantageous in many different ways. Peptides and the like containing D-amino acids exhibit increased stability in vitro or in vivo compared to their counterparts containing L-amino acids.Thus, constructing peptides and the like that incorporate D-amino acids can be particularly useful when greater intracellular stability is desired or required. More specifically, when such properties are desirable, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby improving the bioavailability of the molecule and extending its in vivo longevity. Furthermore, D-peptides and the like cannot be efficiently processed due to major histocompatibility complex class II-restricted presentation to T helper cells and are therefore less likely to induce a humoral immune response in the whole organism.

[0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0055] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also represents all possible silent variations of that nucleic acid. Those of skill in the art will understand that each codon in a nucleic acid can be modified to result in a functionally identical molecule (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan). Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0056] With respect to amino acid sequences, those skilled in the art will understand that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where such changes result in the deficiency of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0057] Conservative substitution tables providing functionally similar amino acids are known to those of skill in the art. Each of the following eight groups contains amino acids that can be considered conservative substitutions for one another: Alanine (A), glycine (G); aspartic acid (D), glutamic acid (E); Asparagine (N), Glutamine (Q); Arginine (R), Lysine (K); Isoleucine (I), leucine (L), methionine (M), valine (V); Phenylalanine (F), tyrosine (Y), tryptophan (W); and serine (S), threonine (T); (See, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)).

[0058] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences being the same. Sequences are "substantially identical" or "substantially similar" if the percentage of amino acid residues or nucleotides are the same when the sequences are compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the sequence comparison algorithms below (or other algorithms available to those of skill in the art), or manually aligned and visually inspected. This definition also refers to the complement of a test sequence. The identity may exist over a region at least about 50 amino acids or nucleotides in length, or over a region 75-100 amino acids or nucleotides in length, or, if not specified, over the entire sequence of the polynucleotide or polypeptide. Polynucleotides encoding polypeptides of the antigen-binding polypeptide constructs described herein, including homologs from species other than human, can be obtained by a process comprising screening a library under stringent hybridization conditions with a labeled probe having the polynucleotide sequence of the antigen-binding polypeptide constructs described herein, or a fragment thereof, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art.

[0059] Examples of suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST™ and BLAST™ 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST™ analyses is publicly available through the National Center for Biotechnology Information (see the internet at www.ncbi.nlm.nih.gov). Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from the maximum achieved; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. Exemplary algorithm parameters for the BLASTN program (for nucleotide sequences) are a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, exemplary algorithm parameters for the BLASTP program are a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

[0060] A derivative or variant of a polypeptide is said to share "homology" or be "homologous" to a peptide if the amino acid sequence of the derivative or variant has at least 50% identity over a sequence that is 100 amino acids in length from the original peptide. In certain embodiments, a derivative or variant is at least 75% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 85% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of a derivative is at least 90% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of a derivative is at least 95% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 99% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative.

[0061] As used herein, an "isolated" polypeptide or construct means a construct or polypeptide that has been identified and separated and / or recovered from components of its natural cell culture environment, which contaminant components are materials that would typically interfere with diagnostic and therapeutic uses for the heteromultimer, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0062] In certain embodiments, an "isolated" antigen-binding polypeptide construct, as used herein, describes an antigen-binding polypeptide construct that has been identified and separated and / or recovered from a component of its natural cell culture environment. For example, the isolated bispecific antigen-binding polypeptide constructs described herein comprise a heterodimer pair or "isolated" heterodimer pair, and include heterodimers or heterodimer pairs that have been identified and separated and / or recovered from a component of their natural cell culture environment. Contaminant components of their natural environment are substances that would interfere with diagnostic and therapeutic uses of the heterodimer or antigen-binding polypeptide construct, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0063] Heterodimers and antigen-binding polypeptide constructs can be purified to substantial homogeneity. The terms "substantially homogeneous," "substantially homogeneous form," and "substantial homogeneity" are used to indicate that a correctly paired product is substantially devoid of by-products resulting from undesired polypeptide combinations (e.g., homodimers or mismatched heterodimers). In the context of the LCCA design set (H1L1L2), a correctly paired product is a heterodimer comprising H1 and L1 (H1L1). In the context of the LCCA design set (H2L1L2), a correctly paired product is a heterodimer comprising H2 and L2 (H2L2). In one embodiment, in the context of a bispecific antigen-binding polypeptide construct, when H1, L1, H2, and L2 are expressed, a correctly paired product is a heterodimer pair comprising a correctly paired H1L1 and H2L2 (H1L1H2L2). In some embodiments, in the context of a bispecific antigen-binding polypeptide construct, when H1, L1, H2, and L2 are expressed, correctly paired products may include additional products showing correct pairing in at least one Fab region, such as, for example, H1L1H2L1 or H1L2H2L2, or, when a "half antibody" is produced, H1L1 or H2L2. When expressed in terms of purity, in one embodiment, substantial homogeneity means that the amount of completely mismatched by-products does not exceed 20% of the total LC-MS intensity from all species present in the mixture, e.g., less than 10%, less than 5%, less than 1%, or less than 0.5%, where the % reflects the results from mass spectrometry analysis.

[0064] Each term understood by those skilled in the art of antibody technology is given its art-acquired meaning unless expressly defined otherwise herein. Antibodies are known to have variable regions, hinge regions, and constant domains. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0065] As used herein, the terms "antibody" and "immunoglobulin" or "antigen-binding polypeptide construct" are used interchangeably. An "antigen-binding polypeptide construct" refers to a polypeptide substantially encoded by an immunoglobulin gene(s), or one or more fragments thereof, that specifically binds to an analyte (antigen). Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin isotypes IgG, IgM, IgA, IgD, and IgE, respectively. Furthermore, antibodies can belong to one of many subclasses; for example, IgG can belong to the IgG1, IgG2, IgG3, or IgG4 subclasses.

[0066] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one immunoglobulin "light" chain (approximately 25 kD) and one immunoglobulin "heavy" chain (approximately 50-70 kD). This type of immunoglobulin or antibody structural unit is considered "naturally occurring." The term "light chain" includes full-length light chains and fragments thereof having sufficient variable domain sequence to confer binding specificity. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The light chain variable domain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains. The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable domain, VH, and three constant domains, CH1, CH2, and CH3. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxyl terminus, and CH3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, IgD, and IgE. The term "variable region" or "variable domain" generally refers to the portion of the antibody light and / or heavy chain involved in antigen recognition, typically comprising about 120-130 amino-terminal amino acids in the heavy chain (VH) and about 100-110 amino-terminal amino acids in the light chain (VL).

[0067] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) help maintain the proper conformation of the CDRs to facilitate binding between the antigen-binding region and the antigen. Structurally, framework regions may be located between the CDRs in an antibody. Variable regions typically exhibit the same general structure: relatively conserved framework regions (FRs) connected by three hypervariable region CDRs. The CDRs of the two chains of each pair are typically aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both light-chain and heavy-chain variable regions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain typically follow the definition in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)).

[0068] A "multispecific antigen-binding polypeptide construct" or "multispecific antibody" targets or binds to more than one different antigen or epitope. A "bispecific," "dual-specific," or "bifunctional" antigen-binding polypeptide construct or antibody is a type of multispecific antigen-binding polypeptide construct that targets or binds to two different antigens or epitopes. Generally, a bispecific antigen-binding polypeptide construct may have two different antigen-binding domains. The two antigen-binding domains of a bispecific antigen-binding polypeptide construct or antibody bind to two different epitopes that may be present on the same or different molecular targets. In one embodiment, the bispecific antigen-binding polypeptide construct is in a naturally occurring form. In other words, the bispecific antigen-binding polypeptide construct has the same form as a naturally occurring IgG, IgA, IgM, IgD, or IgE antibody.

[0069] An antibody heavy chain pairs with an antibody light chain, and they abut or contact each other at one or more "connecting portions." A "connecting portion" comprises one or more "contact" amino acid residues in a first polypeptide that interact with one or more "contact" amino acid residues in a second polypeptide. For example, connecting portions exist between the two CH3 domains of a dimerized Fc region, between the CH1 domain of the heavy chain and the CL domain of the light chain, and between the VH domain of the heavy chain and the VL domain of the light chain. The "connecting portion" may be derived from an IgG antibody, for example, a human IgG1 antibody.

[0070] As used herein, the term "amino acid modification" includes, but is not limited to, amino acid insertions, deletions, substitutions, chemical modifications, physical modifications, and rearrangements.

[0071] The amino acid residues of immunoglobulin heavy and light chains are determined by the sequence of amino acids in Kabat (Kabat and Wu, 1991; Kabat et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication no. 91-3242, p 647 (1991)), IMGT (Lefranc, M.-P., et al., IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark), Nucl. Acids Res, 37, D1006-D1012 (2009), and Lefranc, M.-P., IMGT, the International ImMunoGeneTics Information System, Cold Spring Harb Protoc. 2011 Jun 1; 2011 (6)), 1JPT (Katja Faelber, Daniel Kirchhofer, Leonard Presta, Robert F Kelley, Yves A Muller, The Domains may be numbered according to several conventions, including EU (as described in "1.85 Å resolution crystal structures of tissue factor in complex with humanized fab d3h44 and of free humanized fab d3h44: revisiting the solvation of antigen combining sites1, Journal of Molecular Biology, Volume 313, Issue 1, Pages 83-97") and EU (according to the EU index similar to Kabat, which refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85)). Kabat numbering is used herein for the VH, CH1, CL, and VL domains unless otherwise indicated.EU numbering is used herein for the CH3 and CH2 domains and hinge region unless otherwise indicated. Table 22A provides a correspondence table showing the amino acid numbering of selected positions in IgG1 heavy chain polypeptides using the IMGT, Kabat, 1JPT, and EU numbering systems. Table 22B provides a correspondence table showing the amino acid numbering of selected positions in lambda light chain polypeptides using the IMGT and Kabat numbering systems. Table 22C provides a correspondence table showing the amino acid numbering of selected positions in kappa light chain polypeptides using the IMGT, 1JPT, and Kabat numbering systems.

[0072] Antigen-binding polypeptide constructs The antigen-binding polypeptide constructs (i.e., antibodies) described herein may be multispecific or bispecific. A multispecific antigen-binding polypeptide construct may comprise at least one first heterodimer (H1L1) having a first immunoglobulin heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) forming a first Fab region, and at least one second heterodimer (H2L2) having an immunoglobulin heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) forming a second Fab region, wherein H1 and H2 are different from each other. In one embodiment, the bispecific antigen-binding polypeptide construct comprises a first heterodimer (H1L1) having a first immunoglobulin heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) forming a first Fab region, and a second heterodimer (H2L2) having an immunoglobulin heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) forming a second Fab region, wherein H1 and H2 are different from each other. In one embodiment, each heterodimer comprises a single Fab region. The term "Fab region," as used herein, refers to the region resulting from the pairing of one immunoglobulin heavy chain polypeptide sequence with one immunoglobulin light chain polypeptide sequence, and consists of the VH and CH1 domains of the immunoglobulin heavy chain polypeptide sequence and the VL and CL domains of the immunoglobulin light chain polypeptide sequence. In some embodiments, the first Fab region binds a first antigen, and the second Fab region binds a second antigen. The first and second antigens can be the same or different. One or more of the immunoglobulin heavy and light chains can contain amino acid modifications that promote preferential pairing of correctly paired heavy and light chains when co-expressed or co-produced.

[0073] When the antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct (ie, a bispecific antibody), it may also be referred to as a "heterodimeric pair."

[0074] For purposes of illustration, the first heterodimer of the antigen-binding polypeptide construct is referred to as H1L1 and comprises a first immunoglobulin heavy chain polypeptide sequence (H1) paired with an immunoglobulin lambda light chain polypeptide sequence (L1), and the second heterodimer is referred to as H2L2 and comprises a second immunoglobulin heavy chain polypeptide sequence (H2) paired with an immunoglobulin kappa light chain polypeptide sequence (L2). However, this designation is arbitrary and should be understood as meaning only to specify that one heterodimer comprises an immunoglobulin kappa light chain and the other comprises an immunoglobulin lambda light chain. The Fab region of the first heterodimer, H1L1, may also be referred to herein as a "lambda Fab"; whereas, the Fab region of the second heterodimer, H2L2, may also be referred to herein as a "kappa Fab."

[0075] Parent antibody The immunoglobulin heavy chain polypeptide sequence (also referred to as the "heavy chain") and immunoglobulin light chain polypeptide sequence (also referred to as the "light chain") of each heterodimer can be derived from one or more parent antibodies, where at least one parent antibody comprises a kappa light chain and at least one other parent antibody comprises a lambda light chain, and amino acid modifications that promote preferential pairing are engineered into these heavy and light chains. The parent immunoglobulin heavy chain and immunoglobulin light chain sequences that lack the amino acid modifications that promote preferential pairing are referred to as wild-type immunoglobulin heavy chain polypeptide sequences, wild-type immunoglobulin kappa light chain polypeptide sequences, and wild-type immunoglobulin lambda light chain polypeptide sequences. In one embodiment, the heavy and light chains of the heterodimers of the antigen-binding polypeptide construct are derived from two parent antibodies. Generally, the two parent antibodies are different from each other; however, this is not always necessarily the case. In one embodiment, the antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct, where each heterodimer is derived from a different parent antibody. In another embodiment, the antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct, in which both parent antibodies bind to the same antigen but target different epitopes on the same antigen. In one embodiment, at least one parent antibody is monospecific, i.e., capable of binding to only one epitope. In another embodiment, at least one parent antibody is capable of binding to more than one epitope.

[0076] The heavy and light chains of each heterodimer of the antigen-binding polypeptide construct pair to form a Fab region that specifically binds to the same antigen as the parent antibody from which it is derived. For example, if a bispecific antigen-binding polypeptide construct is prepared based on the parent antibodies CAT-2200 (which contains a lambda light chain and binds IL-17A) and D3H44 (which contains a kappa light chain and binds tissue factor), the heavy and light chains of one heterodimer pair to form a Fab region that binds IL-17A, and the heavy and light chains of the second heterodimer pair to form a Fab region that binds tissue factor.

[0077] The parent antibody may be derived from any species including, but not limited to, human, mouse, rat, rabbit, sheep, cow, goat, or camel. In one embodiment, the parent antibody may be derived from a human or mouse.

[0078] Parent antibodies may also include those prepared from hybridomas using standard monoclonal antibody production protocols, such as those described by Kohler and Milstein (Nature, 256:495-497, 1975).

[0079] Antibodies that bind to specific targets may be identified by many different strategies, including phage display, in vitro display, and other methods. These strategies can result in scFv, Fab, or full-length IgG forms. A review of these strategies can be found in Chapter 4 of Therapeutic Antibody Engineering by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1907568379, October 2012. In one embodiment, the parent antibody includes an antibody identified by phage display or in vitro display. Antibodies identified in forms other than Fab or full-length IgG can be converted into the same forms known in the art. Methods for converting scFvs into Fabs are well known in the art (see, for example, Steinwand et al., Mabs 6:204-218, or Zuberbuhler et al., Protein Engineering, Design & Selection 22:169-174). In one embodiment, an antibody may be used as a parent antibody even if it was originally identified as an scFv, but the scFv has been converted to a Fab form and engineered into the form of a traditional or naturally occurring antibody.

[0080] In one embodiment, the heavy and light chains of each heterodimer of the antigen-binding polypeptide construct can be derived from a parent antibody that is a humanized antibody. A humanized antibody can be obtained by substituting the complementarity-determining regions (CDRs) of an antibody derived from a non-human mammal, such as a mouse, with the CDRs of a human antibody. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342:877). General recombinant DNA techniques suitable for this purpose are also known (see European Patent Application Publication No. EP 125023, WO 96 / 02576). For example, the CDRs of a mouse antibody can be determined by known methods, and DNA can be prepared to encode an antibody in which the CDRs are ligated to the framework regions (FRs) of a human antibody. The humanized antibody can then be produced using a system using a conventional expression vector. Such DNA can be synthesized by PCR using several oligonucleotides designed to have overlapping portions at both ends of the CDR and FR regions as primers (see the method described in WO98 / 13388). The human antibody FRs linked via the CDRs are selected so that the CDRs form an appropriate antigen-binding site. If necessary, amino acids in the FRs of the antibody variable region can be modified to enable the CDRs of the reshaped human antibody to form an appropriate antigen-binding domain (Sato, K. et al., Cancer Res. (1993) 53:851-856). Amino acid residues in the FR that can be modified include those that bind directly to the antigen via non-covalent bonds (Amit et al., Science (1986) 233:747-53), those that affect or influence the CDR structure to some extent (Chothia et al., J. Mol. Biol. (1987) 196:901-17), and those that are involved in VH-VL interactions (EP239400 Patent Publication).

[0081] In one embodiment, the heavy and light chains of each heterodimer of the antigen-binding polypeptide construct can be derived from a parent antibody that is a chimeric antibody. Chimeric antibodies are antibodies prepared by combining sequences from different animals. For example, a chimeric antibody can be generated by combining heavy and light chain variable domains from a mouse antibody with heavy and light chain constant domains from a human antibody. Chimeric antibodies can be prepared by known methods. To obtain such a chimeric antibody, for example, DNA encoding an antibody variable domain can be ligated with nucleic acid encoding a human antibody constant domain; the resulting ligation product can be inserted into an expression vector; the construct can be introduced into host cells to produce the chimeric antibody.

[0082] The heavy and light chains of the heterodimer can be derived from many parent antibodies known in the art. Almost any antibody can serve as a parent antibody, provided it contains an immunoglobulin heavy chain polypeptide sequence that pairs with an immunoglobulin light chain polypeptide sequence to form an antigen-binding Fab region. In one embodiment, at least one parent antibody is a therapeutic antibody, i.e., an antibody used to treat a disease. Non-limiting examples of suitable therapeutic antibodies containing a kappa light chain and an antigen that binds to the antibody are identified in Table A below:

[0083] TIFF2025124635000002.tif214170TIFF2025124635000003.tif233170TIFF2025124635000004.tif233170 TIFF2025124635000005.tif237170TIFF2025124635000006.tif233170TIFF2025124635000007.tif220170

[0084] Non-limiting examples of suitable therapeutic antibodies comprising a lambda light chain and an antigen that binds to the antibody are identified in Table B below:

[0085] TIFF2025124635000008.tif152170

[0086] Immunoglobulin subclasses The immunoglobulin heavy chain of the parent antibody is within the following classes: IgA1, IgA2, IgM, IgD, IgE, IgG1, IgG2, IgG3, and IgG4. In one embodiment, the first and second heterodimers of the antigen-binding polypeptide construct comprise an IgG heavy chain. In one embodiment, the first and second heterodimers of the antigen-binding polypeptide construct comprise an IgG1 heavy chain. The immunoglobulin light chain of the parent antibody is either a kappa light chain or a lambda light chain.

[0087] The antigen-binding polypeptide constructs described herein comprise at least one heterodimer having an immunoglobulin heavy chain polypeptide sequence and an immunoglobulin kappa light chain polypeptide sequence, and at least another heterodimer having an immunoglobulin heavy chain polypeptide sequence and an immunoglobulin lambda light chain polypeptide sequence. In one embodiment, the antigen-binding polypeptide construct comprises one heterodimer having an IgG heavy chain polypeptide sequence and an immunoglobulin kappa light chain polypeptide sequence, and another heterodimer having an IgG heavy chain polypeptide sequence and an immunoglobulin lambda light chain polypeptide sequence.

[0088] In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a VH domain selected from VH domain germline groups IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, or IGHV7. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a VH domain from germline subgroup IGHV3. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a J segment selected from J segment germline genes IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, or IGHJ6. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a J segment from germline subgroup IGHJ4. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a CH1 domain selected from CH1 domain germline subgroups IGHG1, IGHG2, IGHG3, or IGHG4. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a CH1 domain from germline subgroup IGHG1.

[0089] In heterodimers comprising lambda light chain polypeptide sequences, the lambda light chain may comprise a CL-lambda domain selected from the germline gene IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer with a lambda light chain comprising a CL-lambda domain from germline subgroup IGLC2. The lambda light chain may comprise a VL-lambda domain selected from germline subgroups IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10, or IGLV11. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer with a lambda light chain having a VL-lambda domain from germline subgroup IGLV6. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a lambda light chain with a lambda J segment selected from the J segment germline genes IGLJ1, IGLJ2, IGLJ3, IGLJ6, or IGLJ7, hi another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a lambda J segment from germline subgroup IGLJ2.

[0090] In heterodimers containing a kappa light chain polypeptide sequence, the kappa light chain is a CL germline allele IGKC * 01.IGKC * 02.IGKC * 03.IGKC * 04, or IGKC * 05. In one embodiment, the antigen-binding polypeptide construct may comprise a CL-kappa domain selected from germline subgroup IGKC. *and heterodimers having a kappa light chain comprising a CL-kappa domain from IGKV1. The kappa light chain may comprise a VL-kappa domain selected from germline subgroup IGKV1, IGKV1D, IGKV2, IGKV3, IGKV4, IGKV5, or IGKV6. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a kappa light chain with a VL-kappa domain from germline subgroup IGKV1. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a kappa light chain with a J segment selected from J segment germline genes IGKJ1, IGKJ2, IGKJ3, IGKJ4, or IGKJ5. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a kappa light chain with a J segment from germline subgroup IGKJ1 or IGKJ2.

[0091] An immunoglobulin heavy chain typically comprises at least one variable (VH) domain and three constant domains, CH1, CH2, and CH3. In one embodiment, each heavy chain of the first and second heterodimers of the antigen-binding polypeptide construct comprises a VH domain, a CH1 domain, a CH2 domain, and a CH3 domain. In another embodiment, each heavy chain of the first and second heterodimers comprises a VH domain, a CH1 domain, and a CH3 domain. In yet another embodiment, each heavy chain of the first and second heterodimers comprises a VH domain and a CH1 domain. An immunoglobulin light chain typically comprises one variable (VL) domain and one constant (CL) domain. In one embodiment, the light chain of each heterodimer comprises a VL domain and a CL domain.

[0092] As noted above, in some embodiments, the immunoglobulin heavy chain and immunoglobulin light chain polypeptide sequences of each heterodimer can be derived from known therapeutic antibodies or antibodies that bind to various target molecules or cancer antigens. The amino acid and nucleotide sequences of many such molecules are readily available (e.g., GenBank Accession No. AJ308087.1 (humanized anti-human tissue factor antibody D3H44 light chain variable region and CL domain); GenBank Accession No. AJ308086.1 (humanized anti-human tissue factor antibody D3H44 heavy chain variable region and CH1 domain); GenBank Accession No. HC359025.1 (pertuzumab Fab light chain gene module); GenBank Accession No. HC359024.1 (pertuzumab Fab heavy chain gene module); GenBank Accession No. GM685465.1 (antibody trastuzumab (=Herceptin)-wild type; light chain ... See NCBI Accession No. GM685463.1 (antibody trastuzumab (=Herceptin)-wild-type; heavy chain); GenBank Accession No. GM685466.1 (antibody trastuzumab (=Herceptin)-GC-optimized light chain); and GenBank Accession No. GM685464.1 (antibody trastuzumab (=Herceptin)-GC-optimized heavy chain). The sequence of each of the above-mentioned polypeptides is available from the NCBI website as of November 28, 2012, and each is incorporated herein by reference in its entirety for all purposes. The amino acid and nucleotide sequences of cetuximab are also known in the art; see, for example, the Drug Bank website supported by the Canadian Institutes of Health Research, Alberta Innovates-Health Solutions, and The Metabolomics Innovation Centre (TMIC), accession number DB00002.

[0093] Amino acid modifications that promote preferential pairing One or more of the heavy and light chains H1, L1, H2, and L2 comprise amino acid modifications that promote preferential pairing between the heavy and light chains engineered into the heavy and light chains of the parent antibody. In one embodiment, two of the heavy and light chains H1, L1, H2, and L2 comprise amino acid modifications that promote preferential pairing between the heavy and light chains. In one embodiment, three of the heavy and light chains H1, L1, H2, and L2 comprise amino acid modifications that promote preferential pairing between the heavy and light chains.

[0094] In some embodiments, the amino acid modifications may be asymmetric, such that the amino acid positions that are modified are different between H1 and H2, and between L1 and L2.

[0095] In one embodiment, H2 and L2 contain amino acid modifications that promote preferential pairing between heavy and light chains, while H1 and L1 do not contain amino acid modifications that promote preferential pairing between heavy and light chains. In one embodiment, H1, L1, and H2 contain amino acid modifications that promote preferential pairing between heavy and light chains, while L2 does not contain amino acid modifications that promote preferential pairing between heavy and light chains. In one embodiment, H1, H2, and L2 contain amino acid modifications that promote preferential pairing between heavy and light chains, while L1 does not contain amino acid modifications that promote preferential pairing between heavy and light chains. In one embodiment, L1, H2, and L2 contain amino acid modifications that promote preferential pairing between heavy and light chains, while H1 does not contain amino acid modifications that promote preferential pairing between heavy and light chains.

[0096] In one embodiment, the one or more amino acid modifications comprise one or more amino acid substitutions. When H1 or H2 are co-expressed with L1 and L2, or when H1, L1, H2, and L2 are co-expressed, the amino acid modifications promote preferential pairing of L1 with H1 and preferential pairing of L2 with H2. As noted above, for purposes of illustration, heterodimers of antigen-binding polypeptide constructs will be identified as follows: an H1L1 heterodimer comprises a lambda light chain, L1, and an H2L2 heterodimer comprises a kappa light chain, L2.

[0097] As used herein, "Mab design" or "Mab design set" refers to a specific set of amino acid modifications that promote preferential pairing, also identified as H1L1H2L2, present in one set of H1, L1, H2, and L2. The amino acid modifications in one or more of H1, L1, H2, and L2 that promote preferential pairing are referred to and presented as a Mab design or Mab design set (i.e., H1L1H2L2). Mab design sets are first tested as LCCA design sets (i.e., H1L1L2 or H2L1L2) to determine the strength of pairing specificity when H1 and H2 are independently co-expressed with L1 and L2.

[0098] In one embodiment, amino acid modifications can be made to one or more amino acids that are part of the junction between the light and heavy chains. In one embodiment, the amino acid modifications introduced into the immunoglobulin heavy chain polypeptide sequence and the immunoglobulin light chain polypeptide sequence are complementary to each other. Complementarity at the junction between the heavy and light chains can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of these and various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock-and-key fits, knobs-into-holes, protrusions-and-cavities, donors-and-acceptors, etc., all of which imply structural and chemical pairing between the two interacting surfaces. In one embodiment, at least one of the heterodimers contains amino acid modifications introduced into the immunoglobulin heavy chain and immunoglobulin light chain that introduce new hydrogen bonds across the light and heavy chains at the junction. In one embodiment, at least one of the heterodimers comprises an amino acid modification introduced into an immunoglobulin heavy chain and an immunoglobulin light chain that introduces a new salt bridge across the light and heavy chains at the interface.

[0099] In one embodiment, the amino acid modifications of the Mab design set promote preferential pairing primarily through electrostatic attraction and repulsion. In one embodiment, the amino acid modifications of the Mab design set promote preferential pairing primarily through steric mechanisms. Such Mab designs are included in Tables 4A, 4B, 7A, and 7B, examples of which include those with unique identifiers 10771-11335, 10771-11360, and 10780-11417. In one embodiment, the amino acid modifications of the Mab design set promote preferential pairing using both steric and electrostatic mechanisms.

[0100] In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, where H1 and L1 do not comprise an amino acid modification that promotes preferential pairing, and H2 and L2 each comprise at least one amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, where one or more of H1, L1, and H2 comprise at least one amino acid modification that promotes preferential pairing, and L2 does not comprise an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, where one or more of H1, L1, and L2 comprise at least one amino acid modification that promotes preferential pairing, and H2 does not comprise an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, wherein one or more of H1, H2, and L2 comprise at least one amino acid modification that promotes preferential pairing, and L1 does not comprise an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, wherein one or more of L1, H2, and L2 comprise at least one amino acid modification that promotes preferential pairing, and H1 does not comprise an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 comprise an amino acid modification, wherein each of L1, H2, and L2 comprises at least one amino acid modification that promotes preferential pairing.

[0101] The amino acid modifications may be in one or more constant and / or variable domains of H1, L1, H2, and L2. In one embodiment, the amino acid modifications may be in the CH1 domain of H1 and H2, the CL-lambda domain of L1, and the CL-kappa domain of L2. In another embodiment, the amino acid modifications may be in the CH1 and VH domains of H1 and H2, the CL-lambda and VL-lambda domains of L1, and the CL-kappa and VL-kappa domains of L2. In another embodiment, the amino acid modifications may be in the VH domains of H1 and H2, the VL-lambda domain of L1, and the VL-kappa domain of L2.

[0102] In one embodiment, the amino acid modification may be in one or more of the framework regions of H1, L1, H2, and L2. In one embodiment, the amino acid modification is limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. For example, Almagro [Frontiers In Bioscience (2008) 13:1619-1633] provides definitions of framework residues based on the Kabat, Chotia, and IMGT numbering schemes.

[0103] The number of amino acid modifications in each Mab design or Mab design set can vary. In one embodiment, H1 includes 0-8 amino acid modifications, 0-7 amino acid modifications, 0-6 amino acid modifications, 0-5 amino acid modifications, 0-4 amino acid modifications, 0-3 amino acid modifications, 0-2 amino acid modifications, 1 amino acid modification, or no amino acid modifications. In one embodiment, L1 includes 0-8 amino acid modifications, 0-7 amino acid modifications, 0-6 amino acid modifications, 0-5 amino acid modifications, 0-4 amino acid modifications, 0-3 amino acid modifications, 0-2 amino acid modifications, 1 amino acid modification, or no amino acid modifications. In one embodiment, H2 includes 0-8 amino acid modifications, 0-7 amino acid modifications, 0-6 amino acid modifications, 0-5 amino acid modifications, 0-4 amino acid modifications, 0-3 amino acid modifications, 0-2 amino acid modifications, 1 amino acid modification, or no amino acid modifications. In one embodiment, L2 includes 0 to 8 amino acid modifications, 0 to 7 amino acid modifications, 0 to 6 amino acid modifications, 0 to 5 amino acid modifications, 0 to 4 amino acid modifications, 0 to 3 amino acid modifications, 0 to 2 amino acid modifications, 1 amino acid modification, or no amino acid modifications.

[0104] In one embodiment, the total number of amino acid modifications in H1, L1, H2, and L2 is less than 20, less than 15, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. In one embodiment, the total number of amino acid modifications in H1, L1, H2, and L2 is 2.

[0105] In one embodiment, amino acid modifications can be specifically designed for the kappa-lambda system, where one parent antibody comprises a kappa light chain polypeptide sequence and one parent antibody comprises a lambda light chain polypeptide sequence. Such amino acid modifications or designs are referred to herein as KL designs. Examples of such amino acid modifications or KL designs are shown in Table 4A, Table 7A, and Tables 10-A1 to 10-A12.

[0106] In another embodiment, amino acid modifications can be first identified for a kappa-kappa system (KK design), where both parent antibodies contain kappa light chain polypeptide sequences, and then grafted into a kappa-lambda system. Those skilled in the art will understand how these designs can be grafted into a kappa-lambda system. For example, the heavy and light chains of the kappa and lambda parent antibodies can be aligned to determine the equivalent lambda light chain positions corresponding to the KK design. The equivalent lambda light chain positions can then be modified to conform to the KK design. Such amino acid modifications or designs, referred to herein as KK-derived KL designs, can be classified into the following groups: a) those in which no design changes are necessary and the amino acid residue modifications in the kappa-kappa system are identical to those in the kappa-lambda system; b) those containing silent modifications, where at least one modification made in the kappa-kappa system is unnecessary in the kappa-lambda system because the modification is naturally present in the lambda light chain polypeptide sequence; c) those containing an amino acid modification in at least one amino acid residue at the same relative position in the kappa and lambda light chain polypeptide sequences, but where the first amino acid residue at that position differs between the kappa and lambda light chain polypeptide sequences, resulting in the same amino acid modification at that position; and d) those containing at least one additional amino acid modification in the kappa-lambda system compared to the kappa-kappa system. Examples of such KK-derived KL designs are provided in Tables 4B, 7B, and 10-B1 to 10-B10. Specific examples of group a) are marked with an asterisk in Table 4B. A specific example of group b) is represented by the Mab design set with unique identifier 10689-10707. The silent modification is in L1 (Q160E is absent in wild-type lambda because the residue at position 160 is E and not Q). A specific example of group c) is represented by the Mab design set with unique identifier 10652-10734, where in L1, amino acid residue 124 is E in wild-type lambda and Q in wild-type kappa. A specific example of group d) is represented by the Mab design set with unique identifier 10684-10706, which includes the amino acid modification K129T.

[0107] In one embodiment, one or more of H1, L1, H2, and L2 comprise amino acid modifications that promote preferential pairing of L1 with H1 relative to L2 and promote preferential pairing of L2 with H2 relative to L1, and the amino acid modifications comprise conservative amino acid substitutions of the Mab design sets provided in Table 4A, Table 4B, Table 7A, Table 7B, Tables 10-A1 to 10-A12, and Tables 10-B1 to 10-B10.

[0108] In one embodiment, the amino acid modification does not introduce a new cysteine ​​residue and does not remove a naturally occurring cysteine ​​residue in an immunoglobulin heavy chain or an immunoglobulin light chain within the same design.

[0109] The combination of amino acid modifications in H1, L1, H2, and L2 that promote preferential pairing is generally referred to as a design. A design can be more specifically referred to as an "LCCA design" (in the context of H1, L1, L2 or H2, L1, L2) or a "Mab design" (in the context of H1, L1, H2, L2). Typically, LCCA designs are engineered with one or more specific complementary LCCA designs based on each heavy chain of the desired bispecific antibody, and therefore are typically presented in a form in which amino acid modifications in all four polypeptide chains of the bispecific antibody are identified (see, e.g., Tables 4A and 4B). While specific amino acid substitutions can be identified throughout, it should be understood that conservative substitutions at each amino acid position are also contemplated. Furthermore, for purposes of illustration, unless otherwise indicated, an H1L1 heterodimer represents a heterodimer containing a lambda light chain, and an H2L2 heterodimer represents a heterodimer containing a kappa light chain. Finally, all amino acid residues or positions are numbered according to the Kabat numbering system unless otherwise indicated.

[0110] The design includes a driver set of amino acid substitutions that promote complementary preferential pairing, and may also include secondary substitutions, which may act to optimize the performance of the driver set.

[0111] One or more driver sets may be used to promote preferential pairing. These driver sets may be used individually or in combination to promote preferential pairing. In one embodiment, the driver set is an electrostatic driver set in which electrostatic attraction and repulsion are believed to be the primary factors promoting preferential pairing. For example, a design in which H1 contains the amino acid substitution 186K, L1 contains the amino acid substitution 133D, H2 contains the amino acid substitution 188D, and L2 contains the amino acid substitution 131K may promote preferential pairing by an electrostatic mechanism. Many other examples of electrostatic drivers can be found throughout the Examples. In one embodiment, one or more electrostatic driver sets may be selected from those identified in Table C:

[0112] TIFF2025124635000009.tif220170TIFF2025124635000010.tif219170

[0113] In one embodiment, the driver set is a disulfide steering driver set, which can act to disfavor disulfide bond formation in mismatched heterodimers. An example of this type of driver set would include 125R in H1, 122D in L1, 228D in H2, and 121K in L2.

[0114] In one embodiment, the driver set may be a steric driver set that can act to promote sterically complementary interactions between correctly matched heterodimers and steric incompatibilities between mismatched heterodimers. Non-limiting examples of steric driver sets are shown in Table D, where "-" indicates the absence of an amino acid substitution that promotes preferential pairing.

[0115] TIFF2025124635000011.tif56170

[0116] In one embodiment, the driver set is a variable design driver set. Such a variable design driver set comprises one or more amino acid modifications in the variable domains of kappa and / or lambda Fab that promote preferential pairing. In one embodiment, the variable design driver set promotes preferential pairing based on a steric mechanism. In one embodiment, the variable design driver set promotes preferential pairing based on an electrostatic mechanism. Non-limiting examples of variable design driver sets are shown in Table E, where "-" indicates the absence of an amino acid substitution in the polypeptide that promotes preferential pairing.

[0117] TIFF2025124635000012.tif51170

[0118] In one embodiment, one or more non-naturally occurring disulfide bonds may be engineered into one or both heterodimers of the antigen-binding polypeptide construct. An example of this type of amino acid modification is one in which the heavy chain contains a 122C substitution paired with a 124C substitution in the kappa light chain.

[0119] Secondary substitutions may be included in a design to optimize the pairing performance of the design. For example, secondary substitutions may A) optimize the number of contact points between the heavy chain and a correctly paired light chain, B) act to provide a conductive environment for the driver, C) optimize the hydrogen bond network for the driver set, or D) provide steric adaptation for the driver. Non-limiting examples of these types of secondary substitutions are shown in Table F, where "Lk" designates a kappa light chain-specific substitution, "L1" designates a lambda light chain-specific substitution, "L" designates a light chain-specific substitution in either a kappa or lambda light chain, and "H" designates a heavy chain-specific substitution.

[0120] TIFF2025124635000013.tif136170

[0121] Antigen-binding polypeptide constructs may be engineered with different combinations of amino acid modifications corresponding to the driver sets and secondary substitutions described above. Non-limiting examples of such combinations, grouped into clusters based on common characteristics, are described below. As described herein, combinations of amino acid modifications at multiple positions within a single chain are identified with an "_" between each modified position. For example, "124_186" indicates that both positions 124 and 186 are modified in the referenced polypeptide chain. Similarly, "124_133_180" indicates that positions 124, 133, and 180 are all modified in the referenced polypeptide chain.

[0122] KL Cluster 1: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 1, H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; and a) H2 comprises an amino acid substitution at position 188 or 124_186, and L2 comprises an amino acid substitution at position 176_178 or 176_180 or 131; or b) H2 contains an amino acid substitution at position 143 or 186; L2 contains an amino acid substitution at positions 124_133 or 124_133_180.

[0123] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 125, 145, and 179; L1 further comprises an amino acid substitution at one or more of positions 122, 124, and 133; H2 further comprises an amino acid substitution at position 228; and / or L2 further comprises an amino acid substitution at position 121.

[0124] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 1, wherein H1 comprises amino acid substitutions at positions 125, 143, 145; L1 comprises amino acid substitutions at positions 122, 124, 131; H2 comprises an amino acid substitution at position 228, and L2 comprises amino acid substitutions at positions 121, 133. In some embodiments, H1 further comprises an amino acid substitution at position 179, L1 further comprises an amino acid substitution at position 133, H2 further comprises an amino acid substitution at one or more of positions 124, 143, 186, and 188, and L2 further comprises an amino acid substitution at one or more of positions 124, 131, 176, 178, and 180.

[0125] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 1, wherein H1 comprises an amino acid substitution at position 125_143_145, or 125_143_145_179; L1 comprises an amino acid substitution at position 122_124_131, or 122_124_131_133; H2 comprises an amino acid substitution at position 124_186_228, 143_228, 143_186_228, 186_228, or 188_228; and L2 comprises an amino acid substitution at position 121_124_133, 121_124_133_180, 121_131_133_178, 121_133_176_178, or 121_133_176_180.

[0126] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 1, wherein the amino acid substitutions in H1 are selected from 125R, 145T, 143D, 143E, 179E, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 124Q, 122D, 131K, 131R, 133S, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 228D, 124R, 1431, 143R, 186K, 186R, 188K, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 124E, 121K, 131D, 176D, 178E, 178F, 180D, 180E, 133D, 133G, 1331, and conservative substitutions thereof.

[0127] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 1, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000014.tif21170

[0128] In one embodiment, H1 comprises 125R_143E_145T_179E and L1 comprises 122D_124Q_131R. In another embodiment, H1 comprises 125R_143E_145T and L1 comprises 122D_124Q_131R. In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 1, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000015.tif43170

[0129] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, H1 comprises 125R_143E_145T_179E, L1 comprises 122D_124Q_131R, H2 comprises 188K_228D, and L2 comprises 121K_133I_176D_178E. In another embodiment, H1 comprises 125R_143D_145T, L1 comprises 122D_124Q_131R, H2 comprises 143R_228D, and L2 comprises 121K_124E_133D.

[0130] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 1, including a disulfide steering driver set.

[0131] In one embodiment, the KL cluster 1 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0132] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 1 set forth in one or more of the designs in Table 10-A1.

[0133] KL Cluster 2: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; and c) H2 contains an amino acid substitution at position 186 or 124_186 and L2 contains an amino acid substitution at position 133 or 133_160 or 124_133 or 176_180; or d) H2 contains an amino acid substitution at position 188; L2 contains an amino acid substitution at position 131; e) H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at positions 124_133 or 124_133_180;

[0134] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 139, 145, 174, and 179; L1 further comprises an amino acid substitution at one or more of positions 116, 124, 133, and 176; H2 further comprises an amino acid substitution at one or more of positions 190, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 135, 178, 38, and 44.

[0135] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, wherein H1 comprises an amino acid substitution at positions 143-145; L1 comprises an amino acid substitution at position 131; H2 comprises an amino acid substitution at position 143, 186, or 188, and L2 comprises an amino acid substitution at position 133. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 139, 174, and 179; L1 further comprises an amino acid substitution at one or more of positions 116, 124, 133, and 176; H2 further comprises an amino acid substitution at one or more of positions 124, 190, 39, and 45; and L2 further comprises an amino acid substitution at one or more of positions 124, 131, 135, 160, 176, 178, 180, 38, 44.

[0136] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, wherein H1 comprises an amino acid substitution at position 139_143_145, 143_145, 143_145, 143_145_174, or 143_145_179; L1 comprises an amino acid substitution at position 116_124_131_176, 124_131, 124_131_133, 124_131_133_176, 131, or 131_133; H2 comprises an amino acid substitution at position 124_186_190, 124_191, 124_192, 124_193, 124_194, 124_195, 124_196, 124_197, 124_198, 124_199, 124_200, 124_201, 124_202, 124_203, 124_204, 124_205, 124_206, 124_207, 124_210, 124_211, 124_212, 124_213, 124_214, 124_215, 124_216, 124_217, 124_218, 124_2199, 124_220, 124_221, 124_222, 124_223, 124_224, 124_225, 124_226, 124_227 L2 contains an amino acid substitution at position 124_133, 124_133_135, 124_133_135_180, 124_133_180, 131_133_135_178, 131_133_178, 133, 133_135_176_180, 133_160, 38_124_133, or 44_124_133.

[0137] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, wherein the amino acid substitutions in H1 are selected from 139W, 143D, 145T, 174G, 179E, and conservative substitutions thereof; and the amino acid substitutions in L1 are selected from 124Q, 176F, 116F, 131K, 131R, 133S, and conservative substitutions thereof; The amino acid substitutions in H2 are selected from 124R, 143I, 143K, 143R, 186K, 188K, 190F, 39E, 45P, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 124E, 131D, 131E, 133D, 133G, 135A, 135W, 160E, 176D, 178F, 180D, 180E, 38R, 44F, and conservative substitutions thereof.

[0138] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 2, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000016.tif73170

[0139] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 2, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000017.tif98170

[0140] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000018.tif25170

[0141] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2 with one or more of conformation 1, conformation 2, variable domain conformation, or variable domain electrostatic driver sets.

[0142] In one embodiment, the KL cluster 2 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0143] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 2 set forth in one or more of the designs in Table 10-A2.

[0144] KL Cluster 3: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3, H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; and a) H2 comprises an amino acid substitution at position 124_186 or 124_179 or 188, and L2 comprises an amino acid substitution at position 176_178 or 176_180 or 131; or b) H2 contains an amino acid substitution at position 143_188 or 143 or 124_143; L2 contains an amino acid substitution at position 124_176_178 or 124_178 or 124_180 or 124_176_180, or 124, or 124_176.

[0145] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 139, 145, 174, and 179; L1 further comprises an amino acid substitution at one or more of positions 116, 124, and 176; H2 further comprises an amino acid substitution at one or more of positions 177, 190, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 133, 135, 178, 38, and 44.

[0146] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3, wherein H1 comprises an amino acid substitution at positions 143-145; L1 comprises an amino acid substitution at positions 124-131; H2 comprises an amino acid substitution at one or more of positions 143, 124, and 188, and L2 comprises an amino acid substitution at positions 133 or 176-178. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 139, 174, and 179, L1 further comprises an amino acid substitution at position 116 or 176, H2 further comprises an amino acid substitution at one or more of positions 177, 179, 186, 190, 39, and 45, and / or L2 further comprises an amino acid substitution at one or more of positions 124, 131, 135, 180, 38, and 44.

[0147] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3, wherein H1 comprises an amino acid substitution at position 139_143_145, 139_143_145_179, 143_145, 143_145_174_179, or 143_145_179; L1 comprises an amino acid substitution at position 116_124_131_176, or 124_131; and H2 comprises an amino acid substitution at positions 124_143, 124_179, 124_186, 143, 143_188, 177_188, 188, 188, 188_1 L2 contains an amino acid substitution at position 90, 39_124_179, or 45_124_179, and L3 contains an amino acid substitution at position 124_133, 124_133_176, 124_133_176_178, 124_133_176_180, 124_133_178, 124_133_180, 131_133_178, 133_135_176_178, 133_135_176_180, 133_176_178, 133_176_180, 176_178, 38_133_176_180, or 44_133_176_180.

[0148] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3, wherein the amino acid substitutions in H1 are selected from 139W, 174G, 145T, 143D, 143E, 179E, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 116F, 124Q, 131K, 131R, 176F, and conservative substitutions thereof; and the amino acid substitutions in H2 are selected from 139W, 174G, 145T, 143D, 143E, 179E, and conservative substitutions thereof. amino acid substitutions in L2 are selected from 24R, 143K, 143R, 177I, 179K, 186K, 186R, 188K, 190F, 39E, 45P, and conservative substitutions thereof; and amino acid substitutions in L2 are selected from 135A, 135W, 44F, 124E, 38R, 131D, 131E, 176D, 176E, 178D, 178E, 178F, 180D, 180E, 133D, 133G, 133I, 133L, and conservative substitutions thereof.

[0149] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 3, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000019.tif31170

[0150] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 3, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000020.tif77170

[0151] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000021.tif30170

[0152] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3 with one or more of conformation 1, conformation 2, variable domain conformation, or variable domain electrostatic driver sets.

[0153] In one embodiment, the amino acid combination of KL cluster 3 comprises one or more secondary substitutions selected from Table F.

[0154] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 3 set forth in one or more of the designs in Table 10-A3.

[0155] KL Cluster 4: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, wherein H1 comprises an amino acid substitution at position 188; L1 comprises an amino acid substitution at position 176-178 or 178; and a) H2 contains amino acid substitutions at positions 177-188 and L2 contains amino acid substitutions at positions 176-178; or b) H2 contains amino acid substitutions at positions 186 or 124 or 124_179; L2 contains amino acid substitutions at positions 176 or 131_176.

[0156] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 125, 139, and 177; L1 further comprises an amino acid substitution at one or more of positions 122, 129, and 133; H2 further comprises an amino acid substitution at one or more of positions 145, 228, 45, and 39; and / or L2 further comprises an amino acid substitution at one or more of positions 135, 44, 38, 121, and 133.

[0157] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, where H1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 188; L1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at positions 176-178; H2 contains an amino acid substitution at positions 188 or 186-188, and L2 contains an amino acid substitution at positions 176-178.

[0158] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, where H1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 188; L1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 178; H2 contains an amino acid substitution at one or more of positions 124, 186, and 188, and L2 contains an amino acid substitution at position 176. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 125, 139, and 177; L1 further comprises an amino acid substitution at one or more of positions 122, 129, 133, and 176; H2 further comprises an amino acid substitution at one or more of positions 145, 228, 45, 177, 179, and 39; and / or L2 further comprises an amino acid substitution at one or more of positions 135, 44, 38, 121, 131, 178, and 133.

[0159] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, wherein H1 does not contain an amino acid substitution that promotes preferential pairing, or contains an amino acid substitution at 125_188, 139_188, 188, or 177_188; L1 does not contain an amino acid substitution that promotes preferential pairing, or contains an amino acid substitution at 129_176_178, 129_178, 122_129_176_178, 176_178, or 133_176_178; and H2 contains an amino acid substitution at 145_186, 145_186_228, 145_177_18 ... containing an amino acid substitution at position 24, 124_145_179, 124_145_179_186_188, 124_145_179_188, 124_186_188, 124_188, 45_124_145_179, 39_124_145_179, or 186_188; L2 is Contains an amino acid substitution at position 31_133_176, 38_131_133_176, 121_131_176, 131_135_176, 131_176, 131_133_176, 131_133_176_178, 176, 176_178, 133_176, or 133_176_178.

[0160] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, wherein the amino acid substitutions in H1 are selected from 125R, 139W, 188A, 188K, and 177I, and conservative substitutions thereof; and the amino acid substitutions in L1 are selected from 129T, 122D, 176A, 176D, 176E, 133I, 133L, 178D, 178E, 178T, and 178W, and conservative substitutions thereof; The amino acid substitutions in H2 are selected from 124E, 145T, 177D, 179E, 186E, 186I, 186L, 188D, 188W, 228D, 39E, and 45P, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 121K, 131K, 131R, 133A, 133G, 135W, 176A, 176K, 176R, 176V, 178A, 178K, 178L, 178R, 38R, and 44F, and conservative substitutions thereof.

[0161] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 4, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000022.tif80170

[0162] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 4, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000023.tif67170

[0163] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000024.tif21170

[0164] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4 with one or more of electrostatic, disulfide steering, steric 3, variable domain steric and variable domain electrostatic driver sets.

[0165] In one embodiment, the KL cluster 4 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0166] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 4 set forth in one or more of the designs in Table 10-A4.

[0167] KL Cluster 5: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 5, H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; and a) H2 comprises an amino acid substitution at position 188 and L2 comprises an amino acid substitution at position 131; or b) H2 contains amino acid substitutions at positions 177-188; L2 contains amino acid substitutions at positions 176-178; or H1 contains amino acid substitutions at positions 124-190; L1 contains amino acid substitutions at position 135; H2 contains amino acid substitutions at positions 124 or 188, and L2 contains amino acid substitutions at positions 176 or 176-178.

[0168] In some embodiments, H1 further comprises an amino acid substitution at position 143 and / or 188, L1 further comprises an amino acid substitution at position 131 and / or 178, H2 further comprises an amino acid substitution at position 143 and / or 145, and / or L2 further comprises an amino acid substitution at position 133 and / or 178.

[0169] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 5, where H1 comprises an amino acid substitution at position 186 or 124; L1 comprises an amino acid substitution at position 133 and / or 135; H2 comprises an amino acid substitution at one or more of positions 188, 177 and 124, and L2 comprises an amino acid substitution at position 176 and / or 131.

[0170] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 143, 188, and 190; L1 further comprises an amino acid substitution at positions 131 and / or 178; H2 further comprises an amino acid substitution at positions 143 and / or 145; and / or L2 further comprises an amino acid substitution at positions 133 and / or 178.

[0171] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 5, wherein H1 comprises an amino acid substitution at position 124_190, 143_186_188, or 186_188; L1 comprises an amino acid substitution at position 131_133_178, 133_135, 133_135_178, 133_178, or 135_178; H2 comprises an amino acid substitution at position 124, 143_188, 145_177_188, or 177_188, and L2 comprises an amino acid substitution at position 131_176_178, 131_178, 133_176, 133_176_178, or 176_178.

[0172] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 5, wherein the amino acid substitutions in H1 are selected from 124E, 143S, 186K, 188T, 190D, 190E, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 131S, 133D, 133I, 135K, 135R, 178F, 178T, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 124R, 143T, 145T, 177D, 177I, 188D, 188K, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 131K, 133G, 133L, 176A, 176D, 176K, 178E, 178K, 178R, 178S, and conservative substitutions thereof.

[0173] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 5, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000025.tif41170

[0174] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 5, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000026.tif38170

[0175] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 heterodimer and the H2L2 heterodimer is such that H1 comprises 186K_188T, L1 comprises 133D_178T, H2 comprises 145T_177D_188D, and L2 comprises 176K_178K.

[0176] In one embodiment, the amino acid combination of KL cluster 5 comprises one or more secondary substitutions selected from Table F.

[0177] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 5 set forth in one or more of the designs in Table 10-A5.

[0178] KL Cluster 6: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 6, H1 contains amino acid substitutions at positions 177-188; L1 contains amino acid substitutions at positions 176-178; and a) H2 contains an amino acid substitution at position 188, and L2 contains an amino acid substitution at positions 176-178 or 131; b) H2 contains an amino acid substitution at position 186; L2 contains an amino acid substitution at positions 133 or 124_160_180; c) H2 contains an amino acid substitution at position 124 or 124_179 or 124_186; L2 contains an amino acid substitution at position 176 or 176_178 or 176_180; or d) H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at positions 133 or 124-133.

[0179] In some embodiments, H1 further comprises an amino acid substitution at positions 145 and / or 146, and / or H2 further comprises an amino acid substitution at one or more of positions 143 and / or 177.

[0180] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 6, H1 contains amino acid substitutions at positions 177-188; L1 contains amino acid substitutions at positions 176-178; H2 contains amino acid substitutions at one or more of positions 124, 143, 179, 186, and 188, and L2 contains amino acid substitutions at one or more of positions 133, 176, and 178.

[0181] In some embodiments, H1 further comprises an amino acid substitution at positions 145 and / or 146, H2 further comprises an amino acid substitution at position 177, and / or L2 further comprises an amino acid substitution at one or more of positions 124, 131, 160, and 180.

[0182] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 6, wherein H1 comprises an amino acid substitution at position 145_177_188, or 146_177_188; L1 comprises an amino acid substitution at position 176_178; H2 comprises an amino acid substitution at positions 124, 124_179, 124_186, 143, 143_186_1 L1 contains an amino acid substitution at position 88, 177_188, 179, 186, 186_188, or 188, and L2 contains an amino acid substitution at position 124_133, 124_160_176_178_180, 124_160_180, 131_133_178, 133, 133_176, 133_176_178, 133_176_180, or 176_178.

[0183] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 6, wherein the amino acid substitutions in H1 are selected from 145T, 146T, 177D, 188D, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 176K, 178K, 178L, 178R, and conservative substitutions thereof; and the amino acid substitutions in H2 are 124R, the amino acid substitutions in L2 are selected from 124E, 131D, 131E, 133D, 133G, 133I, 133L, 160E, 176A, 176D, 176E, 178A, 178D, 178E, 178F, 180E, and conservative substitutions thereof.

[0184] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 6, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000027.tif22170

[0185] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 6, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000028.tif80170

[0186] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000029.tif16170

[0187] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 6 with a steric driver set.

[0188] In one embodiment, the KL cluster 6 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0189] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 6 set forth in one or more of the designs in Table 10-A6.

[0190] KL Cluster 7: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 7, H1 comprises an amino acid substitution at position 188; L1 comprises an amino acid substitution at position 178; H2 comprises an amino acid substitution at position 124 or 188, and L2 comprises an amino acid substitution at position 176-178 or 176-180 or 176. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 125, 139, 145, and 177; L1 further comprises an amino acid substitution at position 122; H2 further comprises an amino acid substitution at one or more of positions 143, 177, 179, 186, 228, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 121, 124, 133, 135, 160, 38, and 44.

[0191] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 7, where H1 comprises amino acid substitutions at positions 145-188; L1 comprises an amino acid substitution at position 178; H2 comprises an amino acid substitution at positions 124 and / or 188, and L2 comprises an amino acid substitution at one or more of positions 124, 133, and 178.

[0192] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 125, 139, and 177; L1 further comprises an amino acid substitution at position 122; H2 further comprises an amino acid substitution at one or more of positions 143, 177, 179, 186, 228, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 121, 135, 160, 176, 180, 38, and 44.

[0193] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 7, wherein H1 comprises an amino acid substitution at position 125_145_188, 139_145_188, 145_177_188, or 145_188; L1 comprises an amino acid substitution at position 122_178, or 178; H2 comprises an amino acid substitution at position 124, L2 contains amino acid substitutions at positions 124_143, 124_179, 124_186, 124_186_228, 124_188, 124_228, 143_188, 177_188, 179_188, 186_188, 188, 188_228, 39_124_179, or 45_124_179, and L3 contains amino acid substitutions at positions 121_133_176, 121_133_176_180, 121_176_178, 124_133_176, 124_133_176_178, 124_133_176_178, and containing an amino acid substitution at position 180, 124_133_176_180, 124_133_178, 124_160_176_178, 124_160_176_178_180, 124_176_178_180, 124_176_180, 133_135_176, 133_135_176_180, 133_176, 133_176_178, 133_176_180, 135_176_178, 176_178, 38_133_176_180, or 44_133_176_180.

[0194] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 7, wherein the amino acid substitutions in H1 are selected from 125R, 139W, 145T, 177T, 188E, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 122D, 178K, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 124K, 124R, 143K, 143R, 177I, 179K, 186R, 188K, 228D, 39E, 45P, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 121K, 124E, 133D, 133G, 133L, 135W, 160E, 176D, 176E, 178D, 178E, 180E, 38R, 44F, and conservative substitutions thereof.

[0195] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 7, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000030.tif27170

[0196] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 7, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000031.tif135170

[0197] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000032.tif22170

[0198] In some embodiments, the KL cluster 7 antigen-binding polypeptide construct comprises a combination of amino acid substitutions with one or more driver sets selected from a disulfide steering driver set, a stereo2 driver set, and a variable domain driver set.

[0199] In one embodiment, the KL cluster 7 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0200] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 7 set forth in one or more of the designs set forth in Table 10-A7.

[0201] KL Cluster 8: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8, H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at position 124; and a) H1 contains an amino acid substitution at position 186 or 179, and L1 contains an amino acid substitution at position 180; b) H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; c) H1 comprises an amino acid substitution at position 143; L1 comprises an amino acid substitution at position 133; or d) H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 178.

[0202] In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 124, 139, 177, and 190; L1 further comprises an amino acid substitution at one or more of positions 129, 131, 135, and 176; H2 further comprises an amino acid substitution at one or more of positions 122, 124, 145, 179, 186, 188, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 129, 133, 135, 160, 176, 178, 38, and 44.

[0203] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8, where H1 comprises an amino acid substitution at position 143, 186, 179 and / or 188; L1 comprises an amino acid substitution at position 129 and / or 178; H2 comprises an amino acid substitution at position 143; and L2 comprises an amino acid substitution at position 124. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 124, 139, 177, and 190; L1 further comprises an amino acid substitution at one or more of positions 131, 133, 135, 176, and 180; H2 further comprises an amino acid substitution at one or more of positions 122, 124, 145, 179, 186, 188, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 129, 133, 135, 160, 176, 178, 38, and 44.

[0204] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8, wherein H1 is 124_143, 139_143, 139_143, 139_143_186, 139_186, 139_188, 143, 143_179, 143_186, 143_186_188, 143_190, 177_188, 179, 179_190, 186, or 18 L1 contains amino acid substitutions at positions 129_131_133, 129_133, 129_133_135, 129_133_135_180, 129_133_178, 129_133_180, 129_176_178, 129_176_178_180, 129_178, 129_178_180, 129_180, 133_176_178, 133_178, or or 176_178; H2 contains an amino acid substitution at positions 122_143_145, 122_143_145_179, 124_143_145, 124_143_145_179, 143_145, 143_145_179, 143_145_179, 143_145_179_186_188, 143_145_179_188, 143_145_188, 39_143_145_179, or 45_143_ L1 contains an amino acid substitution at position 145_179, and L2 contains an amino acid substitution at position 124_129_160_178, 124_129_178, 124_133_178, 124_135_160_178, 124_135_178, 124_160_176_178, 124_160_178, 124_176_178, 124_178, 38_124_178, or 44_124_178.

[0205] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8, wherein the amino acid substitutions in H1 are selected from 124K, 139W, 143A, 143I, 143K, 143S, 177I, 179K, 186K, 186R, 188K, 188T, 190K, and conservative substitutions thereof; and the amino acid substitutions in L1 are selected from 129T, 131D, 131E, 133D, 133L, 133W, 135S, 176A, 176D, 176 the amino acid substitution in H2 is selected from 122C, 124W, 143E, 145T, 179E, 186I, 188L, 188W, 39E, 45P, and conservative substitutions thereof; and the amino acid substitution in L2 is selected from 124C, 124K, 124R, 129K, 133A, 135W, 160K, 160R, 176A, 178R, 38R, 44F, and conservative substitutions thereof.

[0206] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 8, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000033.tif118170

[0207] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 8, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000034.tif130170

[0208] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000035.tif40170

[0209] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8 with one or more of conformation 2, conformation 3, conformation 4, and a variable domain driver set. In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8 that introduce a non-naturally occurring disulfide bond.

[0210] In one embodiment, the KL cluster 8 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0211] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 8 set forth in one or more of the designs in Table 10-A8.

[0212] KL Cluster 9: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 9, H1 comprises an amino acid substitution at position 179, 186, 143, and / or 188; L1 comprises an amino acid substitution at position 180, 133, and / or 176-178; H2 comprises an amino acid substitution at position 143, and L2 comprises an amino acid substitution at position 131 and / or 124. In some embodiments, H1 further comprises an amino acid substitution at position 125, L1 further comprises an amino acid substitution at position 122 or 129, H2 further comprises an amino acid substitution at one or more of positions 145, 179, and 228, and / or L2 further comprises an amino acid substitution at one or more of positions 121, 129, 135, 160, and 178.

[0213] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 9, wherein H1 comprises an amino acid substitution at position 125; L1 comprises an amino acid substitution at positions 122-129; H2 comprises an amino acid substitution at position 145, and L2 comprises an amino acid substitution at position 121 and / or 124. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 143, 179, 186, and 188; L1 further comprises an amino acid substitution at one or more of positions 133, 176, 178, and 180; H2 further comprises an amino acid substitution at one or more of positions 143, 179, and 228; and / or L2 further comprises an amino acid substitution at one or more of positions 129, 131, 135, 160, and 178.

[0214] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 9, wherein H1 comprises an amino acid substitution at position 125_143, 125_179, 125_186, or 125_188; L1 comprises an amino acid substitution at position 122_129_133, 122_129_176_178, or 122_129_180; and H2 comprises , 143_145, 143_145_179, 143_145_179_228, 143_145_228, or 145_179_228, and L2 contains an amino acid substitution at position 121_124_160_178, 121_124_178, 121_129_131, 121_131, 124_135_160_178, or 124_135_178.

[0215] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 9, wherein the amino acid substitutions in H1 are selected from 125R, 143K, 179K, 186R, 188K, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 122D, 129T, 133D, 176D, 176E, 178E, 178T, 180E, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 143E, 145T, 179E, 228D, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 135W, 124R, 160K, 121K, 131K, 129K, 178R, and conservative substitutions thereof.

[0216] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 9, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000036.tif31170

[0217] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 9, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000037.tif55170

[0218] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000038.tif20170

[0219] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 9 with a disulfide steering driver set.

[0220] In one embodiment, the KL cluster 9 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0221] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 9 set forth in one or more of the designs in Table 10-A9.

[0222] KL Cluster 10: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 10, wherein H1 comprises an amino acid substitution at position 174, 179 or 186; L1 comprises an amino acid substitution at position 176 or 180; H2 comprises an amino acid substitution at position 143 or 190, and L2 comprises an amino acid substitution at position 131, 135 or 124; or H1 comprises an amino acid substitution at position 174; L1 comprises an amino acid substitution at position 176; H2 comprises an amino acid substitution at position 190; and L2 does not comprise an amino acid substitution that promotes preferential pairing, or comprises an amino acid substitution at position 135. In some embodiments, H1 further comprises an amino acid substitution at position 143; L1 further comprises an amino acid substitution at one or more of positions 116, 129, and 133; H2 further comprises an amino acid substitution at one or more of positions 145, 179, and 188; and / or L2 further comprises an amino acid substitution at one or more of positions 133, 160, and 178.

[0223] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 10, wherein H1 comprises an amino acid substitution at position 174 and / or 186; L1 comprises an amino acid substitution at position 176 and / or 180; H2 comprises an amino acid substitution at position 145, 190 and / or 188, and L2 comprises an amino acid substitution at position 135, 131, 178, or 133, or does not comprise an amino acid substitution that promotes preferential pairing. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 143 and / or 179, L1 further comprises an amino acid substitution at one or more of positions 116, 129, and 133, H2 further comprises an amino acid substitution at position 143 and / or 179, and / or L2 further comprises an amino acid substitution at position 124 and / or 160.

[0224] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 10, wherein H1 comprises an amino acid substitution at position 143_174, 174, 174_179, 174_186, or 186; L1 comprises an amino acid substitution at position 116_129_133_176, 116_129_176_180, 116_176, 129_180, or 176; H2 comprises an amino acid substitution at position 143_145_17 L1 contains an amino acid substitution at position 124_135_160_178, 124_135_178, 131, 131_135, 133, 135, 135_178, 178, or does not contain an amino acid substitution that promotes preferential pairing.

[0225] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 10, wherein the amino acid substitutions in H1 are selected from 143K, 174G, 179K, 186R, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 116F, 129T, 133D, 176F, 180E, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 143E, 143I, 145T, 179E, 188F, 190F, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 124K, 124R, 131K, 133A, 135A, 160K, 178F, 178R, and conservative substitutions thereof.

[0226] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 10, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000039.tif37170

[0227] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 10, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000040.tif79170

[0228] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000041.tif16170

[0229] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 10 with a stereo1 driver set.

[0230] In one embodiment, the amino acid combination of KL cluster 10 comprises one or more secondary substitutions selected from Table F.

[0231] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 10 as set forth in one or more of the designs in Table 10-A10.

[0232] KL Cluster 11: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 11, wherein H1 comprises amino acid substitutions at positions 143-190; L1 comprises an amino acid substitution at position 133; H2 comprises an amino acid substitution at position 124, and L2 comprises amino acid substitutions at positions 131-135. In some embodiments, H1 further comprises an amino acid substitution at position 125, L1 further comprises an amino acid substitution at one or more of positions 122, 129, and 135, H2 further comprises an amino acid substitution at one or more of positions 139, 145, 190, and 228, and / or L2 further comprises an amino acid substitution at position 121.

[0233] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 11, H1 contains amino acid substitutions at positions 143_190; L1 contains amino acid substitutions at positions 129_133_135; H2 contains amino acid substitutions at positions 124_145, and L2 contains amino acid substitutions at positions 131_135.

[0234] In some embodiments, H1 further comprises an amino acid substitution at position 125, L1 further comprises an amino acid substitution at position 122, H2 further comprises an amino acid substitution at one or more of positions 139, 190 and 228, and / or L2 further comprises an amino acid substitution at position 121.

[0235] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 11, wherein H1 comprises an amino acid substitution at position 125_143_190, or 143_190; L1 comprises an amino acid substitution at position 122_129_133_135, or 129_133_135; H2 comprises an amino acid substitution at position 124_139_145_190, 124_139_145_190_228, or 124_145, and L2 comprises an amino acid substitution at position 121_131_135, or 131_135.

[0236] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 11, wherein the amino acid substitutions in H1 are selected from 125R, 143K, 190K, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 122D, 129T, 133D, 135S, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 124E, 139I, 145T, 190I, 228D, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 121K, 131K, 135K, and conservative substitutions thereof.

[0237] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 11, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000042.tif17170

[0238] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 11, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000043.tif23170

[0239] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 and H2L2 heterodimers is such that H1 comprises 143K_190K, L1 comprises 129T_133D_135S, H2 comprises 124E_145T, and L2 comprises 131K_135K.

[0240] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 11 with a disulfide steering driver set.

[0241] In one embodiment, the amino acid combination of KL cluster 11 comprises one or more secondary substitutions selected from Table F.

[0242] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 11 set forth in one or more of the designs in Table 10-A11.

[0243] KL Cluster 12: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 12, wherein H1 comprises an amino acid substitution at position 143 and / or 186; L1 comprises an amino acid substitution at position 133; H2 comprises an amino acid substitution at position 124, and L2 comprises an amino acid substitution at position 131. In some embodiments, H1 further comprises an amino acid substitution at one or more of positions 124, 125, 139, and 188; L1 further comprises an amino acid substitution at one or more of positions 122, 129, 131, and 178; H2 further comprises an amino acid substitution at one or more of positions 143, 145, 179, 186, 188, 228, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of positions 121, 133, 135, 176, 178, 38, and 44.

[0244] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 12, wherein H1 comprises an amino acid substitution at position 124_143, 125_143, 125_143_186, 125_186, 125_186, 125_186_188, 139_143, 139_143_186, 139_186, 139_186_188, 143, or 186_188; and L1 comprises an amino acid substitution at position 122_129_133, 122_129_133_178, 122_133_178, 129_131_133, 129_133, 129_133_178, or 133_178. H2 contains an amino acid substitution at position 124_143_145, 124_145_179, 124_145_179_186_188, 124_145_179_188, 124_145_179_228, 124_145_186, 39_124_145_179, or 45_124_145_179 , L2 contains an amino acid substitution at position 121_131_133_176, 131_133_135, 131_133_135_176, 131_133_135_178, 131_133_176, 131_133_176_178, 38_131_133_176, or 44_131_133_176.

[0245] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 12, wherein the amino acid substitutions in H1 are selected from 124K, 125R, 139W, 143I, 143K, 186K, 188T, and conservative substitutions thereof; and the amino acid substitutions in L1 are selected from 122D, 129T, 131D, 131E, 133D, 178T, and conservative substitutions thereof. the amino acid substitutions in H2 are selected from 124E, 143E, 145T, 179E, 186E, 186I, 188W, 228D, 39E, 45P, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 121K, 131K, 131R, 133G, 133S, 133T, 135K, 135W, 176R, 178A, 178S, 38R, 44F, and conservative substitutions thereof.

[0246] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KL cluster 12, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000044.tif77170

[0247] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KL cluster 12, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000045.tif56170

[0248] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of H1L1 and H2L2 heterodimers is one of the following: TIFF2025124635000046.tif22170

[0249] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 12 with one or more of disulfide steering, steric 2, steric 3, variable domain electrostatic and variable domain steric driver sets.

[0250] In one embodiment, the KL cluster 12 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0251] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL Cluster 12 set forth in one or more of the designs in Table 10-A12.

[0252] KK Cluster 1: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1, where H1 comprises an amino acid substitution at position 143-179; L1 comprises an amino acid substitution at position 124-178; H2 comprises an amino acid substitution at position 186, and L2 comprises an amino acid substitution at position 178-180 or 160-180. In some embodiments, H1 further comprises an amino acid substitution at position 145.

[0253] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1, where H1 comprises amino acid substitutions at positions 143-145-179; L1 comprises amino acid substitutions at positions 124-178; H2 comprises an amino acid substitution at position 186; and L2 comprises an amino acid substitution at position 180. In some embodiments, L2 further comprises an amino acid substitution at positions 160 and / or 178.

[0254] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1, where H1 comprises amino acid substitutions at positions 143_145_179; L1 comprises amino acid substitutions at positions 124_178; H2 comprises amino acid substitutions at position 186, and L2 comprises amino acid substitutions at positions 178_180 or 160_180.

[0255] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1, wherein the amino acid substitutions in H1 are selected from 143E, 145T, 179E, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 124K, 178R, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 186R, or conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 160E, 178E, 180E, and conservative substitutions thereof.

[0256] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 1, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 143E_145T_179E and L1 comprises 124K_178R. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 1, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 124K_178R and L2 comprises 178E_180E or 160E_180E. In some embodiments, the antigen-binding polypeptide construct comprises these combinations of H1L1 and H2L2 heterodimers.

[0257] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1 set forth in Table 10-B1.

[0258] KK Cluster 2: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 2, where H1 comprises an amino acid substitution at position 143; L1 comprises an amino acid substitution at position 124; H2 comprises an amino acid substitution at position 179 or 186, and L2 comprises amino acid substitutions at positions 124, 160, 180. In some embodiments, H1 further comprises an amino acid substitution at position 145 and / or H2 further comprises an amino acid substitution at position 146.

[0259] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 2, where H1 comprises an amino acid substitution at positions 143-145; L1 comprises an amino acid substitution at position 124; H2 comprises an amino acid substitution at position 179 or 186, and L2 comprises amino acid substitutions at positions 124, 160, 180. In some embodiments, H2 further comprises an amino acid substitution at position 146.

[0260] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 2, where H1 comprises an amino acid substitution at positions 143-145; L1 comprises an amino acid substitution at position 124; H2 comprises an amino acid substitution at positions 186, 179, or 146-179, and L2 comprises an amino acid substitution at positions 124-160-180.

[0261] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 2, wherein the amino acid substitution in H1 is selected from 143E, 145T, and conservative substitutions thereof; the amino acid substitution in L1 is 124R or a conservative substitution thereof; the amino acid substitution in H2 is selected from 186R, 179K, 146G, and conservative substitutions thereof; and the amino acid substitution in L2 is selected from 124E, 160E, 180E, and conservative substitutions thereof.

[0262] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 2, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 143E_145T and L1 comprises 124R. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 2, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 186R, 179K or 146G_179K and L2 comprises 124E_160E_180E. In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 heterodimer and the H2L2 heterodimer is such that H1 comprises 143E_145T, L1 comprises 124R, H2 comprises 179K, and L2 comprises 124E_160E_180E.

[0263] In one embodiment, the KK cluster 2 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0264] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK Cluster 2 set forth in one or more of the designs in Table 10-B2.

[0265] KK Cluster 3: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 3, H1 comprises an amino acid substitution at position 186; L1 comprises an amino acid substitution at positions 180 or 178-180; H2 comprises an amino acid substitution at positions 143 and / or 179, and L2 comprises an amino acid substitution at positions 124-178 or 131. In some embodiments, H2 further comprises an amino acid substitution at position 145.

[0266] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 3, wherein H1 comprises an amino acid substitution at position 186; L1 comprises an amino acid substitution at position 180; H2 comprises an amino acid substitution at position 145, and L2 comprises an amino acid substitution at position 124 or 131. In some embodiments, L1 further comprises an amino acid substitution at position 178, H2 further comprises an amino acid substitution at positions 143 and / or 179, and / or L2 further comprises an amino acid substitution at position 178.

[0267] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 3, where H1 comprises an amino acid substitution at position 186; L1 comprises an amino acid substitution at position 180 or 178_180; H2 comprises an amino acid substitution at position 143_145, 143_145_179, or 145_179, and L2 comprises an amino acid substitution at position 131 or 124_178.

[0268] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 3, wherein the amino acid substitution in H1 is selected from 186R and conservative substitutions thereof; the amino acid substitution in L1 is selected from 178E, 180E, and conservative substitutions thereof; the amino acid substitution in H2 is selected from 143E, 145T, 179E, and conservative substitutions thereof; and the amino acid substitution in L2 is selected from 124K, 131K, 178R, and conservative substitutions thereof.

[0269] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 3, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 186R and L1 comprises 178E_180E or 180E. In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 3, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000047.tif28170

[0270] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 and H2L2 heterodimers is such that H1 comprises 186R, L1 comprises 180E, H2 comprises 143E_145T_179E, and L2 comprises 124K_178R.

[0271] In one embodiment, the KK cluster 3 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0272] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 3 set forth in one or more of the designs in Table 10-B3.

[0273] KK Cluster 4: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 4, wherein H1 comprises an amino acid substitution at position 179; L1 comprises an amino acid substitution at position 180; H2 comprises an amino acid substitution at position 143, and L2 comprises an amino acid substitution at position 124. In some embodiments, H1 further comprises an amino acid substitution at position 146, H2 further comprises an amino acid substitution at position 145, and / or L2 further comprises an amino acid substitution at positions 160 and / or 178.

[0274] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 4, where H1 comprises an amino acid substitution at position 179; L1 comprises an amino acid substitution at position 180; H2 comprises amino acid substitutions at positions 143-145, and L2 comprises an amino acid substitution at position 124. In some embodiments, H1 further comprises an amino acid substitution at position 146, and / or L2 further comprises an amino acid substitution at positions 160 and / or 178.

[0275] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 4, where H1 comprises an amino acid substitution at position 146_179 or 179; L1 comprises an amino acid substitution at position 180; H2 comprises an amino acid substitution at positions 143_145, and L2 comprises an amino acid substitution at positions 124 or 124_160_178.

[0276] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 4, wherein the amino acid substitution in H1 is selected from 146G, 179K, and conservative substitutions thereof; the amino acid substitution in L1 is 180E or a conservative substitution thereof; the amino acid substitution in H2 is selected from 143E, 145T, and conservative substitutions thereof; and the amino acid substitution in L2 is selected from 124R, 160K, 178R, and conservative substitutions thereof.

[0277] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 4, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 179K or 146G_179K, and L1 comprises 180E. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 4, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 143E_145T, and L2 comprises Q124R_Q160K_T178R or Q124R. In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 heterodimer and the H2L2 heterodimer is such that H1 comprises 179K, L1 comprises 180E, H2 comprises 143E_145T, and L2 comprises 124R_160K_178R.

[0278] In one embodiment, the KK cluster 4 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0279] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK Cluster 4 set forth in one or more of the designs in Table 10-B4.

[0280] KK Cluster 5: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 5, wherein H1 comprises an amino acid substitution at position 143 or 186; L1 comprises an amino acid substitution at position 180 or does not comprise an amino acid substitution that promotes preferential pairing; H2 comprises amino acid substitutions at positions 143-145, and L2 comprises an amino acid substitution at position 124. In some embodiments, L2 further comprises an amino acid substitution at one or more of positions 160 and / or 178. In further embodiments, L2 comprises an amino acid substitution at positions 124, 124-178, or 124-160-178.

[0281] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 5, wherein the amino acid substitutions in H1 are selected from 186R, 143R, 143K, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 180E, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 143E, 145T, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 124R, 160K, 178R, and conservative substitutions thereof.

[0282] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 5, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000048.tif25170

[0283] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 5, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000049.tif24170

[0284] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer.

[0285] In one embodiment, the KK cluster 5 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0286] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 5 set forth in one or more of the designs in Table 10-B5.

[0287] KK Cluster 6: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 6, where H1 comprises an amino acid substitution at position 39 or does not contain an amino acid substitution that promotes preferential pairing; L1 comprises an amino acid substitution at position 38 or does not contain an amino acid substitution that promotes preferential pairing; H2 comprises an amino acid substitution at position 39 and L2 comprises an amino acid substitution at position 38.

[0288] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 6, wherein the amino acid substitutions in H1 are selected from 39D, 39E, 39K, 39R, and conservative substitutions thereof; the amino acid substitutions in L1 are selected from 38D, 38E, 38K, 38R, and conservative substitutions thereof; the amino acid substitutions in H2 are selected from 39D, 39E, 39K, 39R, and conservative substitutions thereof; and the amino acid substitutions in L2 are selected from 38D, 38E, 38K, 38R, and conservative substitutions thereof.

[0289] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 6, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000050.tif36170

[0290] In a further embodiment, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 6, wherein the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF2025124635000051.tif30170

[0291] In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 and H2L2 heterodimers is such that H1 comprises 39R, L1 comprises 38E, H2 comprises 39D, and L2 comprises 38R.

[0292] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK Cluster 6 set forth in one or more of the designs in Table 10-B6. In one embodiment, the KK Cluster 6 design is not a design corresponding to LCCA unique identifiers 10674-10749 or 10679-10744.

[0293] KK Cluster 7: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 7, where H1 does not contain an amino acid substitution that promotes preferential pairing; L1 contains an amino acid substitution at position 135; H2 contains an amino acid substitution at position 139, and L2 contains an amino acid substitution at position 116. In some embodiments, L2 further contains an amino acid substitution at position 135.

[0294] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 7, wherein the amino acid substitution in L1 is 135W or a conservative substitution thereof; the amino acid substitution in H2 is 139W or a conservative substitution thereof; and the amino acid substitution in L2 is selected from 116A, 135V, and conservative substitutions thereof.

[0295] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 7, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 does not comprise an amino acid substitution that promotes preferential pairing, and L1 comprises 135W. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 7, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 139W, and L2 comprises 116A or 116A_1335V. In some embodiments, the antigen-binding polypeptide construct comprises a combination of one H2L2 heterodimer and one H1L1 heterodimer.

[0296] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK Cluster 7 as set forth in either of the designs in Table 10-B7.

[0297] KK Cluster 8: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 8, where H1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 45; L1 does not contain an amino acid substitution that promotes preferential pairing; H2 contains an amino acid substitution at position 45, and L2 contains an amino acid substitution at position 44.

[0298] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 8, wherein the amino acid substitution in H1 is 45F or a conservative substitution thereof; the amino acid substitution in H2 is 45P, 45A or a conservative substitution thereof; and the amino acid substitution in L2 is 44F or a conservative substitution thereof.

[0299] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 8, wherein the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: H1 does not comprise an amino acid substitution that promotes preferential pairing or comprises 45F, and L1 does not comprise an amino acid substitution that promotes preferential pairing. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 8, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 45A or 45P, and L2 comprises 44F. In some embodiments, the antigen-binding polypeptide construct comprises a combination of H1L1 and H2L2 heterodimers, wherein H1 and L1 do not comprise an amino acid substitution that promotes preferential pairing, H2 comprises 45A, and L2 comprises 44F.

[0300] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK Cluster 8 set forth in one or more of the designs in Table 10-B8.

[0301] KK Cluster 9: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 9, where H1 comprises an amino acid substitution at position 139; L1 comprises an amino acid substitution at position 116; H2 does not comprise an amino acid substitution that promotes preferential pairing, and L2 comprises an amino acid substitution at position 135.

[0302] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 9, wherein the amino acid substitution in H1 is 139W or a conservative substitution thereof; the amino acid substitution in L1 is 116A or a conservative substitution thereof; and the amino acid substitution in L2 is 135W or a conservative substitution thereof.

[0303] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 9, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 139W and L1 comprises 116A. In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 9, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 does not comprise an amino acid substitution that promotes preferential pairing, and L2 comprises 135W.

[0304] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 9 set forth in Table 10-B9.

[0305] KK Cluster 10: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 10, wherein H1 comprises an amino acid substitution at position 124; L1 comprises an amino acid substitution at position 176; H2 comprises an amino acid substitution at position 124 and L2 comprises an amino acid substitution at position 176. In some embodiments, L1 and / or L2 further comprise an amino acid substitution at position 133.

[0306] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 10, where H1 comprises an amino acid substitution at position 124; L1 comprises an amino acid substitution at positions 133-176; H2 comprises an amino acid substitution at position 124 and L2 comprises amino acid substitutions at positions 133-176.

[0307] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 10, wherein the amino acid substitution in H1 is 124E or a conservative substitution thereof; the amino acid substitution in L1 is 133G, 176R or a conservative substitution thereof; the amino acid substitution in H2 is 124R or a conservative substitution thereof; and the amino acid substitution in L2 is 133G, 176D or a conservative substitution thereof.

[0308] In some embodiments, the antigen-binding polypeptide construct comprises an H1L1 heterodimer comprising a combination of amino acid substitutions according to KK cluster 10, wherein the H1L1 heterodimer comprises the following set of amino acid substitutions: H1 comprises 124E and L1 comprises 133G_176R. In further embodiments, the antigen-binding polypeptide construct comprises an H2L2 heterodimer comprising a combination of amino acid substitutions according to KK cluster 10, wherein the H2L2 heterodimer comprises the following set of amino acid substitutions: H2 comprises 124R and L2 comprises 133G_176D. In some embodiments, the antigen-binding polypeptide construct comprises these combinations of H1L1 and H2L2 heterodimers.

[0309] In one embodiment, the KL cluster 9 amino acid combination comprises one or more secondary substitutions selected from Table F.

[0310] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 10 set forth in Table 10-B10.

[0311] Preferential matching in LCCA design sets. One or more of H1, L1, H2, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2, and promotes preferential pairing of L2 with H2 relative to L1. Generally, in the absence of amino acid modifications and any naturally occurring biases, a wild-type immunoglobulin heavy chain sequence (H1), when co-expressed with two different wild-type immunoglobulin light chain sequences (L1 and L2), will statistically pair equally with both light chains, resulting in an approximately 50:50 mixture of H1 paired with L1 (H1L1, correctly paired) and H1 paired with L2 (H1L2, mispaired). Similarly, when wild-type H2 is co-expressed with wild-type L1 and L2, the heavy chain statistically pairs equally with both light chains, resulting in an approximately 50:50 mixture of H2 paired with L1 (H2L1, mispaired) and H2 paired with L2 (H2L2, correctly paired). The term "preferential pairing" is used herein to describe the pairing specificity or preference of one immunoglobulin light chain polypeptide sequence with an immunoglobulin heavy chain polypeptide sequence compared to another immunoglobulin light chain polypeptide sequence. In this context, for example, when H1 is co-expressed with both L1 and L2, preferential pairing occurs between H1 and L1 if the amount of H1L1 heterodimer is greater than the amount of H1L2 heterodimer. Similarly, for example, when H2 is co-expressed with both L1 and L2, if the amount of H2L2 heterodimer is greater than the amount of H2L1 heterodimer, then preferential pairing occurs between H1 and L1.

[0312] However, in some cases, there is an inherent pairing bias observed in wild-type heavy and light chain polypeptide sequences derived from parent antibodies. This inherent pairing bias can be observed in the context of an LCCA design set in which a wild-type parent H1 or H2 is co-expressed with wild-type parent L1 and L2, and one of the light chains preferentially pairs with the heavy chains of both parent antibodies. In one embodiment, preferential pairing occurs when there are more amino acid modifications in one or more of H1, L1, H2, and L2 that promote preferential pairing than occurs in the corresponding wild-type system.

[0313] The degree of preferential pairing or design strength is a measure of the ability of an amino acid modification to promote preferential pairing. The degree of preferential pairing can be assessed as described elsewhere herein and in the Examples and is based on measuring correctly paired heterodimers (i.e., H1L1 and H2L2) compared to mismatched heterodimers (i.e., H1L2 and H2L1). The degree of preferential pairing can be assessed in the context of an LCCA design set (H1L1L2, or H2L1L2) in which one heavy chain is co-expressed with two unique light chains, or a Mab design set (H1L1H2L2) in which the heavy and light chains of parent antibodies are co-expressed.

[0314] The following embodiments relate to the context of an LCCA design set. In all of the embodiments in this section, the term "about" means ±5% of the specified ratio, and unless otherwise indicated, the preferred pairing is compared to wild-type. In one embodiment, one or more of H1, H2, L1, and L2 contain an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 60:40. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 60:40.

[0315] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 65:35. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 65:35.

[0316] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 70:30. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 70:30.

[0317] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 75:25. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 75:25.

[0318] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 80:20. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 80:20.

[0319] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 85:15. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 85:15.

[0320] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 90:10. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 to H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 to H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 90:10.

[0321] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 95:5. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 95:5.

[0322] In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 99: 1. In one embodiment, one or more of H1, H2, L1, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2 to form H1L1, or promotes preferential pairing of L2 with H2 relative to L1 to form H2L2, wherein the ratio of H2L2:H2L1 is at least about 40:60 and the ratio of H1L1:H1L2 is at least about 99: 1.

[0323] In other embodiments, one or more of H1, H2, L1, and L2 comprise amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 to form H1L1, or that promote preferential pairing of L2 to H2 relative to L1 to form H2L2, such that the amount of H1L1 or H2L2 is about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more.

[0324] In one embodiment, preferential pairings are measured by LCCA as described in the Examples. LCCA results generally predict outcomes in the context of preferential pairings in Mab design sets (described below) where H1, L1, H2, and L2 are co-expressed.

[0325] In one embodiment, one or more of H1, H2, L1, and L2 contain amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 to form H1L1, or that promote preferential pairing of L2 to H2 relative to L1 to form H2L2, such that the relative pairing of at least one of H1L1 or H2L2 is at least about 10% greater than wild type and the relative pairing of the other is within about 10% of wild type or at least about 10% greater than wild type.

[0326] In one embodiment, one or more of H1, H2, L1, and L2 contain amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 to form H1L1, or promote preferential pairing of L2 to H2 relative to L1 to form H2L2, such that the relative pairing of at least one of H1L1 or H2L2 is at least about 20% greater than wild type and the relative pairing of the other is within about 10% of wild type or at least about 10% greater than wild type.

[0327] In one embodiment, one or more of H1, H2, L1, and L2 contain amino acid modifications that promote preferential pairing of L1 to H1 relative to L2 to form H1L1, or promote preferential pairing of L2 to H2 relative to L1 to form H2L2, such that the relative pairing of at least one of H1L1 or H2L2 is at least about 30% greater than wild type and the relative pairing of the other is within about 10% of wild type or at least about 10% greater than wild type.

[0328] Preferred pairings in Mab design sets Preferential pairing can also be assessed in the context of a Mab design set in which H1, L1, H2, and L2 are co-expressed, and one or more of H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing of L1 with H1 and promote preferential pairing of L2 with H2, forming a bispecific antigen-binding polypeptide construct comprising a first correctly paired heterodimer (H1L1) and a second correctly paired heterodimer (H2L2). In this type of embodiment, as shown in Figure 8, when two different immunoglobulin heavy chain polypeptide sequences are co-expressed with two different immunoglobulin light chain polypeptide sequences, many possible products can be obtained, 14 of which are shown in Figure 8, of which only one is the desired or correctly paired bispecific antibody H1L1H2L2 (antibody species A in Figure 8). However, in the context of assessing correct pairing between heavy and light chains based on the Mab design set, some of the additional products contain heterodimers that are correctly paired at the Fab level (see, e.g., antibody species E, H, K, and M in Figure 8), and therefore may also be considered to represent correct pairing in the context of the Fab region. In some embodiments, the Fc portion of the antigen-binding polypeptide construct comprises asymmetric amino acid modifications that promote the formation of heterodimeric Fc. In these embodiments, the number and abundance of species E through J are expected to be reduced.

[0329] For LCCA design sets, in the context of a Mab design set co-expressing all four immunoglobulin polypeptide sequences, H1, L1, H2, and L2, there may in some cases be an inherent bias in pairing resulting from one of the light chains (either L1 or L2) preferentially pairing with both H1 and H2. Thus, when determining the strength of a Mab design in the context of a bispecific antigen-binding polypeptide construct, it may be necessary to assess the degree of pairing with the amino acid modifications of the Mab design compared to the amount of correct pairing observed in the corresponding wild-type parental system (the H1, L1, H2, L2 polypeptide sequences without the amino acid modifications of the Mab design). Thus, in one embodiment, a Mab design is considered to exhibit preferential pairing if the amount of correctly paired bispecific antigen-binding polypeptide construct is greater than the amount of correctly paired bispecific antibody observed in the corresponding wild-type parental system. Alternatively, a Mab design is considered to exhibit preferential pairing if the percentage of correctly paired bispecific antigen-binding polypeptide constructs in the total expression product is greater than the amount of correctly paired bispecific antibody obtained in the total expression product in the corresponding wild-type parental line. In one embodiment, the total expression product may include antibody species A-N of Figure 8. In one embodiment, the total expression product may be exclusively antibody species having two heavy chains and two light chains (antibody species A-J of Figure 8). In the latter embodiment, preferential pairing is measured as the percentage of total bispecific antibody excluding half antibodies, such as species K-N of Figure 8.

[0330] In another embodiment, a Mab design is considered to exhibit preferential pairing if the amount of correct pairing is increased in heterodimers of bispecific antigen-binding polypeptide constructs that show a high degree of mismatching in the corresponding wild-type parental line. In another embodiment, a Mab design is considered to exhibit preferential pairing if the total amount of correct pairing between H1 and L1 and H2 and L2 is greater than that observed in the corresponding wild-type parental line. For example, with reference to Figure 8, species A, B, H, I, and M are considered to be correctly paired to H1L1, and species A, C, E, F, and K are considered to be correctly paired to H2L2. In this embodiment, preferential pairing is measured as a percentage of total pairings.

[0331] In one embodiment, preferential pairing is measured by SMCA as described herein.

[0332] In some embodiments, a Mab design is considered to promote preferential pairing if the change in the total amount of correct pairing, as measured by the sum of H1L1 and H2L2 pairings, is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% compared to the pairing of the corresponding H1, L1, H2, and L2 polypeptide chains without the amino acid substitutions in the Fab region that promote preferential pairing.

[0333] In some embodiments, a Mab design is considered to promote preferential pairing if the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of species other than the half antibodies produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of the corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing.

[0334] In one embodiment, a Mab design is considered to promote preferential pairing if the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of all species produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of the corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing.

[0335] Thermal stability of the Fab region Amino acid modifications in one or more of the H1, L1, H2, and L2 polypeptide sequences promote preferential pairing of L1 with H1 relative to L2, and promote preferential pairing of L2 with H2 relative to L1, minimizing the effect on the thermal stability of each heterodimer of the antigen-binding polypeptide construct. The effect of amino acid modifications on each heterodimer is determined by measuring the thermal stability of the Fab region formed by H1 and L1 or the Fab region formed by H2 and L2 and comparing it to the thermal stability of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences (wild-type first Fab region) or the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences (wild-type second Fab region). The terms "corresponding wild-type H1 and L1 polypeptide sequences" and "corresponding wild-type H2 and L2 polypeptide sequences" are meant to describe the corresponding H1, L1, H2, and L2 polypeptide sequences without the preferential pairing-promoting amino acid modifications described herein.

[0336] Thermal stability can be measured by a variety of methods known in the art and described herein, including differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF), the latter method providing a measure of thermal stability in terms of the "melting temperature" or Tm.

[0337] In the context of the following embodiments, the term "about" means ±10% of the stated temperature. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 20°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 15°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 10°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 9°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 8°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 7°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 6°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 5°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 4°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 3°C ​​of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 2°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm within about 1°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a first Fab region having a Tm approximately the same as the Tm of the corresponding wild-type first Fab region.

[0338] In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 20°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 15°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 10°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 9°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 8°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 7°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 6°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 5°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 4°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 3°C ​​of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 2°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm within about 1°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct comprises a second Fab region having a Tm approximately the same as the Tm of the corresponding wild-type second Fab region.

[0339] In one embodiment, the antigen-binding polypeptide construct comprises a first heterodimer and a second heterodimer, wherein the melting temperature (Tm) of the first Fab region is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Tm of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences for the first antigen (wild-type first Fab region), and / or the melting temperature (Tm) of the second Fab region is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Tm of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences for the second antigen (wild-type second Fab region).

[0340] Furthermore, in some embodiments, the Tm of the first or second Fab region is greater than the Tm of the corresponding wild-type first Fab or the corresponding wild-type second Fab. Thus, in one embodiment, the Tm of the first or second Fab region is increased by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5.0°C or more compared to the corresponding wild-type first Fab region or the corresponding wild-type second Fab region.

[0341] In one embodiment, the antigen-binding polypeptide construct comprises amino acid substitutions corresponding to KL Design Nos. 2979, 3018, 3041, 3102, 3898, and / or 3947. In one embodiment, the antigen-binding polypeptide construct comprises amino acid substitutions corresponding to KL Design Nos. 3025, 3109, 3113, 3878, 3890, 3910, 3931, 3954, 3967, 4010, and / or 4040.

[0342] Ability of the Fab region to bind to antigen Amino acid modifications in one or more of the H1, L1, H2, and L2 polypeptide sequences promote preferential pairing of L1 with H1 relative to L2, and promote preferential pairing of L2 with H2 relative to L1, minimizing the effect on the ability of each heterodimer of the antigen-binding polypeptide construct to bind its antigen. The effect of amino acid modifications on each heterodimer is determined by measuring the ability of the Fab region formed by H1 and L1 or the Fab region formed by H2 and L2 to bind its respective antigen and comparing it to the ability of the corresponding wild-type first Fab region or the corresponding wild-type second Fab region to bind its respective antigen.

[0343] The ability of the Fab regions to bind to their respective antigens can be measured by many methods known in the art, some of which are described elsewhere herein. For example, surface plasmon resonance (SPR) or whole cell binding assays may be used to assess the ability of a first Fab region to bind to a first antigen and a second Fab region to bind to a second antigen. The latter two methods measure the ability of the Fab regions to bind to their respective antigens by determining the affinity of the Fab region for that antigen.

[0344] In one embodiment, the affinity of the first Fab region for the first antigen is within about 100-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 50-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 40-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 30-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 20-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 10-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 9-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 8-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 7-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 6-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 5-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within 4-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 3-fold of the affinity of the wild-type first Fab region for the first antigen. In one embodiment, the affinity of the first Fab region for the first antigen is within about 2-fold of the affinity of the wild-type first Fab region for the first antigen.In one embodiment, the affinity of the first Fab region for the first antigen is approximately the same as the affinity of the wild-type first Fab region for the first antigen.

[0345] In one embodiment, the affinity of the second Fab region for the second antigen is within about 100-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 50-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 40-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 30-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 20-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 10-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 9-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 8-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 7-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 6-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 5-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within 4-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 3-fold of the affinity of the wild-type second Fab region for the second antigen. In one embodiment, the affinity of the second Fab region for the second antigen is within about 2-fold of the affinity of the wild-type second Fab region for the second antigen.In one embodiment, the affinity of the second Fab region for the second antigen is about the same as the affinity of the wild-type second Fab region for the second antigen.

[0346] Transferability of amino acid modifications or design sets The amino acid modifications or design sets described herein can be used to prepare bispecific antigen-binding polypeptide constructs in which the immunoglobulin heavy chain polypeptide sequence and the immunoglobulin light chain polypeptide of each heterodimer can be derived from one or more parent antibodies, where at least one parent antibody comprises a kappa light chain and at least one other parent antibody comprises a lambda light chain. Based on the discussion below, Mab design sets can be applied to the majority of such bispecific antigen-binding polypeptide constructs.

[0347] The VH:VL and CH1:CL interface residues at the interface between immunoglobulin heavy and light chains are relatively well conserved (Padlan et al., 1986, Mol. Immunol. 23(9):951-960). This sequence conservation, a result of evolutionary constraints, increases the likelihood that a functionally active antibody-binding domain will form upon pairing of a light and heavy chain combination. As a result of this sequence conservation, the Mab design set described herein can be transferred to other parent antibody kappa Fabs and lambda Fabs for preferential pairing based on modeling of the structures of the D3H44 kappa Fab and CAT-2200 lambda Fab, which result in preferential pairing, because this region shows high sequence conservation across antibodies. Furthermore, when sequence divergence occurs, it is usually distal to the CH1:CL interface. This is particularly the case for the CH1 and CL domains. In certain embodiments, the antigen-binding polypeptide constructs described herein comprise heterodimers in which a kappa Fab comprises one or more amino acid modifications in the CL and / or CH1 domains that promote preferential pairing. In certain embodiments, the antigen-binding polypeptide constructs described herein comprise heterodimers in which a lambda Fab comprises one or more amino acid modifications in the CL and / or CH1 domains that promote preferential pairing.

[0348] However, there are some sequence changes to the CDR (complementarity determining region) loop residues (and length), particularly in the antigen-binding site relative to CDR-H3. Thus, in one embodiment, the antigen-binding polypeptide constructs described herein comprise heterodimers in which the kappa Fab comprises one or more amino acid modifications in the VH and / or VL domains distal to the CDR loops, where the amino acid sequence of the antigen-binding site differs significantly from that of the D3H44 antibody. In another embodiment, the antigen-binding polypeptide constructs described herein comprise heterodimers in which the lambda Fab comprises one or more amino acid modifications in the VH and / or VL domains that promote preferential pairing and that are distal to the CDR loops, where the amino acid sequence of the antigen-binding site differs significantly from that of the CAT-2200 antibody. In another embodiment, the antigen-binding polypeptide constructs described herein include heterodimers in which a kappa Fab contains one or more amino acid modifications in the VH and / or VL domains that promote preferential pairing when the amino acid sequence of the antigen-binding site is substantially similar to that of the D3H44 antibody and that are proximal or distal to the CDR loops. In another embodiment, the antigen-binding polypeptide constructs described herein include heterodimers in which a lambda Fab contains one or more amino acid modifications in the VH and / or VL domains that promote preferential pairing when the amino acid sequence of the antigen-binding site is substantially similar to that of the CAT-2200 antibody and that are proximal or distal to the CDR loops. In certain embodiments, the antigen-binding polypeptide constructs described herein include heterodimers in which a kappa Fab contains one or more amino acid modifications in the CL and / or CH1 domains and modifications in the VH and / or VL domains that promote preferential pairing. In certain embodiments, the antigen-binding polypeptide constructs described herein comprise heterodimers in which the lambda Fab comprises one or more amino acid modifications in the CL and / or CH1 domains and modifications in the VH and / or VL domains that promote preferential pairing.

[0349] In one embodiment, amino acid modifications in one or more of H1, L1, H2, and L2 of an antigen-binding polypeptide construct can promote preferential pairing in an antigen-binding polypeptide construct in which the kappa Fab of one parent antibody is human or humanized IgG1 / κ. Non-limiting examples of such parent antibodies include ofatumumab (human) or trastuzumab, or bevacizumab (humanized). In one embodiment, amino acid modifications in one or more of H1, L1, H2, and L2 of an antigen-binding polypeptide construct can promote preferential pairing in an antigen-binding polypeptide construct in which the lambda Fab of one parent antibody is human or humanized IgG1 / lambda. Non-limiting examples of such human antibodies include briakinumab or sifalimumab, while an example of a humanized antibody is brontixutuzumab.

[0350] In another embodiment, the amino acid modifications described herein can be transferred to the immunoglobulin heavy and light chains of antibodies that use the commonly used VH and VL subgroups.

[0351] In one embodiment, the amino acid modifications described herein can be transferred to the immunoglobulin heavy and light chains of an antibody with a germline-like framework, such as obinutuzumab.

[0352] In one embodiment, the amino acid modifications described herein can be transferred to the immunoglobulin heavy and light chains of an antibody having a VH:VL interdomain angle that approximates the average observed for heavy and light chain pairs. An example of this type of antibody includes, but is not limited to, pertuzumab. In another embodiment, the amino acid modifications described herein can be transferred to the immunoglobulin heavy and light chains of an antibody having canonical CL and CH1 domains. A suitable example of such an antibody includes, but is not limited to, trastuzumab.

[0353] The examples, figures, and tables show that amino acid modifications (e.g., in one or more Fab fragments comprising variable and constant regions) that can promote preferential pairing can be transferred to other immunoglobulin heavy and light chains, resulting in a similar pattern of preferential pairing of one immunoglobulin heavy chain with one of two immunoglobulin light chains.

[0354] scaffold The heterodimer of the antigen-binding polypeptide construct can be linked to a scaffold. The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance. The heterodimer of the antigen-binding polypeptide construct can be linked to either the N-terminus or C-terminus of the polypeptide scaffold. In one embodiment, the scaffold is an albumin polypeptide.

[0355] In another embodiment, the scaffold is an immunoglobulin Fc (Fc), or a portion thereof. In some embodiments, the Fc comprises at least one or two CH3 domain sequences. In some embodiments, the Fc further comprises at least one or two CH2 domain sequences. In some embodiments, the antigen-binding polypeptide construct comprises an Fc linked to a first heterodimer and / or a second heterodimer, with or without one or more linkers. In some embodiments, the Fc is a human Fc. In some embodiments, the Fc is a human IgG or IgG1 Fc. In some embodiments, the Fc is a heterodimeric Fc. In some embodiments, the Fc is a single-chain polypeptide. In some embodiments, the Fc is multiple peptides, e.g., two polypeptides.

[0356] In some embodiments, the Fc comprises one or more amino acid modifications in at least one of the CH3 domain sequences. Amino acid modifications can be made to an immunoglobulin heavy chain Fc to induce preferential pairing between heterodimeric CH3 domain sequences over homodimeric CH3 domain sequences. Such amino acid modifications are known in the art and include, for example, those described in U.S. Patent Publication No. 2012 / 0149876. Alternative strategies for inducing preferential pairing between heterodimeric CH3 domain sequences over homodimeric CH3 sequences include, for example, "knobs-into-holes," charged residues via ionic interactions, and strand-exchange engineered domain (SEED) technology can also be used. The latter strategy has been described in the art and is outlined above in Klein et al. Further discussion of the Fc domain follows.

[0357] In some embodiments, the Fc is an Fc described in patent application PCT / CA2011 / 001238, filed November 4, 2011, or PCT / CA2012 / 050780, filed November 2, 2012, the entire disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0358] In some embodiments, the antigen-binding polypeptide constructs described herein comprise a heterodimeric Fc comprising a modified CH3 domain that is asymmetrically modified. The heterodimeric Fc can comprise two heavy chain constant domain polypeptides, i.e., a first Fc polypeptide and a second Fc polypeptide, which can be used interchangeably, provided that the Fc comprises one first heavy chain polypeptide and one second heavy chain polypeptide. Generally, the first heavy chain polypeptide comprises a first CH3 sequence, and the second heavy chain polypeptide comprises a second CH3 sequence.

[0359] Two CH3 sequences containing one or more asymmetrically introduced amino acid modifications generally result in a heterodimeric Fc rather than a homodimer when the two CH3 sequences dimerize. As used herein, "asymmetric amino acid modification" refers to any modification in which an amino acid at a specific position on a first CH3 sequence differs from an amino acid at the same position on a second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer rather than a homodimer. This heterodimerization can be the result of modification of only one of the two amino acids at the same individual amino acid position on each sequence; or modification of both amino acids at the same individual position on each of the first and second CH3 sequences. The first and second CH3 sequences of a heterodimeric Fc can contain one or more asymmetric amino acid modifications.

[0360] Table X provides the amino acid sequence of the human IgG1 Fc sequence corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain.

[0361] Typically, an Fc can comprise two consecutive heavy chain sequences (A and B) that can dimerize. In some embodiments, one or both sequences of the Fc comprise one or more mutations or modifications at the following positions, using EU numbering: L351, F405, Y407, T366, K392, T394, T350, S400, and / or N390. In some embodiments, an Fc comprises a mutant sequence shown in Table X. In some embodiments, an Fc comprises mutations of variants 1A-B. In some embodiments, an Fc comprises mutations of variants 2A-B. In some embodiments, an Fc comprises mutations of variants 3A-B. In some embodiments, an Fc comprises mutations of variants 4A-B. In some embodiments, an Fc comprises mutations of variants 5A-B.

[0362] TIFF2025124635000052.tif97170

[0363] In some embodiments, the Fc may contain one or more amino acid modifications in at least one of the CH2 domain sequences. Numerous mutations in the heavy chain sequence of the Fc that selectively alter the affinity of antibody Fc for different Fcγ receptors are known in the art. In some embodiments, the Fc contains one or more modifications to alter the binding of the Fcγ receptor to the antigen-binding polypeptide construct.

[0364] The CH2 domain corresponds to amino acids 231-340 of the sequence shown in Table X. Exemplary non-limiting amino acid modifications that alter the ability of the Fc of an antigen-binding polypeptide construct to bind to an Fc-γ receptor are listed below: S298A / E333A / K334A, S298A / E333A / K334A / K326A(Lu Y, Vernes JM, Chiang N, et al.J Immunol Methods.2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L(StavenhagenJB,Gorlatov S,Tuaillon N,et al.Cancer Res.2007 Sep 15;67(18):8882-90;Nordstrom JL, Gorlatov S, Zhang W, et al.Breast Cancer Res.2011 Nov 30;13(6):R123);F243L(Stewart R,Thom G,LevensM,et al.Protein Eng Des Sel.2011 Sep;24(9):671-8),S298A / E333A / K334A(Shields RL, Namenuk AK, Hong K, et al. J Biol. Chem.2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E(Lazar GA,Dang W,Karki S,et al.Proc Natl Acad Sci USA.2006 Mar 14;103(11):4005-10);S239D / S267E,S267E / L328F(Chu SY,Vostiar I,Karki S,et al.Mol Immunol.2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135, which are incorporated herein by reference. Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) describes on page 283 additional modifications to Fc that affect binding of Fc to Fc-gamma receptors.

[0365] Additional modifications to improve effector function In some embodiments, the Fc of the antigen-binding polypeptide constructs described herein may be modified to improve effector function. Such modifications are known in the art and include afucosylation or engineering the affinity of the Fc portion towards activating receptors, primarily FCGR3a for ADCC and C1q for CDC. Table Y below summarizes various designs reported in the literature for engineering effector function.

[0366] TIFF2025124635000053.tif94170

[0367] Thus, in one embodiment, the antigen-binding polypeptide constructs described herein may comprise a dimeric Fc comprising one or more amino acid modifications as set forth in the table above that result in improved effector function, hi another embodiment, the antigen-binding polypeptide constructs may be afucosylated to improve effector function.

[0368] FcRn binding and PK parameters As is known in the art, binding to FcRn recycles internalized antibodies from endosomes back into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766). This process, coupled with exclusion of antibodies from renal filtration due to the large size of the full-length molecule, results in a favorable antibody serum half-life ranging from 1 to 3 weeks. Binding of Fc to FcRn also plays an important role in antibody transport. Thus, in one embodiment, the Fc contains one or more amino acid modifications that alter or enhance the ability of the Fc to bind to FcRn.

[0369] Linker The constructs described herein may comprise one or more heterodimers described herein operably coupled to an Fc described herein. In some embodiments, the Fc is coupled to one or more heterodimers with or without one or more linkers. In some embodiments, the Fc is directly coupled to one or more heterodimers. In some embodiments, the Fc is coupled to one or more heterodimers with one or more linkers. In some embodiments, the Fc is coupled to the heavy chain of each heterodimer by a linker.

[0370] In some embodiments, the one or more linkers are one or more polypeptide linkers. In some embodiments, the one or more linkers comprise one or more antibody hinge regions. In some embodiments, the one or more linkers comprise one or more IgG1 hinge regions.

[0371] Further optional modifications In one embodiment, the immunoglobulin heavy and light chains of the antigen-binding polypeptide constructs described herein can be further modified (i.e., by the covalent attachment of various types of molecules) so that the covalent attachment does not interfere with the preferential pairing between the heavy and light chains, or affects the ability of the heterodimer to bind its antigen, or affects its stability. Such modifications include, but are not limited to, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to intracellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical degradation, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0372] In another embodiment, the immunoglobulin heavy and light chains of the antigen-binding polypeptide constructs described herein can be conjugated (directly or indirectly) to a therapeutic agent or drug moiety that modifies a predetermined biological response. In certain embodiments, the antigen-binding polypeptide construct is conjugated to a drug, such as a toxin, a chemotherapeutic agent, an immunomodulator, or a radioisotope. Several methods for preparing ADCs (antibody-drug conjugates or antigen-binding polypeptide construct-drug conjugates) are known in the art and are described, for example, in U.S. Patent Nos. 8,624,003 (the Pott method), 8,163,888 (one-step method), and 5,208,020 (two-step method). In some embodiments, the drug is selected from maytansine, auristatin, calicheamicin, or a derivative thereof. In other embodiments, the drug is a maytansine selected from DM1 and DM4.

[0373] In some embodiments, the antigen-binding polypeptide construct is conjugated to a cytotoxic agent. The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term is intended to include toxins, such as radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu177), chemotherapeutic agents, and small molecule toxins, or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof).

[0374] Therapeutic agents or drug moieties should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, onconase (or another cytotoxic RNase), Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator; apoptotic agents, e.g., TNF-alpha, TNF-beta, AIMI (see International Publication WO 97 / 33899), AIM II (see International Publication WO 97 / 34911), Fas Ligand (Takahashi et al., J. Immunol. 2002; 2003; 2004; 2005; 2006; 2007; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2029; 2030; 2031; 2032; 2033; 2034; 2035; 2036; 2037; 2038; 2040; 2041; 2042; 2043; 2044; 2045; 2046; 2047; 2048; 2049 al., 1994, J. Immunol., 6:1567), and VEGI (see International Publication WO 99 / 23105); thrombotic or anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as, for example, lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), and granulocyte-colony-stimulating factor ("G-CSF")), or growth factors (e.g., growth hormone ("GH")). Furthermore, in alternative embodiments, the antigen-binding polypeptide constructs may be conjugated to a therapeutic moiety, such as a radioactive material or a macrocyclic chelator useful for conjugating radioactive metal ions (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al. 1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.

[0375] In some embodiments, the immunoglobulin heavy and light chains of the antigen-binding polypeptide construct are expressed as fusion proteins containing tags to facilitate purification and / or testing, etc. As used herein, a "tag" is any additional sequence of amino acids provided to a protein, either at the C-terminus, N-terminus, or internally, that contributes to the identification or purification of the protein. Suitable tags include, but are not limited to, tags known to those skilled in the art that are useful for purification and / or testing, such as albumin-binding domain (ABD), His tag, FLAG tag, glutathione-S-transferase, hemagglutinin (HA), and maltose-binding protein. Such tagged proteins may also be engineered to contain cleavage sites, such as thrombin, enterokinase, or factor X cleavage sites, to simplify tag removal before, during, or after purification.

[0376] Methods for preparing antigen-binding polypeptide constructs As described above, the antigen-binding polypeptide constructs described herein can comprise a first heterodimer and a second heterodimer. The first heterodimer comprises an immunoglobulin heavy chain or fragment thereof having at least a VH and CH1 domain, and an immunoglobulin lambda light chain having a VL domain and a CL domain. The second heterodimer comprises an immunoglobulin heavy chain or fragment thereof having at least a VH and CH1 domain, and an immunoglobulin kappa light chain having a VL domain and a CL domain. The immunoglobulin polypeptide sequences are genetically engineered to incorporate amino acid modifications that promote preferential pairing as described herein. Thus, in the case of bispecific antigen-binding polypeptide constructs, there are typically four distinct polypeptide sequences: two immunoglobulin heavy chain polypeptide sequences or fragments thereof, and two immunoglobulin light chain polypeptide sequences that comprise the antigen-binding polypeptide construct. The immunoglobulin heavy chain polypeptide sequences and immunoglobulin light chain polypeptide sequences of the antigen-binding polypeptide construct can be readily prepared using recombinant DNA techniques known in the art.Standard techniques, such as those described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001), Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2nd ed., 1989), Short Protocols in Molecular Biology (Ausubel et al., John Wiley and Sons, New York, 4th ed., 1999), and Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, DC, 2nd ed., 1998), can be used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture, transgene integration, and recombinant protein expression.

[0377] The polynucleotide and amino acid sequences of the immunoglobulin heavy and light chains of the parent antibodies that make up the antigen-binding polypeptide construct are either known in the art or can be readily determined using nucleic acid and / or protein sequencing methods.

[0378] Thus, a polynucleotide or set of polynucleotides encoding the immunoglobulin heavy and light chains of the antigen-binding polypeptide construct is also provided. Such polynucleotides include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Polynucleotides include full-length genes or cDNA molecules, as well as combinations of fragments thereof.

[0379] Polynucleotides encoding the engineered immunoglobulin heavy and light chain polypeptides described herein can be prepared by site-specifically mutating nucleotides within the DNA encoding the polypeptides using cassette mutagenesis or PCR mutagenesis or other techniques well known in the art to generate DNA encoding the engineered immunoglobulin heavy and light chain polypeptides, followed by expression of the recombinant DNA in cell culture as outlined herein. However, polynucleotides encoding the engineered immunoglobulin heavy and light chain polypeptides may also be prepared by in vitro gene synthesis, using established techniques.

[0380] As will be apparent to one of skill in the art, due to the degeneracy of the genetic code, a large number of polynucleotides can be generated, all of which will encode the engineered immunoglobulin heavy and light chain polypeptides described herein. Thus, after identifying a particular amino acid sequence, one of skill in the art can generate any number of different polynucleotides by simply modifying the sequence of one or more codons without changing the amino acid sequence of the encoded protein.

[0381] Further provided are expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that contain at least one polynucleotide as described above. Also provided are host cells containing such expression systems or constructs.

[0382] Typically, expression vectors used in host cells contain sequences for plasmid maintenance and sequences for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to in certain embodiments as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a polynucleotide encoding an expressed polypeptide, and a selectable marker element. A vector can be multicistronic, i.e., express two or more polynucleotides encoding the immunoglobulin heavy and light chains of an antigen-binding polypeptide construct, or the antigen-binding polypeptide construct can be expressed by a vector set, each vector expressing one or more of the polynucleotides. An antigen-binding polypeptide construct can also be expressed using a vector set comprising a combination of a multicistronic vector and a vector comprising a single polynucleotide encoding one of the immunoglobulin heavy and light chains.

[0383] In some embodiments, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (e.g., hexaHis) or another "tag" such as FLAG, HA (influenza virus hemagglutinin), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. If desired, the tag can then be removed from the purified polypeptide by various means, such as peptidase cleavage.

[0384] A vector typically contains a promoter recognized by a host organism and operably linked to a polynucleotide encoding a polypeptide. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene (generally about 100-1000 bp) that controls the transcription of the structural gene. Promoters are traditionally classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature. Constitutive promoters, on the other hand, unilaterally transcribe the gene to which they are operably linked, i.e., they exert little control over gene expression. Many promoters recognized by a variety of potential host cells are well known.

[0385] Promoters suitable for use with yeast, bacterial, and insect hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, but are not limited to, promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0386] A vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include EASE elements (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and matrix attachment regions (MARs). MARs mediate chromatin structural organization and can protect the integrated vector from "position" effects. Therefore, MARs are particularly useful when the vector is used to generate stable transfectants. Many natural and synthetic MAR-containing nucleic acids are known in the art, e.g., U.S. Patent Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; and 7,129,062.

[0387] After the vector is constructed and the polynucleotide is inserted into the appropriate site of the vector, the completed vector may be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell can be accomplished by well-known methods, such as transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will, in part, be a function of the type of host cell used. The host cell can be transiently transfected, or the host cell can be stably transfected. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., 2001, supra.

[0388] For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express engineered heavy and light chains of an antigen-binding polypeptide construct can be generated. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the exogenous DNA or polynucleotide, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the plasmid to stably integrate into the cell's chromosome and grow to form foci that can then be cloned and expanded into cell lines.

[0389] Many selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) can be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

[0390] When cultured under appropriate conditions, the host cells will produce an antigen-binding polypeptide construct, which can then be recovered from the culture medium (if the host cells secrete the antigen-binding polypeptide construct into the medium) or directly from the host cells that produce it (if the antigen-binding polypeptide construct is not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) desired or necessary for activity, and the ease of folding into a biologically active molecule. The host cell can be eukaryotic or prokaryotic. For example, expression in bacterial systems will produce a non-glycosylated product, while expression in yeast will produce a glycosylated product. Eukaryotic host cells that possess the cellular machinery for appropriate processing of the primary transcript of the gene product (e.g., glycosylation or phosphorylation) can be used.

[0391] Mammalian cell lines that can be used as expression hosts are well known in the art, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Any cell line used in an expression system known in the art can be used to produce the recombinant polypeptides described herein. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the antigen-binding polypeptide construct. Host cells that can be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms (e.g., Escherichia coli or Bacillus). Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the monkey kidney cell line COS-7 (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives (e.g., Veggie CHO) and related cell lines that grow in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, the BHK (ATCC CRL 10) cell line, and the African green monkey kidney cell line CVI (ATCC CCL 163) described in McMahan et al., 1991, EMBO J. 10:2821. Examples of suitable yeast cell lines include the CV1 / EBNA cell line derived from Escherichia coli (E.g., Escherichia coli 70), human embryonic kidney cells (e.g., 293, 293EBNA, or MSR293), human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells. Alternatively, polypeptides can be produced in lower eukaryotes (e.g., yeast) or prokaryotes (e.g., bacteria). Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides.Suitable bacterial species include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any species capable of expressing heterologous polypeptides.

[0392] When antigen-binding polypeptides are produced in yeast or bacteria, it may be desirable to modify the product produced therein to obtain a functional product, for example, by phosphorylation or glycosylation of appropriate sites. Such covalent attachments can be achieved using known chemical or enzymatic methods. Antigen-binding polypeptide constructs can also be produced using an insect expression system by operably linking a set of polynucleotides to appropriate control sequences in one or more insect expression vectors. Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, for example, Invitrogen Corporation (San Diego, CA) (MaxBac® kit); such methods are well known in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio / Technology 6:47 (1988). Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0393] In certain embodiments, cell-free protein expression systems can be used to co-express polypeptides (e.g., heavy and light chain polypeptides) from a set of polynucleotides without the use of living cells. Instead, all components required for transcribing DNA into RNA and translating RNA into protein (e.g., ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided in solution for in vitro use. In certain embodiments, in vitro expression requires a reaction solution containing (1) a genetic template (mRNA or DNA) encoding the heavy and light chain polypeptides and (2) the essential molecular machinery for transcription and translation. In certain embodiments, cell extracts substantially supply the components of the reaction solution, such as RNA polymerase for mRNA transcription, ribosomes, tRNAs, amino acids, enzyme cofactors, energy source, and cellular components essential for proper protein folding, for polypeptide translation. Cell-free protein expression systems can be prepared using lysates derived from bacterial cells, yeast cells, insect cells, plant cells, mammalian cells, human cells, or combinations thereof. Such cell lysates can provide the correct composition and ratios of enzymes and components required for translation. In some embodiments, the cell membrane is removed, leaving only the cytoplasm and organelle components of the cell.

[0394] Several cell-free protein expression systems are known in the art, as reviewed in Carlson et al. (2012) Biotechnol. Adv. 30:1185-1194. For example, cell-free protein systems are available based on prokaryotic or eukaryotic cells. Examples of prokaryotic cell-free expression systems include those derived from E. coli. Eukaryotic cell-free protein expression systems are available based on extracts from rabbit reticulocytes, wheat germ, and insect cells. Such prokaryotic and eukaryotic cell-free protein expression systems are commercially available from companies such as Roche, Invitrogen, Qiagen, and Novagen. Those skilled in the art will be able to easily select an appropriate cell-free protein expression system capable of producing polypeptides (e.g., heavy and light chain polypeptides) that can pair with each other. Furthermore, cell-free protein expression systems can also be supplemented with chaperones (e.g., BiP) and isomerases (e.g., disulfide isomerase) to improve the efficiency of IgG folding.

[0395] Co-expression of heavy and light chains The engineered immunoglobulin heavy and light chains of the antigen-binding polypeptide constructs described herein can be co-expressed in mammalian cells, as described above. In one embodiment, the immunoglobulin heavy and light chains of the antigen-binding polypeptide construct are co-expressed in a host cell. Thus, in the case of a bispecific antigen-binding polypeptide construct, two immunoglobulin heavy chains and two immunoglobulin light chains are co-expressed in a host cell. However, other methods for producing bispecific antigen-binding polypeptide constructs that do not rely on the use of a single clonal or transient cell line expressing all four chains are known in the art (Gramer, et al. (2013) mAbs 5,962; Strop et al. (2012) J Mol Biol 420,204). These methods rely on arm exchange after production of the two pairs of light and heavy chains involved in forming the bispecific antibody under redox conditions (redox generation). In this approach, the H1L1 and H2L2 heterodimers are expressed in two different cell lines to allow for the independent generation of two heterodimers, which are then mixed under selective redox conditions to achieve reassociation of the two unique heavy chains, H1 and H2, to form a bispecific antigen-binding polypeptide construct comprising H1L1H2L2.

[0396] Although preferential pairing is primarily driven by the incorporation of Mab design set amino acid modifications into the immunoglobulin heavy and light chain polypeptides, the amount of correctly paired heterodimers may be further optimized by varying the ratio of polynucleotides encoding each polypeptide relative to one another, as shown in the Examples.

[0397] Testing of antigen-binding polypeptide constructs As described above, the antigen-binding polypeptide construct comprises a first heterodimer comprising H1 and L1 and a second heterodimer comprising H2 and L2, where L1 is a lambda light chain and L2 is a kappa light chain, and H1 and H2 are different from each other. One or more of H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing of L1 with H1 relative to L2 and promote preferential pairing of L2 with H2 relative to L1. The first Fab region of the H1L1 heterodimer and the second Fab region of the H2L2 heterodimer can bind to antigen with affinity similar to that of the corresponding wild-type first Fab region or wild-type second Fab region. The first Fab region of the H1L1 heterodimer and the second Fab region of the H2L2 heterodimer also exhibit thermal stability comparable to that of the corresponding wild-type first Fab region or wild-type second Fab region.

[0398] The affinity of each heterodimer of a heterodimer pair for its respective antigen can be tested as described below. The thermal stability of each heterodimer of a heterodimer pair can also be tested as described below.

[0399] In one embodiment, one heavy chain is co-expressed with two different light chains in an LCCA design set as described above, where the heavy chain preferentially pairs with one of the two light chains. In another embodiment, two unique heavy chains are co-expressed with two unique light chains, where each heavy chain preferentially pairs with one of the light chains.

[0400] Methods for measuring preferential pairing The degree of preferential matching can be assessed, for example, by using the methods described below and in the Examples. Preferential matching can be assessed in the context of the LCCA design set (H1L1L2, or H2L1L2) or the Mab design set (H1L1H2L2).

[0401] In one embodiment, a light chain competition assay (LCCA) can be used to assess preferential pairing in the context of an LCCA design set. Co-filed patent application PCT / US2013 / 063306, filed October 3, 2013, describes various embodiments of LCCA and is incorporated herein by reference in its entirety for all purposes. This method allows for quantitative analysis of heavy chain and specific light chain pairing within a mixture of co-expressed proteins and can be used to determine whether one specific immunoglobulin heavy chain preferentially associates with any one of two immunoglobulin light chains when the heavy and light chains are co-expressed. The method is briefly described as follows: At least one heavy chain and two different light chains are co-expressed in cells in a ratio such that the heavy chain is the reactant that constrains pairing. The heavy and light chains may be tagged for easy detection. Secreted proteins may be isolated from the cells, and immunoglobulin light chain polypeptides that bind to the heavy chain are separated from other secreted proteins to generate a fraction paired with the isolated heavy chain. The amount of each different light chain is then detected in the isolated heavy chain fraction, and the relative amount of each different light chain in the isolated heavy chain fraction is analyzed to determine the ability of the heavy chain to selectively pair with one of the light chains. Further details regarding embodiments of this method are described in the Examples.

[0402] In another embodiment, preferential pairing is evaluated in the context of a Mab design set, where H1, L1, H2, and L2 are co-expressed. In this embodiment, one or more of H1, L2, H2, and L2 may be tagged to facilitate detection and analysis. The resulting species of paired heavy and light chains are evaluated using LCMS (liquid chromatography-mass spectrometry) based on the molecular weight difference between each species. Antigen activity assays can also be used to quantify the relative heterodimer populations containing each light chain, thereby allowing the degree of binding measured (compared to a control) to be used to estimate the relative heterodimer populations of each.

[0403] thermal stability The thermal stability of heterodimers can be determined according to methods known in the art. The melting temperature of each heterodimer indicates its thermal stability. The melting point of heterodimers can be measured using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of heterodimers can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

[0404] Affinity for antigen The binding affinity of the heterodimer to each antigen and the off-rate of the interaction can be determined by competitive binding assays according to methods well known in the art. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (a heterodimer described herein (e.g., 3H or 125I) and a molecule of interest) in the presence of increasing amounts of unlabeled antibody and detecting the molecule bound to the labeled ligand. The affinity and binding off-rate of the heterodimer for the antigen can be determined from saturation data by Scatchard plot analysis.

[0405] Also, surface plasmon resonance (SPR)-based assays known in the art can be used to determine the kinetic parameters of the heterodimers described herein (BIAcore kinetic analysis).For the overview of SPR-based technology, see Mullet et al., 2000, Methods 22:77-91; Dong et al., 2002, Review in Mol.Biotech., 82:303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9:97-101; Rich et al., 2000, Current Opinion in Biotechnology 11:54-61. Additionally, any of the SPR devices and SPR-based methods for measuring protein-protein interactions described in U.S. Patent Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; and 6,268,125 are contemplated in the methods of the present invention. FACS can also be used to measure affinity, as is known in the art.

[0406] Pharmaceutical Compositions Also provided herein are pharmaceutical compositions comprising the antigen-binding polypeptide constructs described herein. Such compositions comprise a therapeutically effective amount of the antigen-binding polypeptide construct and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or approved in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent may be administered. Such pharmaceutical carriers can be sterile liquids (e.g., water and oils), including, but not limited to, oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a preferred carrier when the pharmaceutical composition is administered intravenously. In addition, saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions may be formulated as suppositories with traditional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers (e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.). Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.The formulation should suit the mode of administration.

[0407] In certain embodiments, a composition comprising an antigen-binding polypeptide construct is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical-grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0408] In certain embodiments, the compositions described herein are formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0409] The amount of the compositions described herein that will be effective in treating, inhibiting, and preventing diseases or disorders associated with abnormal expression and / or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The appropriate dose to be employed in the formulation will depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0410] Uses of antigen-binding polypeptide constructs As mentioned above, the antigen-binding polypeptide constructs described herein are derived from parent antibodies, where each heterodimer of the antigen-binding polypeptide construct corresponds to one of the parent antibodies and has been genetically engineered to incorporate amino acid modifications that promote preferential pairing of the immunoglobulin heavy and light chains that make up the heterodimer. Thus, the antigen-binding polypeptide constructs described herein can be used to treat or prevent the same diseases, disorders, or infections as the parent antibodies or combinations of parent antibodies are used.

[0411] In another embodiment, the antigen-binding polypeptide constructs described herein may also be utilized in combination with other therapeutic agents known in the art for the treatment or prevention of cancer, autoimmune diseases, inflammatory disorders, or infectious diseases. In certain embodiments, the antigen-binding polypeptide constructs described herein may be used in combination with, for example, monoclonal or chimeric antibodies, lymphokines, or hematopoietic growth factors (e.g., IL-2, IL-3, and IL-7) that interact with the molecule and act to increase the number or activity of effector cells that enhance the immune response. The antigen-binding polypeptide constructs described herein may also be utilized in combination with one or more drugs used to treat a disease, disorder, or infection, such as, for example, an anti-cancer agent, an anti-inflammatory agent, or an anti-viral agent.

[0412] Generation of bispecific antibodies using Mab design set libraries In one embodiment, the Mab design sets described herein can be used to generate bispecific antigen-binding polypeptide constructs. The Mab design sets described herein can be utilized in the form of a Mab design set library, which comprises Mab design sets that exhibit utility in promoting preferential pairing to form bispecific antigen-binding polypeptide constructs. In one embodiment, the Mab design set library is represented by the Mab design sets contained in Tables 4A and 4B. In one embodiment, the Mab design set library is represented by the Mab design sets in one or more of Tables 10-A1 through 10-A12 and 10-B1 through 10-B10. In another embodiment, the Mab design set library is represented by one or more of Tables 10-A1 through 10-A12. In one embodiment, the Mab design set library is represented by one or more of Tables 10-B1, 10-B2, 10-B3, 10-B4, 10-B6, 10-B8, and 10-B10. The Mab design set library can be used to generate antigen-binding polypeptide constructs starting from two parent antibodies (i.e., Mab1 and Mab2) as follows: For illustrative purposes, Mab1 comprises a lambda Fab and comprises an immunoglobulin heavy chain polypeptide H1 and an immunoglobulin light chain polypeptide L1, while Mab2 comprises a kappa Fab and comprises an immunoglobulin heavy chain polypeptide H2 and an immunoglobulin light chain polypeptide L2.

[0413] The Mab design set amino acid modifications (H1L1H2L2) of a Mab design set library are introduced into the immunoglobulin heavy and light chains (H1 and L2) of Mab1 and the immunoglobulin heavy and light chains (H2 and L2) of Mab2. H1, L1, H2, and L2 are then co-expressed, and the amount of correctly paired bispecific antigen-binding polypeptide constructs is determined. One or more Mab design sets of a Mab design set library may be individually tested or screened to determine which provides the desired amount of bispecific antigen-binding polypeptide constructs. Each heterodimer of the bispecific antigen-binding polypeptide construct can be further tested to evaluate the ability of each heterodimer to bind antigen or to evaluate its thermal stability as described herein. Additional properties that can be evaluated include the solubility, aggregation, k on and k off rates, and ability to withstand exposure to acid, base, oxidation, freeze / thaw cycles, shaking, pressure, etc. of the bispecific antigen-binding polypeptide construct compared to the parent antibody or the Fab region of the parent antibody. The latter property can be influenced by the complementarity determining regions (CDRs) of the antibody of interest and may therefore be tested for each bispecific antigen-binding polypeptide construct generated.

[0414] In some embodiments, the amount of correctly paired bispecific antigen-binding polypeptide constructs is assessed by LCMS. In some embodiments, the amount of correctly paired bispecific antigen-binding polypeptide constructs is assessed by charge-based separation techniques, such as capillary isoelectric focusing (cIEF) or chromatographic techniques. The procedure for preparing bispecific antigen-binding polypeptide constructs from Mab1 and Mab2 using a library of Mab design sets is shown schematically in Figure 9.

[0415] In one embodiment, the Mab design set library is stored on a computer readable storage medium to facilitate use of the Mab design set library to design bispecific antigen-binding polypeptide constructs.

[0416] Computer implementation In one embodiment, a computer includes at least one processor coupled to a chipset. Also coupled to the chipset are memory, storage, a keyboard, a graphics adapter, a pointing device, and a network adapter. A display is coupled to the graphics adapter. In one embodiment, chipset functionality is provided by a memory controller hub and an I / O controller hub. In another embodiment, memory is coupled directly to the processor rather than to the chipset.

[0417] A storage device is any device capable of holding data, such as a hard drive, compact disc read-only memory (CD-ROM), DVD, or solid-state storage device. Memory holds instructions and data used by the processor. A pointing device may be a mouse, trackball, or other type of pointing device and is used in conjunction with a keyboard to input data into a computer system. A graphics adapter displays images and other information on a display. A network adapter connects a computer system to a local or wide area network.

[0418] As is known in the art, a computer may have different and / or other components than those described above. In addition, a computer may lack certain components. Furthermore, storage devices may be local and / or remote from the computer (e.g., embodied in a storage area network (SAN)).

[0419] As is known in the art, a computer is adapted to execute computer program modules to provide the functionality described herein. As used herein, the term "module" refers to computer program logic used to provide a specified functionality. Thus, a module may be implemented in hardware, firmware, and / or software. A program module may be stored on a storage device, loaded into memory, and executed by a processor.

[0420] It is understood that the examples and embodiments described herein are for illustrative purposes and that in view thereof, various modifications or changes will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. [Example]

[0421] Below are examples of specific embodiments for making and using the antigen-binding polypeptide constructs described herein. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0422] The constructs and methods described herein can be prepared and performed using conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art, unless otherwise indicated, and such techniques are explained fully in the literature. For example, TECreighton, Proteins: Structures and Molecular Properties (WHFreeman and Company, 1993); ALLehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992). Example

[0423] Example 1: Molecular modeling and computer-guided genetic engineering of the Fab connector Using a structure- and computational molecular modeling-guided approach, we generated a kappa-lambda (KL) design library for preparing bispecific antibodies in which one Fab has a kappa light chain and the other has a lambda light chain (i.e., a kappa-lambda system, or KL system). The KL design library contains designs with amino acid modifications in the heavy and light chains of the Fab that promote the preferential formation of the desired bispecific antibody when these heavy and light chains are coexpressed. The KL design library is optimized for the kappa-lambda system to take advantage of the inherent differences between kappa and lambda light chains in bispecific antibodies. The KL design library was generated by studying the structures of representative Fabs, D3H44 (anti-tissue factor antibody) as a representative Fab containing a kappa light chain (kappa Fab) and CAT-2200 (anti-IL-17A antibody) as a representative Fab containing a lambda light chain (lambda Fab). However, the library can be used in the context of other bispecific KL-series antibodies or fragments thereof to identify designs that exhibit the desired pairing specificity in the antibody of interest.

[0424] Representative Fabs were selected based on the criteria listed in Table 1. These criteria included that the Fabs be human or humanized, be commonly used in the VH and VL subgroups, and contain minimal framework region mutations. Additionally, pairwise 3D superpositions were performed with available non-redundant (90% sequence identity threshold) kappa and lambda structures (structures obtained from the RSCB PDB, a database maintained by Rutgers University (Camden, NJ) and University of California, San Diego (San Diego, CA), USA; see the internet at www.rcsb.org). A low HL cross-domain RMSD (root mean square deviation) relative to VH-VL or CH1-CL, along with other parameters listed in Table 1, was used to select representative structures for the kappa and lambda systems, respectively. After selecting D3H44 (PDB ID 1JPT) and CAT-2200 (PDB ID 2VXS) as representative Fabs, we performed in silico structural analysis of these Fab interfaces to identify and understand the residues important for the interaction between the heavy and light chains using a two-pronged approach.

[0425] First, a global analysis of sequence conservation across the Fab variable and constant connecting regions was performed via sequence and structural alignment of known antibodies. Alignment of constant and variable domain sequences from various antibody subgroups compared to the sequences of D3H44 and the anti-HER2 antibodies Pertuzumab (both containing kappa light chains) and CAT-2200 (containing lambda light chains) is shown in Figure 1 . Figures 1A and 1E show the alignment of representative human VH germline subgroups compared to those of D3H44 / Pertuzumab and CAT-2200, respectively. Figure 1B shows the alignment of representative human kappa VL germline subgroups compared to D3H44 / Pertuzumab. Figures 1C and 1G show the alignment of human CH1 allele sequences compared to those of D3H44 / Pertuzumab and CAT-2200, respectively. Figure 1D shows the alignment of D3H44 / Pertuzumab compared to the human kappa allele sequence. Figure 1F shows an alignment of CAT-2200 compared to representative human lambda VL germline subgroups. Figure 1H shows an alignment of CAT-2200 compared to human lambda allele sequences. This analysis revealed that pertuzumab and D3H44 show a high degree of sequence conservation with the kappa constant and variable domain germline sequences, while CAT-2200 shows a high degree of sequence conservation with the lambda constant and variable domain germline sequences. These alignments also demonstrate a high degree of conservation in both the heavy and light chain constant domains, making the constant domain interface design likely to be transferable to other antibodies.

[0426] The second approach involved analyzing the D3H44 and CAT-2200 crystal structure connectors using a number of molecular modeling tools (e.g., ResidueContacts™ and AffinityDecomposition™), as shown in Figure 2. To improve transferability to other antibodies or fragments, the analysis focused on the constant domains with higher sequence conservation (see Figure 1). Using these analyses, we identified differences in hotspot positions (critical connector residues) between representative kappa Fab (D3H44) and lambda Fab (CAT-2200) structures, as shown in Table 2. There are significant conformational differences in the constant domains between the CH1-CL (kappa) and CH1-CL (lambda) structures. Superimposing available Fab structures onto the CH1 domains showed that the CL (kappa) structures adopt very similar conformations and form a tight cluster. However, the CL (lambda) conformations tend to exist in two distinct clusters: one closer to a kappa orientation (kappa-like cluster) and one with a more contradictory orientation (lambda cluster). This analysis reveals that D3H44 represents a typical kappa structure, while CAT-2200 represents a typical lambda structure (lambda cluster). Figure 3 illustrates the differences between typical constant domain kappa-lambda conformations, using D3H44 and CAT-2200 as representative structures. These differences likely stem primarily from the rigid-body motion of the light chain constant domain relative to the CH1 domain, altering the nature of the HL connection in kappa constant domains compared to lambda systems. The identified hotspot discrepancies (Table 2) and the conformational differences described above (Figure 3) served as a starting point for engineering the HL pair design for bispecific systems containing kappa and lambda Fabs. The amino acid numbering in the parental D3H44 and CAT-2200 sequences according to Kabat is provided in Tables 3A and 3B.

[0427] Next, potential mutations at hotspot locations in the 3D crystal structure, as well as at positions adjacent to the hotspots of interest, were simulated and identified through in silico mutagenesis and packing / modeling with Zymepack™. Zymepack™ is a software suite that, given an input structure and a set of mutations, generates a new structure that approximates the physical structure of the mutant protein by changing the residue types in the input structure according to the supplied mutations. In addition, Zymepack™ evaluates the properties of the mutant protein by calculating various quantitative metrics. These metrics include measures of steric and electrostatic complementarity, which can be correlated with the stability, binding affinity, or heterodimer specificity of the mutant protein.

[0428] By exploiting mismatches at the connectors, mutations were introduced to promote selective pairing of desired or preferred polypeptide chains or heterodimers while discouraging the formation of mismatched or mismatched polypeptide chains or heterodimers. For example, to prepare a bispecific antibody with one kappa Fab and one lambda Fab, four polypeptide chains are required. The kappa Fab contains heavy chain 1 (H1) and kappa light chain 1 (L1), while the lambda Fab contains heavy chain 2 (H2) and lambda light chain 2 (L2). In this case, the desired HL pairing is H1L1 and H2L2, while the mismatch is H1L2 and H2L1. Note that the designation / numbering of the polypeptide chains is arbitrary. Therefore, mutations that favor a matched CH1-CL(kappa) connector (H1L1) over a mismatched CH1-CL(lambda) connector (H1L2) and a matched CH1-CL(lambda) connector (H2L2) over a mismatched CH1-CL(kappa) connector (H2L1) were identified to generate kappa Fab-lambda Fab coordinated designs in the constant domain. Using computational methods including Zymepack™, steric complementarity was modeled and calculated based on energy factors such as van der Waals packing, cavitation effects, and close contact of hydrophobic groups. Similarly, electrostatic interaction energies were modeled and estimated based on Coulombic interactions between charge, hydrogen bonding, and desolvation effects. Both the preferred heavy and light chain pair model (H1L1 and H2L2) and the mismatch model (H1L2 and H2L1) obtained by introducing targeted mutations were simulated, and relative steric and electrostatic scores were calculated. This allowed us to determine whether a particular mutation set resulted in favorable energy, i.e., greater steric or electrostatic complementarity, for the preferred heavy-light chain pair compared to the mismatched pair. The calculated steric and electrostatic energies are components of the free energy associated with the pairing of the light and heavy chains. Therefore, greater steric and electrostatic complementarity indicates a greater change in free energy associated with the pairing of the desired pair compared to the mismatched pair.Greater steric and electrostatic complementarity results in preferential (selective) pairing of the desired heavy and light chains compared with the steric penalty and / or electrostatic repulsion of mismatched pairs.

[0429] Example 2: Design Selection and Description Using the approach described in Example 1, heavy chain-light chain heterodimer pairs (i.e., H1L1 and H2L2) are designed that exhibit selective or preferential pairing when one member of the HL heterodimer pair contains a kappa light chain and the other a lambda light chain. The heterodimers are designed in pairs, referred to as "Mab designs" or "Mab design sets," and contain a series of amino acid substitutions on the H1, L1, H2, and L2 chains that promote preferential pairing. Mab design sets are first tested as "LCCA designs," in which one heavy chain of the Mab design set is co-expressed with two light chains of the Mab design set, one kappa and one lambda, to assess relative pairing. The amino acid substitutions described throughout the examples were identified with reference to Tables 3A and 3B (for Pertuzumab and CAT-2200 Fab) using the Kabat numbering system as set forth in Kabat and Wu, 1991; Kabat et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication no. 91-3242, p647 (1991), unless otherwise indicated. However, for reference, Tables 22A, 22B, and 22C provide the identification of selected amino acid positions in the heavy chain, kappa light chain, and lambda light chain using the appropriate IMGT, 1JPT, and EU numbering systems.

[0430] Mab designs were loaded onto molecular models of D3H44 and CAT-2200, and metrics were calculated as described in Example 1. Top designs were then selected based on risk (minimizing impact on stability and immunogenicity) and impact (considering the proposed power of drive pairing specificity). The top designs were then tested by light chain competition assay (LCCA) to experimentally determine their pairing specificity (see Example 4). Mab designs were identified using D3H44 and CAT-2200 as representative Fabs, but were tested in the KL system using pertuzumab as the kappa Fab and CAT-2200 as the lambda Fab (Pertuzumab-CAT-2200 KL system). As shown in Figures 1C and 1D, D3H44 and pertuzumab have identical sequences in the constant domains and can seamlessly interconvert between the two systems. These Mab designs are referred to as KL designs.

[0431] A second set of designs was also tested in the pertuzumab / CAT-2200 KL system. These designs were proposed using representative designs reflecting the diversity of designs from the kappa-kappa (KK) design library described in Table 30 of International Patent Application No. PCT / CA2013 / 050914 (International Patent Publication No. WO2014 / 082179) as a starting point. These representative KK-derived designs included a subset of designs that, where possible, were either grafted unmodified into the KL system or adapted to the kappa-lambda system by modifying amino acid residues as necessary to account for differences in the sequences and structures of the kappa and lambda light chains. Both types of representative KK-derived designs are referred to as KK-derived KL designs.

[0432] The pairing specificity of the KL design and the KK-derived KL design was experimentally evaluated as an LCCA design by LCCA in the kappa-lambda system, as described in Example 4.

[0433] Example 3: Preparation of Fab constructs encoding Pertuzumab IgG heavy chain, Pertuzumab IgG light chain, CAT-2200 IgG heavy chain, and CAT-2200 IgG light chain The wild-type Fab heavy and light chains of the anti-HER2 antibody pertuzumab and the anti-IL17 antibody CAT-2200 were prepared as follows. The protein sequences of the pertuzumab Fab light chain (GenBank accession number HC359025.1, SEQ ID NO: 2) and heavy chain (GenBank accession number HC359024.1, SEQ ID NO: 1) were reverse-translated into DNA, codon-optimized for mammalian expression, and genes were synthesized (SEQ ID NOs: 8 and 7, respectively). The protein sequences of the CAT-2200 Fab light chain (2VXS chain L, SEQ ID NO: 4) and heavy chain (2VXS chain H, SEQ ID NO: 3) were taken from PDB entry 2VXS, reverse-translated into DNA, codon-optimized for mammalian expression, and genes were synthesized (SEQ ID NOs: 10 and 9, respectively). The polypeptide and DNA sequences of these antibody heavy and light chains are shown in Table 3C.

[0434] The light chain vector insert, consisting of 5'-EcoRI restriction site-HLA-A signal peptide-HA or FLAG tag-light chain Ig clone-"TGA or TAA stop"-BamH1 cleavage site-3', was ligated into the pTT5 vector (Durocher Y et al., Nucl. Acids Res. 2002;30, No. 2 e9) to generate the light chain expression vector. The resulting light chain expression vector was sequenced to confirm the correct reading frame and sequence of the encoding DNA. Similarly, the heavy chain vector insert, consisting of 5'-EcoRI restriction site-HLA-A signal peptide-heavy chain clone (ending at T238, see Table 3A)-ABD2-His6 tag-TGA stop-BamH1 cleavage site-3', was ligated into the pTT5 vector (ABD2 = two copies of the albumin-binding domain protein linked in tandem) to generate the heavy chain expression vector. The resulting heavy chain expression vector was also sequenced to confirm the correct reading frame and sequence of the encoding DNA. Various pertuzumab or CAT-2200 Fab constructs containing the amino acid substitutions of the Mab design set were generated either by gene synthesis or site-directed mutagenesis (Braman J, Papworth C & Greener A., ​​Methods Mol. Biol. (1996) 57:31-44).

[0435] The heavy and light chains were tagged at the C- and N-termini, respectively, to facilitate evaluation of prefer...

Claims

1. 1. An antigen-binding polypeptide construct comprising a first heterodimer and a second heterodimer, the first heterodimer (H1L1) comprises a first immunoglobulin heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) that form a first Fab region that specifically binds to a first antigen; the second heterodimer (H2L2) comprises a second immunoglobulin heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) that form a second Fab region that specifically binds to a second antigen; H1 differs from H2 in that H1 and H2 each comprise a heavy chain variable domain (VH domain) and a heavy chain constant domain 1 (CH1 domain); L1 contains a lambda light chain variable (VL-lambda) domain and a lambda light chain constant (CL-lambda) domain, and L2 contains a kappa light chain variable (VL-kappa) domain and a kappa light chain constant (CL-kappa) domain; one or more of H1, H2, L1, and L2 comprise amino acid modifications compared to the corresponding wild-type H1, H2, L1, and L2 polypeptide sequences that promote preferential pairing of L1 with H1 relative to L2 and / or promote preferential pairing of L2 with H2 relative to L1, wherein said amino acid modifications do not remove a naturally occurring cysteine ​​residue. The antigen-binding polypeptide construct.

2. 2. The construct of claim 1, wherein the amino acid modifications promote preferential pairing of L1 to H1 relative to L2 and / or promote preferential pairing of L2 to H2 relative to L1 when H1, H2, L1 and L2 are co-expressed in a cell or mammalian cell, or when H1, H2, L1 and L2 are co-expressed in a cell-free expression system, or when H1 and L1 are produced in a cell and H2 and L2 are produced in a different cell and the products of the two cells are mixed via a redox generating method, or when H1 and L1 are produced in a cell-free expression system and H2 and L2 are produced in a different cell-free expression system and the products of the two cell-free expression systems are mixed.

3. 3. The antigen-binding polypeptide construct of claim 1 or 2, wherein each heterodimer comprises a single Fab.

4. a. H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at position 124; and i. H1 contains an amino acid substitution at position 186 or 179, and L1 contains an amino acid substitution at position 180; ii. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; iii. H1 comprises an amino acid substitution at position 143; L1 comprises an amino acid substitution at position 133; or iv. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 178; b. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; i. H2 contains an amino acid substitution at position 186 or 124_186, and L2 contains 133 or 133_160 or 124_133 or 176_180; or ii. H2 contains an amino acid substitution at position 188; L2 contains an amino acid substitution at position 131; iii. H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at position 124_133 or 124_133_180; c. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; i. H2 comprises an amino acid substitution at position 124_186 or 124_179 or 188, and L2 comprises an amino acid substitution at position 176_178 or 176_180 or 131; or ii. H2 contains an amino acid substitution at position 143_188 or 143 or 124_143; L2 contains an amino acid substitution at position 124_176_178 or 124_178 or 124_180 or 124_176_180, or 124, or 124_176; d. H1 contains an amino acid substitution at positions 179, 186, 143, and / or 188; L1 contains an amino acid substitution at positions 180, 133, and / or 176-178; H2 contains an amino acid substitution at position 143, and L2 contains an amino acid substitution at positions 131 and / or 124; e. H1 contains an amino acid substitution at position 39 or does not contain an amino acid substitution that promotes preferential pairing; L1 contains an amino acid substitution at position 38 or does not contain an amino acid substitution that promotes preferential pairing; H2 contains an amino acid substitution at position 39 and L2 contains an amino acid substitution at position 38; f. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 131; i. H2 comprises an amino acid substitution at position 188 or 124_186, and L2 comprises an amino acid substitution at position 176_178 or 176_180 or 131; or ii. H2 contains an amino acid substitution at position 143 or 186; L2 contains an amino acid substitution at position 124_133 or 124_133_180; g. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 176_178 or 178; and i. H2 comprises an amino acid substitution at position 177_188 and L2 comprises an amino acid substitution at position 176_178; or ii. H2 contains an amino acid substitution at position 186 or 124 or 124_179; L2 contains an amino acid substitution at position 176 or 131_176; h. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; i. H2 comprises an amino acid substitution at position 188 and L2 comprises an amino acid substitution at position 131; or ii. H2 comprises an amino acid substitution at position 177_188; L2 comprises an amino acid substitution at position 176_178; or H1 contains an amino acid substitution at position 124_190; L1 contains an amino acid substitution at position 135; H2 contains an amino acid substitution at position 124 or 188, and L2 contains an amino acid substitution at position 176 or 176_178; i. H1 contains an amino acid substitution at position 177_188; L1 contains an amino acid substitution at position 176_178; and i. H2 contains an amino acid substitution at position 188, and L2 contains an amino acid substitution at position 176_178 or 131; ii. H2 contains an amino acid substitution at position 186; L2 contains an amino acid substitution at positions 133 or 124_160_180; iii. H2 comprises an amino acid substitution at position 124 or 124_179 or 124_186; L2 comprises an amino acid substitution at position 176 or 176_178 or 176_180; or iv. H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at position 133 or 124_133; j. H1 contains an amino acid substitution at position 188; L1 contains an amino acid substitution at position 178; H2 contains an amino acid substitution at position 124 or 188; L2 contains an amino acid substitution at position 176_178 or 176_180 or 176; k. H1 contains amino acid substitutions at positions 145_188; L1 contains amino acid substitutions at position 178; H2 contains amino acid substitutions at positions 124 and / or 188; L2 contains amino acid substitutions at one or more of 124, 133, and 178; l. H1 contains an amino acid substitution at position 174, 179, or 186; L1 contains an amino acid substitution at position 176 or 180; H2 contains an amino acid substitution at position 143 or 190, and L2 contains an amino acid substitution at position 131, 135, or 124; or m. H1 contains an amino acid substitution at position 174; L1 contains an amino acid substitution at position 176; H2 contains an amino acid substitution at position 190; L2 contains no amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 135; n. H1 contains an amino acid substitution at position 143_190; L1 contains an amino acid substitution at position 133; H2 contains an amino acid substitution at position 124; L2 contains an amino acid substitution at positions 131_135; o. H1 contains an amino acid substitution at position 143 and / or 186; L1 contains an amino acid substitution at position 133; H2 contains an amino acid substitution at position 124; L2 contains an amino acid substitution at position 131; p. H1 contains an amino acid substitution at position 143_179; L1 contains an amino acid substitution at position 124_178; H2 contains an amino acid substitution at position 186, and L2 contains an amino acid substitution at position 178_180 or 160_180; q. H1 contains an amino acid substitution at position 143; L1 contains an amino acid substitution at position 124; H2 contains an amino acid substitution at position 179 or 186, and L2 contains an amino acid substitution at positions 124_160_180; r. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 180 or 178; H2 contains an amino acid substitution at position 143 and / or 179, and L2 contains an amino acid substitution at position 124, 178, or 131; s. H1 contains an amino acid substitution at position 179; L1 contains an amino acid substitution at position 180; H2 contains an amino acid substitution at position 143, and L2 contains an amino acid substitution at position 124; t. H1 contains an amino acid substitution at position 143 or 186; L1 contains an amino acid substitution at position 180 or does not contain an amino acid substitution that promotes preferential pairing; H2 contains amino acid substitutions at positions 143-145, and L2 contains an amino acid substitution at position 124; u. H1 does not contain an amino acid substitution that promotes preferential pairing; L1 contains an amino acid substitution at position 135; H2 contains an amino acid substitution at position 139, and L2 contains an amino acid substitution at position 116; v. H1 does not contain an amino acid substitution that promotes preferential pairing or contains an amino acid substitution at position 45; L1 does not contain an amino acid substitution that promotes preferential pairing; H2 contains an amino acid substitution at position 45, and L2 contains an amino acid substitution at position 44; w. H1 contains an amino acid substitution at position 139; L1 contains an amino acid substitution at position 116; H2 does not contain an amino acid substitution that promotes preferential pairing, and L2 contains an amino acid substitution at position 135; or x. The antigen-binding polypeptide construct of any one of claims 1 to 3, wherein H1 comprises an amino acid substitution at position 124; L1 comprises an amino acid substitution at position 176; H2 comprises an amino acid substitution at position 124; and L2 comprises an amino acid substitution at position 176.

5. 5. The antigen-binding polypeptide construct of claim 1, wherein the affinity of the first Fab region for the first antigen is within about 100-fold of the affinity of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences for the first antigen, and / or the affinity of the second Fab region for the second antigen is within about 100-fold of the affinity of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences for the second antigen.

6. 6. The antigen-binding polypeptide construct of any one of claims 1 to 5, wherein the melting temperature (Tm) of the first Fab region is within about 20°C of the Tm of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences for the first antigen, and / or the melting temperature (Tm) of the second Fab region is within about 20°C of the Tm of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences for the second antigen.

7. a. H1 and L1 are wild-type polypeptide sequences, and H2 and L2 each comprise at least one amino acid modification; b. one or more of H1, L1, and H2 comprises at least one amino acid modification, and L2 is a wild-type polypeptide sequence; c. one or more of H1, L1, and L2 comprises at least one amino acid modification, and H2 is a wild-type polypeptide sequence; d. one or more of H1, H2, and L2 comprises at least one amino acid modification, and L1 is a wild-type polypeptide sequence; e. one or more of L1, H2, and L2 comprises at least one amino acid modification, and H1 is a wild-type polypeptide sequence; or f) The antigen-binding polypeptide construct of any one of claims 1 to 6, wherein each of H1, L1, H2, and L2 comprises at least one amino acid modification.

8. the amino acid modification is a. at least two of the CH1 domain of H1, the CH1 domain of H2, the CL-lambda domain of L1, and the CL-kappa domain of L2; b. at least two of the CH1 and VH domains of H1 and H2, the CL-lambda and VL-lambda domains of L1, and the CL-kappa and VL-kappa domains of L2, or c. The antigen-binding polypeptide construct of any one of claims 1 to 7, which is in at least two of the VH domain of H1, the VH domain of H2, the VL-lambda domain of L1, and the VL-kappa domain of L2.

9. The construct of any one of claims 1 to 8, wherein H1, L1, H2, and / or L2 each comprise at least one amino acid modification in the Fab region.

10. 10. The construct of any one of claims 1 to 9, wherein at least one of H1, H2, L1 and L2 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications in at least one constant domain and / or at least one variable domain.

11. 11. The construct of any one of claims 1 to 10, wherein the amino acid modifications promote preferential pairing of L1 and H1 relative to L2 to form H1L1, or promote preferential pairing of L2 and H2 relative to L1 to form H2L2, such that the relative pairing of at least one of H1L1 or H2L2 is at least about 10% greater than wild type and the relative pairing of the other is within about 10% of wild type or at least about 10% greater than wild type.

12. 12. The construct of any one of claims 1 to 11, wherein the thermal stability of the first Fab region is within about 0, 1, 2, or 3°C of the Tm of the Fab region formed by the corresponding wild-type H1 and L1 polypeptide sequences, and / or the thermal stability of the second Fab region is within about 0, 1, 2, or 3°C of the Tm of the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequences.

13. 4. The construct of any one of claims 1 to 3, wherein the amino acid modification is selected from the group consisting of the unique identifier Mab design sets shown in one or more of Tables 10-A1 to 10-A12.

14. 4. The construct of any one of claims 1 to 3, wherein the amino acid modification is selected from the group consisting of the unique identifier Mab design sets shown in any one of Tables 10-B1 to 10-B10.

15. 15. The construct of any one of claims 1 to 14, wherein the construct further comprises a dimeric Fc having two Fc polypeptides, each comprising a CH3 domain sequence, linked, with or without a linker, to one of the first and second Fab regions.

16. 16. The construct of claim 15, wherein the Fc is a human Fc, a human IgG1 Fc, a human IgA Fc, a human IgG Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc.

17. 17. The construct of claim 15 or 16, wherein the Fc comprises one or more modifications compared to wild type in at least one of the CH3 domain sequences that promote the formation of a heterodimeric Fc.

18. The Fc is i) a heterodimeric IgG1 Fc having the modifications L351Y_F405A_Y407V in said first Fc polypeptide and the modifications T366L_K392M_T394W in said second Fc polypeptide; ii) a heterodimeric IgG1 Fc having the modification L351Y_F405A_Y407V in said first Fc polypeptide and the modification T366L_K392L_T394W in said second Fc polypeptide; iii) a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in said first Fc polypeptide and T350V_T366L_K392L_T394W in said second Fc polypeptide; iv) a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in said first Fc polypeptide and the modifications T350V_T366L_K392M_T394W in said second Fc polypeptide; or v) a heterodimeric IgG1 Fc having the modifications T350V_L351Y_S400E_F405A_Y407V in said first Fc polypeptide and the modifications T350V_T366L_N390R_K392M_T394W in said second Fc polypeptide. The construct according to any one of claims 15 to 17, comprising:

19. The construct of any one of claims 15 to 18, wherein the Fc further comprises at least one CH2 domain sequence.

20. When H1, L1, H2, and L2 are co-expressed: a) the change in the total amount of correct pairing, as measured by the sum of H1L1 and H2L2 pairing, is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%, compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in said Fab region that promote preferential pairing; b) the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of species other than half antibodies produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing; or c) the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of all species produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing; 20. The construct of claim 19.

21. 21. The construct of claim 20, wherein the Fc comprises one or more modifications to promote selective binding of Fc-γ receptors, reduce or eliminate binding to Fc-γ receptors, or promote binding to FcRn.

22. The construct of any one of claims 15 to 21, wherein the linker is one or more polypeptide linkers.

23. 23. The construct of claim 22, wherein the linker comprises one or more antibody hinge regions.

24. 24. The construct of claim 23, wherein the linker comprises one or more IgG1 hinge regions.

25. 25. The construct of claim 23 or 24, wherein the one or more polypeptide linkers comprise one or more modifications compared to a wild-type polypeptide linker.

26. The construct of any one of claims 1 to 25, wherein the amino acid modification is an amino acid substitution.

27. The construct of any one of claims 1 to 26, wherein each of the sequences H1, H2, L1 and L2 is derived from a human or humanized sequence.

28. A construct according to any one of claims 1 to 27 conjugated to a therapeutic agent or drug.

29. A polynucleotide or set of polynucleotides encoding the construct of any one of claims 1 to 27.

30. 30. A vector or vector set comprising one or more of the polynucleotides or set of polynucleotides of claim 29.

31. 31. The vector or vector set of claim 30, wherein the vector, or at least one vector in the vector set, is polycistronic.

32. 32. An isolated cell comprising a polynucleotide or set of polynucleotides according to claim 29, or a vector or set of vectors according to claim 30 or 31.

33. 33. The isolated cell of claim 32, wherein the cell is a yeast cell, a bacterial cell, an insect cell, or a mammalian cell.

34. 34. The isolated cell of claim 33, wherein the cell is stably or transiently transfected with the vector or vector set of claim 30 or 31.

35. A pharmaceutical composition comprising the antigen-binding polypeptide construct of any one of claims 1 to 28 and a pharmaceutically acceptable carrier.

36. 36. The pharmaceutical composition of claim 35, further comprising one or more substances selected from the group consisting of buffers, antioxidants, small molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelating agents, stabilizers, and excipients.

37. A method for preparing a construct according to any one of claims 1 to 27, comprising the steps of: (d) obtaining a host cell comprising a polynucleotide or set of polynucleotides encoding said antigen-binding polypeptide construct; (e) culturing the host cells in the host cell culture under conditions that allow expression of the antigen-binding polypeptide construct; and (f) harvesting the antigen-binding polypeptide construct from the host cell culture. The method comprising:

38. 38. The method of claim 37, wherein the host cell is transiently or stably transfected with the polynucleotide or set of polynucleotides.

39. storing a dataset comprising data representing complementary amino acid modifications in a first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin lambda light chain polypeptide sequence (L1); and a second heterodimer comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin kappa light chain polypeptide sequence (L2); H1 and H2 each contain at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain) and are different from each other; Each of L1 and L2 comprises at least a light chain variable domain (VL domain) and a light chain constant domain (CL domain); the complementary amino acid modifications promote preferential pairing of L1 with H1 relative to L2, and promote preferential pairing of L2 with H2 relative to L1; the dataset comprises data representing those modifications or a subset of those modifications set forth in one or more of Tables 10-A1 to 10-A12 or Tables 10-B1 to 10-B10; A computer-readable storage medium.

40. 1. A method for producing a bispecific antigen-binding polypeptide construct, comprising: The construct comprises: a. a first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin lambda light chain polypeptide sequence (L1); and b. a second heterodimer comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin kappa light chain polypeptide sequence (L2); Including, H1 and H2 each contain at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain) and are different from each other; L1 and L2 each comprise a light chain variable domain (VL domain) and a light chain constant domain (CL domain); one or more of H1, L1, H2, and L2 comprise an amino acid modification that promotes preferential pairing of L1 with H1 relative to L2, and promotes preferential pairing of L2 with H2 relative to L1; The method comprises: a. introducing one or more complementary amino acid modifications from the dataset of claim 37 into H1, L1, H2, and / or L2; and b. co-expressing H1, L1, H2, and L2 in a host cell to produce an expression product comprising the bispecific antigen-binding polypeptide construct. The method comprising:

41. 41. The method of claim 40, further comprising determining the amount of said bispecific antigen-binding polypeptide construct in said expression product relative to other polypeptide products, and selecting a preferred subset of complementary amino acid modifications that result in an increased amount of said bispecific antigen-binding polypeptide construct compared to the amount of bispecific antigen-binding polypeptide construct in an expression product obtained from co-expression of wild type H1, L1, H2, and L2.

42. 42. The method of claim 40 or 41, wherein the construct comprises an Fc comprising at least two CH3 domain sequences, and the Fc is linked to the first heterodimer and the second heterodimer with or without one or more linkers.

43. 43. The method of claim 42, wherein the Fc is a heterodimeric Fc comprising one or more amino acid modifications that promote the formation of a heterodimeric Fc over a homodimeric Fc.

44. When H1, L1, H2, and L2 are co-expressed: a) a change in the total amount of correct pairing, as measured by the sum of % H1L1 and % H2L2 produced, compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in said Fab regions that promote preferential pairing, of more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%; b) the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of species other than half antibodies produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing; or c) the change in the total amount of correct pairing, as measured by the amount of bispecific antibody produced as a percentage of all species produced, is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to the pairing of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab regions that promote preferential pairing; 44. The method according to any one of claims 40 to 43.

Images