Antigen-binding polypeptide constructs containing kappa and lambda light chains and their use

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 List This application includes a sequence listing, which is submitted electronically in ASCII format and whose entire contents are incorporated herein by reference. The ASCII copy, created on December 1, 2015, is named 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. The ability to bind to two different epitopes or antigens makes bispecific antibodies an attractive tool for therapeutic applications where targeting more than one antigen or epitope in the treatment of a disease is beneficial. However, because antibody heavy chains have evolved to bind to antibody light chains in a relatively indiscriminate manner, efficiently producing bispecific antibodies in a form similar to naturally occurring antibodies can be challenging. As a result of this indiscriminate pairing, the simultaneous expression of two different heavy chains and two different light chains of 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 bioefficiency.

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

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

[0005] An overview of various approaches to producing bispecific antibodies in a form similar to naturally occurring antibodies from two parent antibodies can be found in Klein et al., (2012) mAbs 4:6, 1-11. International patent application PCT / EP2011 / 056388 (WO2011 / 131746) describes an in vitro method for producing heterodimer proteins in which asymmetric mutations are introduced into the CH3 region of two monospecific starting proteins to facilitate the directional exchange of "Fab arms" or "half-branches" between two monospecific IgG4 or IgG4-like antibodies during incubation under reducing conditions.

[0006] U.S. Patent Publication 2009 / 0182127 (Novo Nordisk, Inc.) describes the production of a bispecific antibody by modifying amino acid residues at the Fc linkage and the CH1:CL linkage of the light-heavy chain pair to reduce the ability of one pair of light chains to interact with the other pair of heavy chains. International Patent Publications WO2014 / 081955 (Amgen) and WO2014 / 150973 (Eli Lilly) describe amino acid residues in the lambda light chain that can be modified to yield the desired pairing specificity. Neither of these publications describes complementary amino acid modifications that can be used to prepare a bispecific antibody from one parent antibody having a kappa light chain and another parent antibody having a lambda light chain. International Patent Publication WO2012 / 131555 (Glenmark) describes replacing the connection between the heavy chain and lambda light chain of an antibody with that of a TCR (T cell receptor) domain connection. [Overview of the Initiative]

[0007] This disclosure provides a multispecific antigen-binding polypeptide comprising an immunoglobulin lambda light chain and an immunoglobulin kappa light chain. In one embodiment, 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) forming 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) forming 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 include a heavy chain variable domain (VH domain) and a heavy chain constant domain 1 (CH1 domain). In one embodiment, L1 includes a variable lambda light chain (VL-lambda) domain and a constant lambda light chain (CL-lambda) domain. In one embodiment, L2 includes a variable kappa light chain (VL-kappa) domain and a constant kappa light chain (CL-kappa) domain. In some embodiments, one or more of H1, H2, L1, and L2 include amino acid modifications compared to the corresponding wild-type H1, H2, L1, and L2 polypeptide sequences, where the amino acid modifications promote preferential pairing of L1 and H1 compared to L2, and / or preferential pairing of L2 and H2 compared 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 includes amino acid modifications that promote preferential pairing of L1 and H1 compared to L2, and / or preferential pairing of L2 and H2 compared to L1, when H1, H2, L1 and L2 are co-expressed in cells or mammalian cells, 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 cell systems are mixed via a redox production 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 contains a single Fab.

[0010] In some embodiments of the antigen-binding polypeptide constructs described herein, 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 contains an amino acid substitution at position 143; L1 contains 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 contains amino acid substitutions at positions 124 and 186 or 124 and 179 or 188, and L2 contains 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 and 176 and 178 or 124 and 178 or 124 and 180 or 124 and 176 and 180, or 124 or 124 and 176; d.H1 contains amino acid substitutions at positions 179, 186, 143 and / or 188; L1 contains amino acid substitutions 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 position 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 contains amino acid substitutions at positions 188 or 124 and 186, and L2 contains 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 an amino acid substitution 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 contains amino acid substitutions at positions 177 and 188, and L2 contains amino acid substitutions at positions 176 and 178; or ii. H2 contains amino acid substitutions at positions 186 or 124 or both 124 and 179; L2 contains amino acid substitutions at positions 176 or both 131 and 176; h. H1 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 133; i. H2 contains an amino acid substitution at position 188 and L2 contains 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 an amino acid substitution at position 135; H2 contains an amino acid substitution at position 124 or 188; L2 contains an amino acid substitution at position 176 or both 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 contains an amino acid substitution at position 188 and L2 contains amino acid substitutions 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 both 124 and 160 and 180; c. H2 contains an amino acid substitution at position 124 or both 124 and 179 or both 124 and 186; L2 contains amino acid substitutions at position 176 or both 176 and 178 or both 176 and 180; or d. H2 contains an amino acid substitution at position 143; L2 contains amino acid substitutions at positions 133 or both 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 position 124 or 188; L2 contains amino acid substitutions at positions 176 and 178 or both 176 and 180 or 176; k. H1 contains amino acid substitutions at positions 145 and 188; L1 contains an amino acid substitution at position 178; H2 contains an amino acid substitution at position 124 and / or 188; L2 contains an amino acid substitution at one or more of positions 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 does not contain an 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 an amino acid substitution at position 133; H2 contains an amino acid substitution 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 position 180 or 178 and 180; H2 contains an amino acid substitution at position 143 and / or 179, and L2 contains an amino acid substitution at position 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 does not contain an 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 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.H1 contains an amino acid substitution at position 124; L1 contains an amino acid substitution at position 176; H2 contains an amino acid substitution at position 124; L2 contains an amino acid substitution at position 176.

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

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

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

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

[0015] In some embodiments, amino acid modification is performed. a. At least two of the following: 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 following: the CH1 and VH domains of H1 and H2, the CL-lambda domain and VL-lambda domain of L1, and the CL-kappa domain and VL-kappa domain of L2, or c. At least two of the following: 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 contain 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 contains 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 include amino acid modifications that promote preferential pairing of L1 and H1 compared to L2 to form H1L1, or promote preferential pairing of L2 and H2 compared to L1 to form H2L2, such that at least one relative pairing of H1L1 or H2L2 is at least about 10% greater than the wild type, and the other relative pairing is within about 10% of the wild type or at least about 10% greater than the wild type.

[0018] In some embodiments, the amino acid modification promotes the preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes the preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 modification promotes the preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes the preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 approximately 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 approximately 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 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 unique identifier Mab design sets shown in one or more of Tables 10-A1 to 10-A12. In some embodiments, the amino acid modification is selected from the group consisting of unique identifier Mab design sets shown in any one of Tables 10-B1 to 10-B10.

[0021] In some embodiments, the construct further comprises a dimer Fc, each comprising a CH3 domain sequence and having two Fc polypeptides linked to one of a first Fab region and a second Fab region, with or without a linker. In some embodiments, Fc is 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, Fc includes one or more modifications compared to the wild type in at least one of the CH3 domain sequences that promote the formation of the heterodimer Fc.

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

[0023] In some embodiments, Fc further comprises at least one CH2 domain sequence. In one embodiment, Fc comprises one or more modifications to promote selective binding to the Fc-γ receptor, reduce or eliminate binding to the 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 pairings, as measured by the sum of H1L1 and H2L2 pairings, is greater than approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% compared to the pairings of the corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab region that promotes preferential pairing, or the change in the total amount of correct pairings, as measured by the amount of bispecific antibodies produced as a percentage of species other than the half-antibodies produced, is greater than the change in the amount of bispecific antibodies that promote preferential pairing. Compared to pairings of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the ab region, the change in the total amount of correct pairings, as measured by the amount of bispecific antibodies produced as a percentage of all species produced, is greater than approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to pairings of corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab region that promotes 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, one or more polypeptide linkers comprise one or more modifications compared to the wild-type polypeptide linker.

[0026] In some embodiments, amino acid modification includes amino acid substitution.

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

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

[0029] In another embodiment, the disclosure provides isolated recombinant polynucleotides or sets of isolated recombinant polynucleotides encoding constructs described herein. In some embodiments, what is provided is a vector or vector set comprising one or more polynucleotides or sets of polynucleotides described herein. In some embodiments, the vector, or at least one vector in the vector set, is polycistronic.

[0030] In another embodiment, the Disclosure provides isolated cells comprising a polynucleotide or polynucleotide set, or a vector or vector set, as described herein. In some embodiments, the cells are yeast cells, bacterial cells, insect cells, or mammalian cells. In some embodiments, the isolated cells are stably or transiently transfected with the vector or vector set described herein.

[0031] In another embodiment, a pharmaceutical composition comprising an antigen-binding polypeptide construct described herein is provided. 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 buffers, antioxidants, low molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelating agents, stabilizers, and excipients.

[0032] In another embodiment, a method for preparing the structures described herein will be described. In some embodiments, the method is (a) A step of obtaining a host cell containing a polynucleotide or set of polynucleotides encoding an antigen-binding polypeptide construct; (b) A step of culturing host cells in a host cell culture under conditions that express an antigen-binding polypeptide construct, and (c) The step includes collecting antigen-binding polypeptide constructs from host cell cultures.

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

[0034] In another embodiment, a computer-readable storage medium is provided. In some embodiments, the computer-readable storage medium stores a dataset containing 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 include at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain), and are distinct from each other. In some embodiments, the L1 and L2 polypeptide sequences stored in the dataset include 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 and H1 compared to L2, and preferential pairing of L2 and H2 compared to L1. In some embodiments, the dataset includes data representing the modifications or subsets thereof listed in Table 4A or Table 4B. In some embodiments, the dataset includes data representing the modifications or subsets thereof listed in one or more of Tables 10-A1 to 10-A12 or Tables 10-B1 to 10-B10.

[0035] In another embodiment, a method for producing a bispecific antigen-binding polypeptide construct is described. In some embodiments, the bispecific antigen-binding polypeptide construct produced by the method is 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 generated by the method include at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain), and are distinct from each other. In some embodiments, the L1 and L2 polypeptide sequences generated by the method include a light chain variable domain (VL domain) and a light chain constant domain (CL domain). In some embodiments, one or more polypeptide sequences among H1, L1, H2, and L2 generated by the method include amino acid modifications that promote preferential pairing of L1 and H1 compared to L2, and promote preferential pairing of L2 and H2 compared to L1.

[0037] In some embodiments, the method for producing a bispecific antigen-binding polypeptide construct is: a. Introducing one or more complementary amino acid modifications from the datasets described herein to H1, L1, H2, and / or L2; and b. The process involves co-expressing H1, L1, H2, and L2 in host cells and producing an expression product containing a bispecific antigen-binding polypeptide construct.

[0038] In some embodiments, the method further includes determining the amount of bispecific antigen-binding polypeptide construct in the expression product compared to other polypeptide products and selecting a preferred subset of complementary amino acid modifications that result in an increase in the amount of bispecific antigen-binding polypeptide construct compared to the amount of bispecific antigen-binding polypeptide construct in the expression product obtained from the 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 more compared to other polypeptide products. In some embodiments, the construct produced by the method comprises an Fc containing at least two CH3 domain sequences, the Fc linking to a first heterodimer and a second heterodimer with or without one or more linkers. In some embodiments, the Fc is a heterodimer Fc containing one or more amino acid modifications that promote the formation of the heterodimer Fc over the homodimer Fc.

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

[0040] [Figure 1]The D3H44, pertuzumab, and CAT-2200 heavy and light chain amino acid sequences are shown aligned to human germline sequences of variable and constant domains. The translated protein sequences for each domain, germline, and allele were obtained by directly querying IMGT / GENE-DB (http: / / www.imgt.org / genedb / query). Closest gene / allele determination was performed using IMGT / DomainGapAlign (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). Consensus sequence identification was performed using 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 amino acid assignments to each domain in Figure 1 were performed 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 domain and Ig superfamily V-like domains" Dev.Comp.Immunol., 27, 55-77 (2003). Figure 1A shows pertuzumab and the D3H44 variable heavy chain (VH) domain aligned with the human IGHV and IGHJ germline subgroups (one representative sequence is shown for each gene and allele).The gene and allele sequences most closely related to pertuzumab IGHV and IGHJ are X92218|IGHV3-66*01 and J00256|IGHJ4*01, respectively. The gene and allele sequences most closely related to 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 with the human kappa IGKV and IGKJ germline subgroups (one representative sequence is shown for each gene and allele). The gene and allele sequences most closely related to pertuzumab IGKV and IGKJ are Y14865|IGKV1-NL1*01 and J00242|IGKJ2*01, respectively. The gene and allele sequences closest 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 with the human CH1 IGHG germline subgroup. The gene and allele sequence closest to pertuzumab and D3H44 IGHG is J00228|IGHG1*01. Figure 1D shows the pertuzumab and D3H44 constant light chain (CL) domains aligned with the human kappa IGKC germline subgroup. The gene and allele sequence closest to pertuzumab and D3H44 IGKC is J00241|IGKC*01. Figure 1E shows the CAT-2200 VH domain aligned with the human IGHV and IGHJ germline subgroups (one representative sequence is shown for each gene and allele). The gene and allele sequences closest 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 with the human lambda IGLV and IGLJ germline subgroups (one representative sequence is shown for each gene and allele). The gene and allele sequences closest 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 with the human CH1 IGHG germline subgroup. The gene and allele sequence closest to CAT-2200 IGHG is J00228|IGHG1*01. Figure 1H shows the CAT-2200 CL domain aligned with the human lambda IGLC germline subgroup. The gene and allele sequence closest to CAT-2200 IGLC is J00253|IGLC2*01. [Figure 2] We identify the connecting residues and present a flowchart for computational modeling of the design using preferential heavy-light chain pairing. [Figure 3] Figure 3A shows the 3D structural alignment between the steady-state domains of D3H44 (PDB ID 1JPT) and CAT-2200 (PDB-ID 2VXS). Figure 3A illustrates the typical conformational differences observed between the kappa and lambda light chains when aligned on their respective heavy chains. Figure 3B shows a diagram of the light chain connection (with the heavy chains removed) of the model presented in Figure 3A to further illustrate the conformational differences. The dotted arrows indicate conformational rearrangement of secondary structural elements at the connection between the heavy and light chains. [Figure 4]This diagram provides a high-level schematic of the engineering requirements for forming bispecific antibodies and the assay requirements necessary for quantifying heavy-chain-light-chain (HL) pairs. The design goal of engineering bispecific antibodies with high purity (i.e., little to no mispairing HL associations) can be achieved by rational engineering the preferential pairing of two intrinsic heavy chains to intrinsic congeneral light chains (through the introduction of specific amino acid mutations). This process is schematicly shown; here, H1 is genetically engineered to preferentially pair with L1 (indicated by a checkmark) rather than L2 (indicated by "X"). Similarly, H2 is genetically engineered to preferentially pair with L2 rather than L1. Arrows on the H1L1 and H2L2 heterodimers represent the ease of pairing between these HL pairs, while arrows on the H1L2 and H2L1 heterodimers represent the disruption of pairing between the latter HL pairs. Experimental screening of designs to promote preferential pairing requires assays capable of simultaneously quantifying H1L1:H1L2 and H2L2:H2L1. These assay requirements can be simplified by assuming that each bispecific Fab arm is independently genetically engineerable. In this case, the assay only needs to quantify either H1L1:H1L2 or H2L2:H2L1, and does not need to quantify both simultaneously. [Figure 5] This diagram provides a schematic representation of how heavy and light chains can be tagged and how preferential pairing is determined. In this diagram, circular boundaries represent cells transfected with three constructs (one heavy chain and two inherent light chains). The expression product is secreted from the cell 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 two light chains competing 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]The following performance filtering criteria are presented based on two LCCA results per design. 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 include positive LCCA results above the neutral zone (defined as the region between 40:60 and 60:40 pairing:mismatching ratios), while other LCCAs must be above the lower limit of the neutral zone (40:60 pairing:mismatching ratio). 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 the neutral zone (not below it). Scenario C represents a design that is excluded because both LCCA results are positive, but neither is above the neutral zone. Even if the other LCCA exceeds the neutral zone (see Scenario D), at least one of the LCCA results will fall below the neutral zone; therefore, Scenarios D and E represent designs that are excluded. This also includes designs with the H1L1 and H2L2 designations reversed. This diagram explains the assumption that the wild-type matchup ratio is 50:50. [Figure 7] This metric represents the performance of selected KL designs and KK-derived KL designs, defined by Design Strength = ΔH1:L1:L2_scalar + ΔH2:L2:L1_scalar (based on LCCA data for the Mab design set in Tables 4A and 4B). This metric is an indicator of overall compatibility success at the design level. [Figure 8] This shows the possible heavy chain-related products that can be expected when two different light chains are co-expressed with two different heavy chains in a cell. [Figure 9] This specification describes a general method for preparing bispecific antigen-binding polypeptide constructs using the Mab design set library provided herein. [Figure 10]Figure 10A shows the minimum and maximum box plots summarizing the performance of all KL designs tested with SMCA in three dual-singular systems by design clusters. Figure 10A shows the performance measured by total dual-singularity calculation (Δ dual-singularity%). Figure 10B shows the performance measured by total syntactic calculation (Δ syntactic%). [Figure 11] The minimum and maximum box plots summarizing the performance of KL designs and KK-derived KL designs per dual singular system by transferability group are shown; "kl 3 / 3" indicates a KL design that is transferable in 3 / 3 dual singular systems, "kl 3 / 3+2 / 3" indicates a KL design that is transferable in at least two dual singular systems, and "kl all" indicates all tested KL designs. Similarly, "kk 3 / 3" indicates a KK-derived KL design that is transferable in 3 / 3 dual singular systems, "kk 3 / 3+2 / 3" indicates a KK-derived KL design that is transferable in at least two dual singular systems, and "kk all" indicates all tested KK-derived KL designs; the results are presented based on total dual singularity calculations (Δ dual singularity%). [Figure 12] The DSC sensorgrams of bispecific antibodies produced using Mab design set 3972 (SMCA design ID) (in each of the three bispecific systems) and wild-type parent antibodies for each 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] The minimum and maximum box plots summarizing the effect of amino acid substitutions in Mab designs on the Tm of the tested Fabs are shown. Results are reported as the change in Fab Tm compared to wild-type, with Tm measured ("all") and shown for all designs isolated by paratope. [Figure 14]The minimum and maximum box plots summarizing the effect of amino acid substitutions in the Mab design on the affinity of the Fab tested for that antigen are shown. The results are reported as the difference in log(KD) of appropriate bispecific Fabs from the wild type (-(log(KD_mutant)-log(KD_wt)). The results are shown for all designs isolated by paratope, with affinity measured ("all"). [Figure 15] Figure 15A shows the ULC-SEC profiles of protein A and the preparatively 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] This document describes the process for selecting wild-type reference values ​​to calculate the "change in total pairings relative to wild-type" and the "change in total bispecificity relative to wild-type" for designs tested in SMCA, when the corresponding wild-type bispecificity construct was not evaluated by SMCA. Figure 16A shows the process for selecting wild-type reference values ​​for the "total pairings" ("%H1L1 and %H2L2 pairings") for each of the three bispecific systems; Figure 16B shows the process for selecting wild-type reference values ​​for the "total bispecificity" ("H1L1_H2L2 and H1L2_H2L1**") for each of the three bispecific systems. [Modes for carrying out the invention]

[0041] Provided herein are genetically engineered antibodies (also referred to herein as multispecific antigen-binding polypeptide constructs) that may comprise a first heterodimer (H1L1) having a first immunoglobulin heavy chain (H1) and an immunoglobulin lambda light chain (L1) that pair to form a first Fab region, and a second heterodimer (H2L2) having an immunoglobulin heavy chain (H2) and an immunoglobulin kappa light chain (L2) that pair to form a second Fab region. The first Fab region typically binds to a first antigen, and the second Fab region typically binds to 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 genetically engineered to include amino acid modifications that promote the preferential pairing of correctly paired heavy and light chains (H1L1 or H2L2) when co-expressed or co-produced. More specifically, 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, the co-expression of H1, L1, H2, and L2 polypeptides can reduce or limit mispairing and produce correctly paired bispecific antibodies, 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 heterodimer Fc region, further reducing the amount of mispaired heavy chains. Amino acid modifications do not significantly affect the thermal stability of correctly paired heterodimers or the binding affinity of correctly paired heterodimers to antigens, compared to heterodimers formed from wild-type H1 and L1 or H2 and L2 polypeptides.

[0042] Furthermore, this specification provides a method for producing 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 those generally understood by those skilled in the art in the field to which the subject matter of the claims pertains. If there are multiple definitions of a term herein, the definition in this section shall prevail. Where references are made by URL or other such identifiers or addresses, it is understood that such identifiers may change, and specific information on the Internet may change, but similar information may be found through an Internet search. In addition, references demonstrate the availability and public dissemination of such information.

[0044] The above general description and the following embodiments for carrying out the invention are merely illustrative and descriptive, and should not be understood as limiting the subject matter of any claim. In this specification, the use of the singular includes the plural unless specifically stated otherwise.

[0045] In the description of this invention, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the enumerated range and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of the integer), unless otherwise specified. As used herein, “about” means ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the indicated range, value, sequence, or structure, unless otherwise specified. As used herein, the terms “a” and “an” should be understood to mean “one or more” of the enumerated components, unless otherwise specified or indicated in that context. The use of options (e.g., “or”) should be understood to mean one of the options, both, or any combination thereof. As used herein, the terms “include” and “comprise” are used synonymously. In addition, individual single-chain polypeptides or immunoglobulin constructs derived from various combinations of structures and substituents described herein should be understood to be disclosed to the same extent as each single-chain polypeptide or heterodimer has been described individually. Accordingly, the selection of specific components for forming individual single-chain polypeptides or heterodimers is within the scope of this disclosure.

[0046] The section titles used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. All documents, or parts thereof, cited herein, including but not limited to patents, patent applications, articles, books, manuals, and papers, are explicitly incorporated herein by reference for any purpose.

[0047] The methods and compositions described herein are not limited to the specific methodologies, protocols, cell lines, constructs, and reagents described herein, and should therefore be understood to be subject to change. The terminology used herein is for the sole purpose of describing specific embodiments and is not intended to limit the scope of the methods and compositions described herein, which is limited only by the appended claims.

[0048] All publications and patents referenced herein, such as constructs and methodologies described in publications, are incorporated herein in their entirety by reference for explanatory and disclosure purposes and may be used in connection with the methods, compositions, and compounds described herein. The publications considered herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment by the inventors described herein that they have no prior authority to such disclosure, either due to prior invention or for any other reason.

[0049] In this application, amino acid names and atomic 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 and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984), along with a modified version of 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), and glycine. This is interpreted as indicating amino acid residues belonging to the group consisting of (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 polymers of amino acid residues. That is, descriptions of polypeptides are equally applicable to descriptions of peptides and proteins, 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 not naturally encoded amino acids. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which amino acid residues are linked by peptide covalent bonds.

[0051] The terms "nucleotide sequence" or "nucleic acid sequence" are intended to describe a continuous extension of two or more nucleotide molecules. A nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or a combination of any of these.

[0052] The terms “cell,” “host cell,” “cell line,” and “cell culture” are used interchangeably herein, and it should be understood that all such terms include offspring resulting from the proliferation and culture of cells. The terms “transformation” and “transfection” are used interchangeably to refer to the process of introducing nucleic acid sequences into cells.

[0053] The term "amino acid" refers to naturally occurring amino acids, as well as amino acid analogs and mimics that function in a similar manner 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 pyrrolidine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., compounds in which carbon is bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. References to amino acids include, for example, naturally occurring proteolytic L-amino acids; D-amino acids, chemically modified amino acids (such as amino acid variants and derivatives); naturally occurring non-proteolytic 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-methyl amino acids (e.g., methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, hydroxyhistidine, homohistidine), amino acids having an extra methylene group in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is substituted with a sulfonic acid group (e.g., cysteic acid). Incorporating synthetic, non-native, substituted, or non-natural amino acids, including one or more D-amino acids, into the proteins of the antigen-binding polypeptide constructs described herein may be advantageous in many different ways. Peptides containing D-amino acids exhibit increased stability in vitro or in vivo compared to their counterparts containing L-amino acids.Therefore, constructing peptides incorporating D-amino acids can be particularly useful when higher intracellular stability is desired or required. More specifically, when such properties are desirable, D-peptides exhibit resistance to endogenous peptidases and proteases, thereby improving molecular bioavailability and extending in vivo lifespan. Furthermore, D-peptides cannot be efficiently processed to present the class II constraint of the major histocompatibility complex to T helper cells, and therefore have a low probability of inducing humoral immune responses in organisms as a whole.

[0054] Amino acids are referred to herein by either the commonly known three-letter or one-letter notation recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their commonly accepted one-letter codes.

[0055] The term "conservatively modified variant" applies to both amino acids and nucleic acid sequences. For a given nucleic acid sequence, a "conservatively modified variant" refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at every position where alanine is identified by the codon, the codon can be changed to one of the corresponding codons listed without altering the encoded polypeptide. Such nucleic acid mutations are "silent mutations" and are a type of conservatively modified mutation. All nucleic acid sequences in this specification that code for polypeptides also represent all possible silent mutations of that nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid can be modified to obtain a functionally identical molecule (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan). Therefore, each silent mutation in the nucleic acid encoding the polypeptide is potentially present 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 the sequences of nucleic acids, peptides, polypeptides, or proteins, which change, add, or delete a single amino acid or a few percent of amino acids in the encoded sequence, are “conservatively modified variants” that result from such changes in amino acid deficiencies, amino acid additions, or amino acid substitutions with chemically similar amino acids. Conservative substitution tables that provide functionally similar amino acids are known to those skilled in the art. Such conservatively modified variants are added to, and not excluded from, the polymorphic variants, interspecies homologs, and alleles of the present invention.

[0057] A table of conserved substitutions that provides functionally similar amino acids is known to those skilled in the art. Each of the following eight groups contains amino acids that can be considered to be conserved substitutions of each other: 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); (For example, see 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 terms “identical” or “percent “identical” refer to two or more sequences or subsequences being the same. Sequences are “substantially identical” or “substantially similar” (i.e., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity across a specified region). This definition also refers to the complement of test sequences. Identity may exist over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75 to 100 amino acids or nucleotides in length, or, if not specified, over the entire sequence of the polynucleotide or polypeptide. A polynucleotide encoding the polypeptide of the antigen-binding polypeptide construct described herein, including homologs from non-human species, can be obtained by a process comprising the steps of screening a library under stringent hybridization conditions using a labeled probe having the polynucleotide sequence or a fragment thereof of the antigen-binding polypeptide construct described herein, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

[0059] Examples of suitable algorithms for determining sequence identity % and sequence similarity % are the BLAST® and BLAST® 2.0 algorithms, 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® analysis is publicly available through the National Center for Biotechnology Information (see www.ncbi.nlm.nih.gov). Cumulative scores are calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of a word hit in each direction is stopped if: the cumulative alignment score decreases by an amount X from the maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. Examples of algorithm parameters for the BLASTN program (for nucleotide sequences) are word length (W) of 11, expected value (E) of 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, examples of algorithm parameters for the BLASTP program are word length of 3, expected value (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 “homologousness” or be “homologous” with a peptide if the amino acid sequence of the derivative or variant has at least 50% identity with the original peptide over a sequence of 100 amino acids in length. In certain embodiments, the derivative or variant is at least 75% identical to any peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 85% identical to any peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% identical to any peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to any peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% identical to any peptide or peptide fragment having the same number of amino acid residues as the derivative.

[0061] As used herein, “isolated” polypeptide or construct means a construct or polypeptide identified, isolated, and / or recovered from components of its natural cell culture environment. Contaminating components of that natural environment are typically substances that interfere with the diagnostic and therapeutic use of heteromultimers and may include enzymes, hormones, and other protein-like or non-protein-like solutes.

[0062] In certain embodiments, as used herein, “isolated” antigen-binding polypeptide constructs describe antigen-binding polypeptide constructs identified, separated, and / or recovered from components of their natural cell culture environment. For example, the isolated bispecific antigen-binding polypeptide constructs described herein include heterodimer pairs or “isolated” heterodimer pairs, and include heterodimers or heterodimer pairs identified, separated, and / or recovered from components of their natural cell culture environment. Contaminating components of their natural environment are substances that interfere with the diagnostic and therapeutic use of the heterodimers or antigen-binding polypeptide constructs, and may include enzymes, hormones, and other protein-like or non-protein-like solutes.

[0063] Heterodimers and antigen-binding polypeptide constructs can be purified until they are substantially homogeneous. The terms “substantially homogeneous,” “substantially homogeneous form,” and “substantially homogeneous” are used to indicate that the correctly paired product substantially lacks by-products derived from undesirable polypeptide combinations (e.g., homodimers or mispaired heterodimers). In the context of the LCCA design set (H1L1L2), the correctly paired product is a heterodimer containing H1 and L1 (H1L1). In the context of the LCCA design set (H2L1L2), the correctly paired product is a heterodimer containing 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, the correctly paired product is a heterodimer pair containing correctly paired H1L1 and H2L2 (H1L1H2L2). In some embodiments, in the context of bispecific antigen-binding polypeptide constructs, when H1, L1, H2, and L2 are expressed, correctly paired products may include additional products that show correct pairing in at least one Fab region, such as H1L1H2L1 or H1L2H2L2, or, if a “half-antibody” is produced, H1L1 or H2L2. When expressed by purity, in one embodiment, substantial homogeneity means that the amount of completely mispaired byproducts 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 percentage reflects the results from mass spectrometry.

[0064] Terms understood by those skilled in the art of antibody technology are given the meanings obtained in that art unless otherwise explicitly defined herein. Antibodies are known to have a variable region, a hinge region, and a constant domain. Immunoglobulin structure and function are outlined, 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, “antibody” and “immunoglobulin” or “antigen-binding polypeptide construct” are used interchangeably. “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 kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, respectively, defining the immunoglobulin isotypes IgG, IgM, IgA, IgD, and IgE, respectively. Furthermore, an antibody may belong to one of many subclasses; for example, IgG may belong to the subclasses IgG1, IgG2, IgG3, or IgG4.

[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 to be "naturally occurring." The term "light chain" includes both the full-length light chain and its fragments that have a sufficient variable domain sequence to give binding specificity. The full-length light chain contains a variable domain, VL, and a constant domain, CL. The variable domain of the light chain is located at the amino terminus of the polypeptide. The light chain contains a kappa chain and a lambda chain. The term "heavy chain" includes both the full-length heavy chain and its fragments that have a sufficient variable domain sequence to give binding specificity. The full-length heavy chain contains a variable domain, VH, and three constant domains, CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, the CH domain is located at the carboxyl terminus, and CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can be 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 a portion of the light and / or heavy chain of an antibody involved in antigen recognition, typically containing 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] The "complementarity-determining region" or "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity. The "framework" region (FR) helps maintain the proper conformation of the CDR, thereby facilitating binding between the antigen-binding region and the antigen. Structurally, the framework region can be located between CDRs in an antibody. The variable region typically exhibits the same general structure as a relatively conserved framework region (FR) linked by three hypervariable region CDRs. The CDRs of the two chains in each pair are typically aligned by the framework region, enabling them to bind to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain typically follow the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) unless otherwise specified.

[0068] A "multispecific antigen-binding polypeptide construct" or "multispecific antibody" targets or binds to one or more different antigens or epitopes. 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, a bispecific antigen-binding polypeptide construct is a naturally occurring form. In other words, a bispecific antigen-binding polypeptide construct has the same form as a naturally occurring IgG, IgA, IgM, IgD, or IgE antibody.

[0069] The antibody heavy chain pairs with the antibody light chain, and they are in contact with or adjacent to each other at one or more "connecting regions." Each "connecting region" contains one or more "contact" amino acid residues in the first polypeptide that interact with one or more "contact" amino acid residues in the second polypeptide. For example, connecting regions exist between two CH3 domains in 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. These "connecting regions" may originate from an IgG antibody, such as 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 the immunoglobulin heavy and light chains are as described 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®, the international ImMunoGeneTics information system®, 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 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 sites 1, Journal of Molecular Biology, Volume 313, Issue 1, Pages 83-97, may be numbered according to several conventions, including EU (following a similar EU index to Kabat, referencing the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85)). Kabat numbering is used herein for VH, CH1, CL, and VL domains unless otherwise specified.EU numbering is used herein for CH3 and CH2 domains and hinge regions unless otherwise specified. Table 22A provides a correspondence table showing amino acid numbering at selected positions in IgG1 heavy chain polypeptides using the IMGT, Kabat, 1JPT, and EU numbering systems. Table 22B provides a correspondence table showing amino acid numbering at selected positions in lambda light chain polypeptides using the IMGT and Kabat numbering systems. Table 22C provides a correspondence table showing amino acid numbering at 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, where H1 and H2 are distinct from each other. In one embodiment, each heterodimer contains a single Fab region. The term "Fab region," as used herein, refers to a region obtained from the pairing of one immunoglobulin heavy chain polypeptide sequence and one immunoglobulin light chain polypeptide sequence, comprising 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 to a first antigen, and the second Fab region binds to a second antigen. The first and second antigens may be the same or different. One or more of the immunoglobulin heavy and light chains may contain amino acid modifications that promote the preferential pairing of correctly paired heavy and light chains when co-expressed or co-produced.

[0073] When an antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct (i.e., a bispecific antibody), it can also be called a "heterodimer pair."

[0074] For illustrative purposes, the first heterodimer of the antigen-binding polypeptide construct is called H1L1 and contains a first immunoglobulin heavy chain polypeptide sequence (H1) paired with an immunoglobulin lambda light chain polypeptide sequence (L1), and the second heterodimer is called H2L2 and contains a second immunoglobulin heavy chain polypeptide sequence (H2) paired with an immunoglobulin kappa light chain polypeptide sequence (L2). However, this designation is optional and should be understood as simply specifying that one heterodimer contains an immunoglobulin kappa light chain and the other contains an immunoglobulin lambda light chain. The Fab region of the first heterodimer, H1L1, may also be referred to herein as "lambda Fab"; on the other hand, the Fab region of the second heterodimer, H2L2, may also be referred to herein as "kappa Fab".

[0075] Parent antibody The immunoglobulin heavy chain polypeptide sequence (also called the "heavy chain") and immunoglobulin light chain polypeptide sequence (also called the "light chain") of each heterodimer can be obtained from one or more parental antibodies, where at least one parental antibody contains a kappa light chain and at least one other parental antibody contains a lambda light chain, and amino acid modifications that promote preferential pairing are genetically engineered into these heavy and light chains. The parental immunoglobulin heavy chain and light chain sequences lacking 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 a heterodimer antigen-binding polypeptide construct are obtained from two parental antibodies. Generally, the two parental antibodies are different from each other; however, this is not always the case. In one embodiment, the antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct, where each heterodimer is obtained from different parental antibodies. In another embodiment, the antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide construct, where 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., can bind to only one epitope. In another embodiment, at least one parent antibody can bind to more than one epitope.

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

[0077] Parental antibodies can be obtained from species including, but are not limited to, humans, mice, rats, rabbits, sheep, cattle, goats, or camels. In one embodiment, parental antibodies can be obtained from humans or mice.

[0078] Parental antibodies may 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 a specific target may be identified by many different strategies, including phage display, in vitro display, and other methods. These strategies yield forms such as scFv form, Fab form, or full-length IgG form. An overview 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, Oct 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 form can be converted to those known in the art. Methods for converting scFvs to Fab 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, the antibody was originally identified as scFv, but may also be used as the parent antibody if the scFv has been converted to a Fab form and genetically engineered into a conventional or naturally occurring antibody form.

[0080] In one embodiment, the heavy and light chains of each heterodimer of an antigen-binding polypeptide construct can be obtained from a parent antibody, which is a humanized antibody. The humanized antibody can be obtained by substituting the complementarity-determining region (CDR) of an antibody derived from a non-human mammal, such as a mouse, with the CDR of a human antibody. Methods for identifying the CDR 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 recombination techniques suitable for this purpose are also known (see European Patent Application Publication No. EP125023, WO96 / 02576). For example, the CDR of a mouse antibody can be determined by known methods, and the DNA can be prepared so that the CDR encodes an antibody that ligates with the framework region (FR) of a human antibody. The humanized antibody can then be produced using a conventional expression vector system. Such DNA can be synthesized by PCR using several oligonucleotides as primers, designed to have overlapping portions at both ends of the CDR and FR regions (see the method described in WO98 / 13388). The human antibody FR ligated via the CDR is selected so that the CDR forms an appropriate antigen-binding site. If necessary, amino acids in the FR of the antibody variable region may be modified so that the CDR of the reconstituted human antibody forms an appropriate antigen-binding domain (Sato, K. et al., Cancer Res. (1993) 53:851-856). Modifiable amino acid residues in FR include parts that bind directly to the antigen via non-covalent bonds (Amit et al., Science (1986) 233:747-53), parts that influence or act to some extent on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196:901-17), and parts related to VH-VL interactions (Patent Publication No. EP239400).

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

[0082] The heavy and light chains of heterodimers can be obtained from many parental antibodies known in the art. Most antibodies can act as parental antibodies, provided they contain an immunoglobulin heavy chain polypeptide sequence that pairs with an immunoglobulin light chain polypeptide sequence to form a Fab region that binds to the antigen. In one embodiment, at least one parental 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] TIFF0007901718000001.tif214170TIFF0007901718000002.tif233170TIFF0007901718000003.tif233170 TIFF0007901718000004.tif237170TIFF0007901718000005.tif233170TIFF0007901718000006.tif220170

[0084] Examples of suitable therapeutic antibodies containing a lambda light chain and an antigen that binds to the antibody are identified in Table B below:

[0085] TIFF0007901718000007.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 contain an IgG heavy chain. In one embodiment, the first and second heterodimers of the antigen-binding polypeptide construct contain 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 include 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 includes 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 the VH domain germline group IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, or IGHV7. In another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a VH domain from the germline subgroup IGHV3. In yet another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a J segment selected from the J segment germline gene IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, or IGHJ6. In yet another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a J segment from the germline subgroup IGHJ4. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a CH1 domain selected from the CH1 domain germline subgroup IHG1, IHG2, IHG3, or IHG4. In one embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a CH1 domain from the germline subgroup IHG1.

[0089] In heterodimers containing a lambda light chain polypeptide sequence, the lambda light chain may contain 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 having a lambda light chain containing a CL-lambda domain from the germline subgroup IGLC2. The lambda light chain may contain a VL-lambda domain selected from the 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 having a lambda light chain having a VL-lambda domain from the 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 gene IGLJ1, IGLJ2, IGLJ3, IGLJ6, or IGLJ7. In yet another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a heavy chain with a lambda J segment from the germline subgroup IGLJ2.

[0090] In heterodimers containing the kappa light chain polypeptide sequence, the kappa light chain is the CL germline allele IGKC * 01, IGKC * 02, IGKC * 03, IGKC * 04, or IGKC * It may contain a CL-kappa domain selected from 05. In one embodiment, the antigen-binding polypeptide construct is the germline subgroup IGKC *The antigen-binding polypeptide construct comprises a heterodimer having a kappa light chain containing a CL-kappa domain from 01. The kappa light chain may contain a VL-kappa domain selected from the germline subgroups 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 the 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 the J-segment germline gene IGKJ1, IGKJ2, IGKJ3, IGKJ4, or IGKJ5. In yet another embodiment, the antigen-binding polypeptide construct comprises a heterodimer having a kappa light chain with a J segment from the germline subgroups 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 described above, in some embodiments, the immunoglobulin heavy chain polypeptide sequences and immunoglobulin light chain polypeptide sequences of each heterodimer can be obtained 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 number AJ308087.1 (humanized anti-human tissue factor antibody D3H44 light chain variable region and CL domain); GenBank accession number AJ308086.1 (humanized anti-human tissue factor antibody D3H44 heavy chain variable region and CH1 domain); GenBank accession number HC359025.1 (pertuzumab Fab light chain gene module); GenBank accession number HC359024.1 (pertuzumab Fab heavy chain gene module); GenBank accession number GM685465.1 (antibody trastuzumab (=herceptin)-wild type; light chain); Ge See nBank accession number GM685463.1 (Antibody trastuzumab (=Herceptin)-wild-type; heavy chain); GenBank accession number GM685466.1 (Antibody trastuzumab (=Herceptin)-GC-optimized light chain); and GenBank accession number GM685464.1 (Antibody trastuzumab (=Herceptin)-GC-optimized heavy chain). The sequences of each of the above polypeptides are 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 publicly known in the art; see, for example, the Drug Bank website supported by 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 chain and light chains H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing between the heavy chain and light chain, which are genetically engineered into the heavy chain and light chain of the parent antibody. In one embodiment, two of the heavy chain and light chains H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing between the heavy chain and light chain. In one embodiment, three of the heavy chain and light chains H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing between the heavy chain and light chain.

[0094] In some embodiments, the amino acid modification may be asymmetrical, such that the modified amino acid positions differ between H1 and H2, and between L1 and L2.

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

[0096] In one embodiment, one or more amino acid modifications include one or more amino acid substitutions. When H1 or H2 is co-expressed with L1 and L2, or when H1, L1, H2, and L2 are co-expressed, the amino acid modifications promote the preferential pairing of L1 with H1 and the preferential pairing of L2 with H2. As described above, for illustrative purposes, heterodimers of antigen-binding polypeptide constructs would be identified as follows: the H1L1 heterodimer contains the lambda light chain L1, and the H2L2 heterodimer contains the 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, present in one set of H1, L1, H2, and L2, and also identified as H1L1H2L2. 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). The Mab design set is first tested as an LCCA design set (i.e., H1L1L2 or H2L1L2) to determine the intensity of pairing specificity when H1 and H2 are co-expressed independently of L1 and L2.

[0098] In one embodiment, amino acid modifications can be applied 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 of the heavy and light chains can be achieved based on steric and hydrophobic contacts, electrostatic / charged interactions, or a combination of these and various other interactions. Complementarity between protein surfaces has been extensively described in the literature in terms of key-and-lock fit, knob-into-hole, protrusion and cavity, donor and acceptor, etc., all of which imply structural and chemically paired properties between two interacting surfaces. In one embodiment, at least one of the heterodimers includes 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 includes an immunoglobulin heavy chain and an immunoglobulin light chain with amino acid modifications introduced into them, which introduce a new salt bridge across the light and heavy chains at the connecting portion.

[0099] In one embodiment, amino acid modifications of a Mab design set promote preferential pairing primarily through electrostatic attraction and repulsion. In another embodiment, amino acid modifications of a Mab design set promote preferential pairing primarily through steric mechanisms. Such Mab designs are included in Tables 4A, 4B, 7A, and 7B, among which examples are selected that have unique identifiers 10771-11335, 10771-11360, and 10780-11417. In another embodiment, amino acid modifications of a 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 include amino acid modifications, H1 and L1 do not include amino acid modifications that promote preferential pairing, and each of H2 and L2 includes at least one amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications, one or more of H1, L1, and H2 include at least one amino acid modification that promotes preferential pairing, and L2 does not include an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications, one or more of H1, L1, and L2 include at least one amino acid modification that promotes preferential pairing, and H2 does not include an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications, one or more of H1, H2, and L2 include at least one amino acid modification that promotes preferential pairing, and L1 does not include an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications, one or more of L1, H2, and L2 include at least one amino acid modification that promotes preferential pairing, and H1 does not include an amino acid modification that promotes preferential pairing. In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications, and each of L1, H2, and L2 includes at least one amino acid modification that promotes preferential pairing.

[0101] The amino acid modifications may be in one or more constant domains and / or variable domains of H1, L1, H2, and L2. In one embodiment, the amino acid modifications may be in the CH1 domains 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 yet 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, amino acid modifications may be in one or more framework regions of H1, L1, H2, and L2. In one embodiment, amino acid modifications are limited to conserved framework residues in 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 set of Mab designs 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, and 1 amino acid modification, or does not include any 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, and 1 amino acid modification, or does not include any 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, and 1 amino acid modification, or does not include any amino acid modifications. In one embodiment, L2 includes amino acid modifications of 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, or 1, or does not include any 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, the amino acid modification can be designed specifically for a kappa-lambda system, where one parent antibody contains a kappa light chain polypeptide sequence and the other parent antibody contains a lambda light chain polypeptide sequence. Such an amino acid modification or design is referred to herein as a KL design. Examples of such amino acid modifications or KL designs are shown in Tables 4A, 7A, and 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 a kappa light chain polypeptide sequence, which can then be transplanted into a kappa-lambda system. Those skilled in the art will understand how these designs can be transplanted 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 position corresponding to the KK design. The equivalent lambda light chain position can then be modified to conform to the KK design. Such amino acid modifications or designs are referred to herein as KK-derived KL designs and can be classified into the following groups: a) a group in which no change in design is required and the amino acid residue modification in the kappa-kappa system is the same as the modification in the kappa-lambda system; b) a group in which silent modifications are included and 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) a group in which at least one amino acid modification is included at the same relative position in the kappa light chain polypeptide sequence and the lambda light chain polypeptide sequence, but 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) a group in which at least one additional amino acid modification is included 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. Specific examples of group b) are shown by Mab design sets with unique identifiers 10689-10707. The silent modification is in L1 (Q160E is absent in the wild type in lambda because the residue at position 160 is E and not Q). Specific examples of group c) are shown by Mab design sets with unique identifiers 10652-10734, where in L1, amino acid residue 124 is E in wild-type lambda and Q in wild-type kappa. Specific examples of group d) are shown by Mab design sets with unique identifiers 10684-10706, which include the amino acid modification K129T.

[0107] In one embodiment, one or more of H1, L1, H2, and L2 include amino acid modifications that promote preferential pairing of L1 and H1 compared to L2, and promote preferential pairing of L2 and H2 compared to L1, and the amino acid modifications include conservative amino acid substitutions of the Mab design sets provided in Tables 4A, 4B, 7A, 7B, 10-A1 to 10-A12, and 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 the immunoglobulin heavy chain or 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. More specifically, a design can be called 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, an LCCA design is genetically engineered with one or more specific complementary LCCA designs based on each heavy chain of a desired bispecific antibody, and is therefore typically presented in a form in which amino acid modifications in all four polypeptide chains of a bispecific antibody are identified (see, e.g., Tables 4A and 4B). It should be understood that while specific amino acid substitutions may be identified through this, conserved substitutions at each amino acid position may also be possible. Furthermore, for illustrative purposes, unless otherwise specified, an H1L1 heterodimer represents a heterodimer containing a lambda light chain, and an H2L2 heterodimer represents a heterodimer containing a kappa light chain. Ultimately, all amino acid residues or positions are numbered according to the Kabat numbering system unless otherwise specified.

[0110] The design includes a driver set of amino acid substitutions that promote complementary and preferential pairing, and may also include secondary substitutions. Secondary substitutions 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 considered to be the primary factors promoting preferential pairing. For example, a design in which H1 contains amino acid substitution 186K, L1 contains amino acid substitution 133D, H2 contains amino acid substitution 188D, and L2 contains amino acid substitution 131K may promote preferential pairing by electrostatic mechanism. Many other examples of electrostatic drivers are found throughout the examples. In one embodiment, one or more electrostatic driver sets may be selected from those identified in Table C:

[0112] TIFF0007901718000008.tif220170TIFF0007901718000009.tif219170

[0113] In one embodiment, the driver set is a disulfide steering driver set, which may act unfavorably to the formation of disulfide bonds in the mispaired heterodimer. Examples of this type of driver set 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 acts to promote sterically complementary interactions between correctly paired heterodimers and steric incompatibility between mispaired heterodimers. Non-limiting examples of steric driver sets are shown in Table D, where "-" indicates the absence of amino acid substitutions that promote preferential pairing.

[0115] TIFF0007901718000010.tif56170

[0116] In one embodiment, the driver set is a variable-design driver set. Such a variable-design driver set includes 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 stereomechanism. In one embodiment, the variable-design driver set promotes preferential pairing based on electrostatic mechanism. Non-limiting examples of variable-design driver sets are shown in Table E, where "-" indicates the absence of amino acid substitutions in the polypeptide that promotes preferential pairing.

[0117] TIFF0007901718000011.tif51170

[0118] In one embodiment, one or more non-naturally occurring disulfide bonds may be genetically engineered into one or both heterodimers of the antigen-binding polypeptide construct. An example of this type of amino acid modification is 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 the design to optimize the pairing performance of the design. For example, secondary substitutions may act to A) optimize the number of contacts between the heavy chain and the correctly paired light chain, B) provide a conductive environment for the driver, C) optimize the hydrogen bonding network for the driver set, or D) result in stereoadaptation for the driver. Non-limiting examples of these types of secondary substitutions are shown in Table F, where "Lk" specifies a kappa light chain specific substitution, "L1" specifies a lambda light chain specific substitution, "L" specifies a light chain specific substitution in either the kappa or lambda light chain, and "H" specifies a heavy chain specific substitution.

[0120] TIFF0007901718000012.tif136170

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

[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 contains an amino acid substitution at position 188 or 124-186, and L2 contains an amino acid substitution at position 176-178, 176-180, or 131; or b) 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.

[0123] In some embodiments, H1 further includes an amino acid substitution at one or more of the positions 125, 145, and 179; L1 further includes an amino acid substitution at one or more of the positions 122, 124, and 133; H2 further includes an amino acid substitution at position 228; and / or L2 further includes 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, where H1 comprises an amino acid substitution at positions 125, 143, and 145; L1 comprises an amino acid substitution at positions 122, 124, and 131; H2 comprises an amino acid substitution at position 228; and L2 comprises an amino acid substitution at positions 121 and 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, where H1 comprises an amino acid substitution at positions 125_143_145 or 125_143_145_179; L1 comprises an amino acid substitution at positions 122_124_131 or 122_124_131_133; H2 comprises an amino acid substitution at positions 124_186_228, 143_228, 143_186_228, 186_228, or 188_228; and L2 comprises an amino acid substitution at positions 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 substitution in H1 is selected from 125R, 145T, 143D, 143E, 179E, and their conserved substitutions; the amino acid substitution in L1 is selected from 124Q, 122D, 131K, 131R, 133S, and their conserved substitutions; the amino acid substitution in H2 is selected from 228D, 124R, 1431, 143R, 186K, 186R, 188K, and their conserved substitutions; and the amino acid substitution in L2 is selected from 124E, 121K, 131D, 176D, 178E, 178F, 180D, 180E, 133D, 133G, 1331, and their conserved substitutions.

[0127] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 1, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000013.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: TIFF0007901718000014.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 amino acid combination of KL cluster 1 includes 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, as described 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 the positions 139, 145, 174, and 179; L1 further comprises an amino acid substitution at one or more of the positions 116, 124, 133, and 176; H2 further comprises an amino acid substitution at one or more of the positions 190, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of the 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, where 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 positions 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, and 44.

[0136] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, where H1 comprises amino acid substitutions at positions 139_143_145, 143_145, 143_145_174, or 143_145_179; L1 comprises amino acid substitutions at positions 116_124_131_176, 124_131, 124_131_133, 124_131_133_176, 131, or 131_133; H2 comprises amino acid substitutions at positions 124_186_190, 1 L2 contains amino acid substitutions at positions 43, 143_186, 143_186_190, 143_190, 186, 186_190, 188, 39_143, or 45_143, and L2 contains amino acid substitutions at positions 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, where the amino acid substitution in H1 is selected from 139W, 143D, 145T, 174G, 179E, and their conserved substitutions; the amino acid substitution in L1 is selected from 124Q, 176F, 116F, 131K, 131R, 133S, and their conserved substitutions; The amino acid substitutions in H2 are selected from 124R, 143I, 143K, 143R, 186K, 188K, 190F, 39E, 45P, and their conserved substitutions; the amino acid substitutions in L2 are selected from 124E, 131D, 131E, 133D, 133G, 135A, 135W, 160E, 176D, 178F, 180D, 180E, 38R, 44F, and their conserved substitutions.

[0138] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 2, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000015.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: TIFF0007901718000016.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000017.tif25170

[0141] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 2, having one or more of stereo1, stereo2, variable domain stereo, or variable domain electrostatic driver sets.

[0142] In one embodiment, the amino acid combination of KL cluster 2 includes 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, as described 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 contains an amino acid substitution at position 124_186, 124_179, or 188, and L2 contains an amino acid substitution at position 176_178, 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 includes an amino acid substitution at one or more of the positions 139, 145, 174, and 179; L1 further includes an amino acid substitution at one or more of the positions 116, 124, and 176; H2 further includes an amino acid substitution at one or more of the positions 177, 190, 39, and 45; and / or L2 further includes an amino acid substitution at one or more of the 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, where 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, where H1 comprises an amino acid substitution at positions 139_143_145, 139_143_145_179, 143_145, 143_145_174_179, or 143_145_179; L1 comprises an amino acid substitution at positions 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_1 L2 contains an amino acid substitution at positions 90, 39_124_179, or 45_124_179, and L2 contains an amino acid substitution at positions 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, where the amino acid substitution in H1 is selected from 139W, 174G, 145T, 143D, 143E, 179E, and their conserved substitutions; the amino acid substitution in L1 is selected from 116F, 124Q, 131K, 131R, 176F, and their conserved substitutions; and the amino acid substitution in H2 is 1 The amino acid substitutions at L2 are selected from 24R, 143K, 143R, 177I, 179K, 186K, 186R, 188K, 190F, 39E, 45P, and their conservative substitutions; the amino acid substitution at L2 is selected from 135A, 135W, 44F, 124E, 38R, 131D, 131E, 176D, 176E, 178D, 178E, 178F, 180D, 180E, 133D, 133G, 133I, 133L, and their conservative substitutions.

[0149] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 3, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000018.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: TIFF0007901718000019.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000020.tif30170

[0152] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 3, having one or more of stereo1, stereo2, variable domain stereo, or variable domain electrostatic driver sets.

[0153] In one embodiment, the amino acid combination of KL cluster 3 includes 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, as described 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, where 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 an amino acid substitution at positions 177-188, and L2 contains an amino acid substitution at positions 176-178; or b) H2 contains an amino acid substitution at position 186, 124, or 124-179; L2 contains an amino acid substitution at position 176 or 131-176.

[0156] In some embodiments, H1 further comprises an amino acid substitution at one or more of the positions 125, 139, and 177; L1 further comprises an amino acid substitution at one or more of the positions 122, 129, and 133; H2 further comprises an amino acid substitution at one or more of the positions 145, 228, 45, and 39; and / or L2 further comprises an amino acid substitution at one or more of the 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, wherein 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 176-178; H2 contains an amino acid substitution at position 188 or 186-188, and L2 contains an amino acid substitution at position 176-178.

[0158] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, wherein H1 does not contain any amino acid substitutions that promote preferential pairing, or contains an amino acid substitution at position 188; L1 does not contain any amino acid substitutions that promote 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 includes an amino acid substitution at one or more of the positions 125, 139, and 177; L1 further includes an amino acid substitution at one or more of the positions 122, 129, 133, and 176; H2 further includes an amino acid substitution at one or more of the positions 145, 228, 45, 177, 179, and 39; and / or L2 further includes an amino acid substitution at one or more of the 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, where H1 does not contain amino acid substitutions that promote preferential pairing, or contains amino acid substitutions in 125_188, 139_188, 188, or 177_188; L1 does not contain amino acid substitutions that promote preferential pairing, or contains amino acid substitutions in 129_176_178, 129_178, 122_129_176_178, 176_178, or 133_176_178; H2 contains amino acid substitutions in 145_186, 145_186_228, 145_177_188, 1 The amino acid substitutions are located at positions 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, and L2 is 44_1 Contains amino acid substitutions at positions 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 substitution in H1 is selected from 125R, 139W, 188A, 188K, and 177I and their conserved substitutions; the amino acid substitution in L1 is selected from 129T, 122D, 176A, 176D, 176E, 133I, 133L, 178D, 178E, 178T, and 178W and their conserved substitutions; The amino acid substitutions in H2 are selected from 124E, 145T, 177D, 179E, 186E, 186I, 186L, 188D, 188W, 228D, 39E, and 45P, and their conserved substitutions; 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 their conserved substitutions.

[0161] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 4, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000021.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: TIFF0007901718000022.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000023.tif21170

[0164] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 4, having one or more of the following: electrostatic, disulfide steering, stereo3, variable domain stereo, and variable domain electrostatic driver set.

[0165] In one embodiment, the amino acid combination of KL cluster 4 includes 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, as described 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 contains an amino acid substitution at position 188, and L2 contains an amino acid substitution at position 131; or b) H2 contains an amino acid substitution at positions 177-188; L2 contains an amino acid substitution at positions 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.

[0168] In some embodiments, H1 further comprises an amino acid substitution at positions 143 and / or 188, 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.

[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 includes an amino acid substitution at one or more of the positions 143, 188, and 190; L1 further includes an amino acid substitution at the 131 and / or 178 position; H2 further includes an amino acid substitution at the 143 and / or 145 position; and / or L2 further includes an amino acid substitution at the 133 and / or 178 position.

[0171] In some embodiments, 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 positions 124_190, 143_186_188, or 186_188; L1 comprises an amino acid substitution at positions 131_133_178, 133_135, 133_135_178, 133_178, or 135_178; H2 comprises an amino acid substitution at positions 124, 143_188, 145_177_188, or 177_188; and L2 comprises an amino acid substitution at positions 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 substitution in H1 is selected from 124E, 143S, 186K, 188T, 190D, 190E, and their conserved substitutions; the amino acid substitution in L1 is selected from 131S, 133D, 133I, 135K, 135R, 178F, 178T, and their conserved substitutions; the amino acid substitution in H2 is selected from 124R, 143T, 145T, 177D, 177I, 188D, 188K, and their conserved substitutions; and the amino acid substitution in L2 is selected from 131K, 133G, 133L, 176A, 176D, 176K, 178E, 178K, 178R, 178S, and their conserved substitutions.

[0173] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 5, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000024.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: TIFF0007901718000025.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 H1L1 and H2L2 heterodimers comprises H1 containing 186K_188T, L1 containing 133D_178T, H2 containing 145T_177D_188D, and L2 containing 176K_178K.

[0176] In one embodiment, the amino acid combination of KL cluster 5 includes 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, as described 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 an amino acid substitution at positions 177-188; L1 contains an amino acid substitution at positions 176-178; and a) H2 contains an amino acid substitution at position 188, and L2 contains an amino acid substitution at position 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, 124_179, or 124_186; L2 contains an amino acid substitution at position 176, 176_178, or 176_180; or d) H2 contains an amino acid substitution at position 143; L2 contains an amino acid substitution at position 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 an amino acid substitution at positions 177-188; L1 contains an amino acid substitution at positions 176-178; H2 contains an amino acid substitution at one or more of positions 124, 143, 179, 186, and 188; and L2 contains an amino acid substitution 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, where H1 comprises an amino acid substitution at positions 145_177_188 or 146_177_188; L1 comprises an amino acid substitution at position 176_178; and H2 comprises an amino acid substitution at positions 124, 124_179, 124_186, 143, 143_186_1 L2 contains an amino acid substitution at positions 88, 177_188, 179, 186, 186_188, or 188, and L2 contains an amino acid substitution at positions 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, where the amino acid substitution in H1 is selected from 145T, 146T, 177D, 188D, and their conserved substitutions; the amino acid substitution in L1 is selected from 176K, 178K, 178L, 178R, and their conserved substitutions; and the amino acid substitution in H2 is 124R, The amino acid substitutions at L2 are selected from 143K, 143R, 143S, 177I, 179K, 186K, 186R, 188K, 188T, 188W, and their conservative substitutions; the amino acid substitution at L2 is selected from 124E, 131D, 131E, 133D, 133G, 133I, 133L, 160E, 176A, 176D, 176E, 178A, 178D, 178E, 178F, 180E, and their conservative substitutions.

[0184] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 6, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000026.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: TIFF0007901718000027.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000028.tif16170

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

[0188] In one embodiment, the amino acid combination of KL cluster 6 includes 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, as described 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 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; and L2 contains an amino acid substitution at position 176-178, 176-180, or 176. In some embodiments, H1 further contains an amino acid substitution at one or more of positions 125, 139, 145, and 177; L1 further contains an amino acid substitution at position 122; H2 further contains an amino acid substitution at one or more of positions 143, 177, 179, 186, 228, 39, and 45; and / or L2 further contains 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 contains an amino acid substitution at positions 145-188; L1 contains an amino acid substitution at position 178; H2 contains an amino acid substitution at positions 124 and / or 188; and L2 contains an amino acid substitution at one or more of positions 124, 133, and 178.

[0192] In some embodiments, H1 further includes an amino acid substitution at one or more of the positions 125, 139, and 177; L1 further includes an amino acid substitution at position 122; H2 further includes an amino acid substitution at one or more of the positions 143, 177, 179, 186, 228, 39, and 45; and / or L2 further includes an amino acid substitution at one or more of the 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, where 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, The amino acid substitutions include 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 L2 includes positions 121_133_176, 121_133_176_180, 121_176_178, 124_133_176, 124_133_176_178, 124_133_176_178_ Contains amino acid substitutions at positions 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 substitution in H1 is selected from 125R, 139W, 145T, 177T, 188E, and their conserved substitutions; the amino acid substitution in L1 is selected from 122D, 178K, and their conserved substitutions; the amino acid substitution in H2 is selected from 124K, 124R, 143K, 143R, 177I, 179K, 186R, 188K, 228D, 39E, 45P, and their conserved substitutions; and the amino acid substitution in L2 is selected from 121K, 124E, 133D, 133G, 133L, 135W, 160E, 176D, 176E, 178D, 178E, 180E, 38R, 44F, and their conserved substitutions.

[0195] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 7, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000029.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: TIFF0007901718000030.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000031.tif22170

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

[0199] In one embodiment, the amino acid combination of KL cluster 7 includes 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, as described in one or more of the designs listed 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 contains an amino acid substitution at position 143; L1 contains 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 the positions 124, 139, 177, and 190; L1 further comprises an amino acid substitution at one or more of the positions 129, 131, 135, and 176; H2 further comprises an amino acid substitution at one or more of the positions 122, 124, 145, 179, 186, 188, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of the 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 amino acid substitutions at positions 143, 186, 179 and / or 188; L1 comprises amino acid substitutions at positions 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 the positions 124, 139, 177, and 190; L1 further comprises an amino acid substitution at one or more of the positions 131, 133, 135, 176, and 180; H2 further comprises an amino acid substitution at one or more of the positions 122, 124, 145, 179, 186, 188, 39, and 45; and / or L2 further comprises an amino acid substitution at one or more of the 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, where H1 is 124_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 6_188, containing an amino acid substitution at position 188; L1 is 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 containing an amino acid substitution at position 176-178; H2 is 122-143-145, 122-143-145-179, 124-143-145, 124-143-145-179, 143-145, 143-145-179, 143-145-179-186-188, 143-145-179-188, 143-145-188, 39-143-145-179, or 45-143- L2 contains an amino acid substitution at positions 145-179, and L2 contains an amino acid substitution at positions 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, where the amino acid substitution in H1 is selected from 124K, 139W, 143A, 143I, 143K, 143S, 177I, 179K, 186K, 186R, 188K, 188T, 190K, and their conservative substitutions; the amino acid substitution in L1 is 129T, 131D, 131E, 133D, 133L, 133W, 135S, 176A, 176D, 176 The amino acid substitutions in H2 are selected from E, 178D, 178E, 178T, 178W, 180E, and their conservative substitutions; the amino acid substitutions in L2 are selected from 122C, 124W, 143E, 145T, 179E, 186I, 188L, 188W, 39E, 45P, and their conservative substitutions; the amino acid substitutions in L2 are selected from 124C, 124K, 124R, 129K, 133A, 135W, 160K, 160R, 176A, 178R, 38R, 44F, and their conservative substitutions.

[0206] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 8, wherein the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000032.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: TIFF0007901718000033.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000034.tif40170

[0209] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8 having one or more stereo2, stereo3, stereo4, and variable domain driver sets. In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 8 introducing a disulfide bond that does not occur naturally.

[0210] In one embodiment, the amino acid combination of KL cluster 8 includes 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, as described 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 includes an amino acid substitution at positions 179, 186, 143 and / or 188; L1 includes an amino acid substitution at positions 180, 133 and / or 176-178; H2 includes an amino acid substitution at position 143; and L2 includes an amino acid substitution at position 131 and / or 124. In some embodiments, H1 further includes an amino acid substitution at position 125, L1 further includes an amino acid substitution at position 122 or 129, H2 further includes an amino acid substitution at one or more of positions 145, 179 and 228, and / or L2 further includes 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, where 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 positions 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, where H1 comprises amino acid substitutions at positions 125_143, 125_179, 125_186, or 125_188; L1 comprises amino acid substitutions at positions 122_129_133, 122_129_176_178, or 122_129_180; and H2 comprises amino acid substitutions according to KL cluster 9. L2 contains amino acid substitutions at positions 143_145, 143_145_179, 143_145_179_228, 143_145_228, or 145_179_228, and L2 contains amino acid substitutions at positions 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 substitution in H1 is selected from 125R, 143K, 179K, 186R, 188K, and their conserved substitutions; the amino acid substitution in L1 is selected from 122D, 129T, 133D, 176D, 176E, 178E, 178T, 180E, and their conserved substitutions; the amino acid substitution in H2 is selected from 143E, 145T, 179E, 228D, and their conserved substitutions; and the amino acid substitution in L2 is selected from 135W, 124R, 160K, 121K, 131K, 129K, 178R, and their conserved substitutions.

[0216] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 9, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000035.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: TIFF0007901718000036.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 heterodimer and H2L2 heterodimer is one of the following: TIFF0007901718000037.tif20170

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

[0220] In one embodiment, the amino acid combination of KL cluster 9 includes 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, as described 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 the KL cluster 10, where 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; L2 does not contain an amino acid substitution that promotes preferential pairing, or contains an amino acid substitution at position 135. In some embodiments, H1 further includes an amino acid substitution at position 143, L1 further includes an amino acid substitution at one or more of positions 116, 129, and 133, H2 further includes an amino acid substitution at one or more of positions 145, 179, and 188, and / or L2 further includes 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 the KL cluster 10, where H1 comprises an amino acid substitution at positions 174 and / or 186; L1 comprises an amino acid substitution at positions 176 and / or 180; H2 comprises an amino acid substitution at positions 145, 190 and / or 188; and L2 comprises an amino acid substitution at positions 135, 131, 178, or 133, or does not contain 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 positions 143 and / or 179; and / or L2 further comprises an amino acid substitution at positions 124 and / or 160.

[0224] In some embodiments, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to the KL cluster 10, where H1 comprises amino acid substitutions at positions 143_174, 174, 174_179, 174_186, or 186; L1 comprises amino acid substitutions at positions 116_129_133_176, 116_129_176_180, 116_176, 129_180, or 176; and H2 comprises amino acid substitutions at positions 143_145_17 The L2 contains an amino acid substitution at positions 9_188_190, 143_145_179_190, 143_145_190, 143_190, 145_179, 145_179_188_190, 188, or 190, and L2 contains an amino acid substitution at positions 124_135_160_178, 124_135_178, 131, 131_135, 133, 135, 135_178, or 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 K-L cluster 10, wherein the amino acid substitutions in H1 are selected from 143K, 174G, 179K, 186R, and their conservative substitutions; the amino acid substitutions in L1 are selected from 116F, 129T, 133D, 176F, 180E, and their conservative substitutions; the amino acid substitutions in H2 are selected from 143E, 143I, 145T, 179E, 188F, 190F, and their conservative substitutions; and the amino acid substitutions in L2 are selected from 124K, 124R, 131K, 133A, 135A, 160K, 178F, 178R, and their conservative substitutions.

[0226] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer comprising a combination of amino acid substitutions according to K-L cluster 10, and the H1L1 heterodimer comprises one of the following sets of amino acid substitutions: TIFF0007901718000038.tif37170

[0227] In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer comprising a combination of amino acid substitutions according to K-L cluster 10, and the H2L2 heterodimer comprises one of the following sets of amino acid substitutions: TIFF0007901718000039.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 the H1L1 heterodimer and the H2L2 heterodimer is one of the following: TIFF0007901718000040.tif16170

[0229] In some embodiments, the antigen-binding polypeptide construct has a combination of amino acid substitutions according to K-L cluster 10 and has a steric 1 driver set.

[0230] In one embodiment, the amino acid combination of the K-L cluster 10 includes one or more secondary substitutions selected from Table F.

[0231] In one embodiment, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to the K-L cluster 10 described in one or more of the designs of Table 10-A10.

[0232] K-L cluster 11: In one embodiment, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to the K-L cluster 11, where H1 includes an amino acid substitution at positions 143_190; L1 includes an amino acid substitution at position 133; H2 includes an amino acid substitution at position 124, and L2 includes an amino acid substitution at positions 131_135. In some embodiments, H1 further includes an amino acid substitution at position 125, L1 further includes an amino acid substitution at one or more of positions 122, 129, and 135, H2 further includes an amino acid substitution at one or more of positions 139, 145, 190, and 228, and / or L2 further includes an amino acid substitution at position 121.

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

[0234] In some embodiments, H1 further includes an amino acid substitution at position 125, L1 further includes an amino acid substitution at position 122, H2 further includes an amino acid substitution at one or more of positions 139, 190, and 228, and / or L2 further includes 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 the KL cluster 11, where 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 the KL cluster 11, wherein the amino acid substitution in H1 is selected from 125R, 143K, 190K, and their conserved substitutions; the amino acid substitution in L1 is selected from 122D, 129T, 133D, 135S, and their conserved substitutions; the amino acid substitution in H2 is selected from 124E, 139I, 145T, 190I, 228D, and their conserved substitutions; and the amino acid substitution in L2 is selected from 121K, 131K, 135K, and their conserved substitutions.

[0237] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KL cluster 11, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000041.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: TIFF0007901718000042.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 heterodimer and the H2L2 heterodimer comprises H1 containing 143K_190K, L1 containing 129T_133D_135S, H2 containing 124E_145T, and L2 containing 131K_135K.

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

[0241] In one embodiment, the amino acid combination of KL cluster 11 includes 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 one or more of the designs in Table 10-A11, specifically those of the KL cluster 11.

[0243] KL Cluster 12: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KL cluster 12, where 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, where H1 comprises an amino acid substitution at positions 124_143, 125_143, 125_143_186, 125_186, 125_186_188, 139_143, 139_143_186, 139_186, 139_186_188, 143, or 186_188; L1 comprises an amino acid substitution at positions 122_129_133, 122_129_133_178, 122_133_178, 129_131_133, 129_133, 129_133_178, or 133_178. H2 contains amino acid substitutions at positions 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 positions 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, where the amino acid substitution in H1 is selected from 124K, 125R, 139W, 143I, 143K, 186K, 188T, and their conserved substitutions; and the amino acid substitution in L1 is selected from 122D, 129T, 131D, 131E, 133D, 178T, and their conserved substitutions. The amino acid substitutions in H2 are selected from 124E, 143E, 145T, 179E, 186E, 186I, 188W, 228D, 39E, 45P, and their conservative substitutions; the amino acid substitutions in L2 are selected from 121K, 131K, 131R, 133G, 133S, 133T, 135K, 135W, 176R, 178A, 178S, 38R, 44F, and their conservative substitutions.

[0246] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer that includes a combination of amino acid substitutions according to K-L cluster 12, and the H1L1 heterodimer includes one of the following sets of amino acid substitutions: TIFF0007901718000043.tif77170

[0247] In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer that includes a combination of amino acid substitutions according to K-L cluster 12, and the H2L2 heterodimer includes one of the following sets of amino acid substitutions: TIFF0007901718000044.tif56170

[0248] In some embodiments, the antigen-binding polypeptide construct includes a combination of one H2L2 heterodimer and one H1L1 heterodimer. In one embodiment, the combination of the H1L1 heterodimer and the H2L2 heterodimer is one of the following: TIFF0007901718000045.tif22170

[0249] In some embodiments, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to K-L cluster 12 that has one or more of disulfide steering, conformation 2, conformation 3, variable domain electrostatics, and variable domain steric drivers.

[0250] In one embodiment, the amino acid combination of K-L cluster 12 includes one or more secondary substitutions selected from Table F.

[0251] In one embodiment, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to K-L cluster 12 described in one or more of the designs of Table 10-A12.

[0252] K-K cluster 1: In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 1, where 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. 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 contains an amino acid substitution at positions 143, 145, and 179; L1 contains an amino acid substitution at positions 124 and 178; H2 contains an amino acid substitution at position 186; and L2 contains 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 contains an amino acid substitution at positions 143-145-179; L1 contains an amino acid substitution at positions 124-178; H2 contains an amino acid substitution at position 186; and L2 contains an amino acid substitution 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 substitution in H1 is selected from 143E, 145T, 179E, and their conserved substitutions; the amino acid substitution in L1 is selected from 124K, 178R, and their conserved substitutions; the amino acid substitution in H2 is selected from 186R or its conserved substitution; and the amino acid substitution in L2 is selected from 160E, 178E, 180E, and their conserved substitutions.

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

[0257] In one embodiment, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to KK cluster 1 as described 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 an amino acid substitution at positions 124, 160, and 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 contains an amino acid substitution at positions 143-145; L1 contains an amino acid substitution at position 124; H2 contains an amino acid substitution at positions 179 or 186; and L2 contains an amino acid substitution 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 their conserved substitutions; the amino acid substitution in L1 is 124R or its conserved substitution; the amino acid substitution in H2 is selected from 186R, 179K, 146G, and their conserved substitutions; and the amino acid substitution in L2 is selected from 124E, 160E, 180E, and their conserved substitutions.

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

[0263] In one embodiment, the amino acid combination of KK cluster 2 includes 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, as described 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 contains an amino acid substitution at position 186; L1 contains an amino acid substitution at position 180 or 178-180; 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. In some embodiments, H2 further contains 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, where 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 their conserved substitutions; the amino acid substitution in L1 is selected from 178E, 180E and their conserved substitutions; the amino acid substitution in H2 is selected from 143E, 145T, 179E and their conserved substitutions; and the amino acid substitution in L2 is selected from 124K, 131K, 178R and their conserved substitutions.

[0269] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KK cluster 3, wherein the H1L1 heterodimer contains the following set of amino acid substitutions: H1 contains 186R, and L1 contains 178E_180E or 180E. In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer containing a combination of amino acid substitutions according to KK cluster 3, wherein the H2L2 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000046.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 H1L1 heterodimer and H2L2 heterodimer comprises H1 containing 186R, L1 containing 180E, H2 containing 143E_145T_179E, and L2 containing 124K_178R.

[0271] In one embodiment, the amino acid combination of KK cluster 3 includes 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, as described 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, where 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 amino acid substitutions 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 contains an amino acid substitution at position 179; L1 contains an amino acid substitution at position 180; H2 contains an amino acid substitution at positions 143-145; and L2 contains an amino acid substitution at position 124. In some embodiments, H1 further contains an amino acid substitution at position 146, and / or L2 further contains amino acid substitutions 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 contains an amino acid substitution at position 146-179 or 179; L1 contains an amino acid substitution at position 180; H2 contains an amino acid substitution at position 143-145; and L2 contains an amino acid substitution at position 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 their conserved substitutions; the amino acid substitution in L1 is 180E or its conserved substitution; the amino acid substitution in H2 is selected from 143E, 145T, and their conserved substitutions; and the amino acid substitution in L2 is selected from 124R, 160K, 178R, and their conserved substitutions.

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

[0278] In one embodiment, the amino acid combination of KK cluster 4 includes 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, as described 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, where 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 an amino acid substitution at position 143-145; and L2 contains 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 contains an amino acid substitution at position 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 substitution in H1 is selected from 186R, 143R, 143K, and their conserved substitutions; the amino acid substitution in L1 is selected from 180E and their conserved substitutions; the amino acid substitution in H2 is selected from 143E, 145T, and their conserved substitutions; and the amino acid substitution in L2 is selected from 124R, 160K, 178R, and their conserved substitutions.

[0282] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KK cluster 5, and the H1L1 heterodimer contains one of the following sets of amino acid substitutions: TIFF0007901718000047.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: TIFF0007901718000048.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 amino acid combination of KK cluster 5 includes 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, as described 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, wherein 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.

[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 substitution in H1 is selected from 39D, 39E, 39K, 39R, and their conserved substitutions; the amino acid substitution in L1 is selected from 38D, 38E, 38K, 38R, and their conserved substitutions; the amino acid substitution in H2 is selected from 39D, 39E, 39K, 39R, and their conserved substitutions; and the amino acid substitution in L2 is selected from 38D, 38E, 38K, 38R, and their conserved substitutions.

[0289] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KK cluster 6, the H1L1 heterodimer containing one of the following sets of amino acid substitutions: TIFF0007901718000049.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: TIFF0007901718000050.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 H1L1 heterodimer and H2L2 heterodimer comprises H1 containing 39R, L1 containing 38E, H2 containing 39D, and L2 containing 38R.

[0292] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 6, as described 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-specific 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 at L1 is 135W or its conserved substitution; the amino acid substitution at H2 is 139W or its conserved substitution; and the amino acid substitution at L2 is selected from 116A, 135V, and their conserved substitutions.

[0295] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KK cluster 7, the H1L1 heterodimer containing the following set of amino acid substitutions: H1 contains no amino acid substitutions that promote preferential pairing, and L1 contains 135W. In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer containing a combination of amino acid substitutions according to KK cluster 7, the H2L2 heterodimer containing the following set of amino acid substitutions: H2 contains 139W, and L2 contains 116A or 116A_1335V. In some embodiments, the antigen-binding polypeptide construct has 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 described in one 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, wherein 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, where the amino acid substitution at H1 is 45F or a conserved substitution thereof; the amino acid substitution at H2 is 45P, 45A or a conserved substitution thereof; and the amino acid substitution at L2 is 44F or a conserved substitution thereof.

[0299] In some embodiments, the antigen-binding polypeptide construct has an H1L1 heterodimer containing a combination of amino acid substitutions according to KK cluster 8, wherein the H1L1 heterodimer contains one of the following sets of amino acid substitutions: H1 contains no amino acid substitutions that promote preferential pairing, or contains 45F, and L1 contains no amino acid substitutions that promote preferential pairing. In further embodiments, the antigen-binding polypeptide construct has an H2L2 heterodimer containing a combination of amino acid substitutions according to KK cluster 8, wherein the H2L2 heterodimer contains the following sets of amino acid substitutions: H2 contains 45A or 45P, and L2 contains 44F. In some embodiments, the antigen-binding polypeptide construct has a combination of H1L1 and H2L2 heterodimers, wherein H1 and L1 do not contain amino acid substitutions that promote preferential pairing, H2 contains 45A, and L2 contains 44F.

[0300] In one embodiment, the antigen-binding polypeptide construct comprises a combination of amino acid substitutions according to KK cluster 8, as described 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 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.

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

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

[0304] In one embodiment, the antigen-binding polypeptide construct includes a combination of amino acid substitutions according to KK cluster 9 as described 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, where H1 contains an amino acid substitution at position 124; L1 contains an amino acid substitution at position 176; H2 contains an amino acid substitution at position 124; and L2 contains an amino acid substitution at position 176. In some embodiments, L1 and / or L2 further contain 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 the KK cluster 10, where H1 contains an amino acid substitution at position 124; L1 contains an amino acid substitution at positions 133-176; H2 contains an amino acid substitution at position 124; and L2 contains an amino acid substitution 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, where the amino acid substitution in H1 is 124E or a conserved substitution thereof; the amino acid substitution in L1 is 133G, 176R or a conserved substitution thereof; the amino acid substitution in H2 is 124R or a conserved substitution thereof; and the amino acid substitution in L2 is 133G, 176D or a conserved substitution thereof.

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

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

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

[0311] Prioritized matching in LCCA design sets One or more of H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing of L1 with H1 compared to L2, and preferential pairing of L2 with H2 compared 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 a mixture of approximately 50:50 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 a mixture of approximately 50:50 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 pairing preference between one immunoglobulin light chain polypeptide sequence and 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, if the amount of H1L1 heterodimer is greater than the amount of H1L2 heterodimer, preferential pairing occurs between H1 and L1. Similarly, for example, when H2 is expressed simultaneously with both L1 and L2, if the amount of H2L2 heterodimer is greater than the amount of H2L1 heterodimer, preferential pairing will occur between H1 and L1.

[0312] However, in some cases, an inherent pairing bias exists in the wild-type heavy and light chain polypeptide sequences obtained from parental antibodies. This inherent pairing bias can be observed in the context of LCCA design sets in which wild-type parental H1 or H2 is co-expressed with wild-type parental L1 and L2, and one of the light chains preferentially pairs with the heavy chains of both parental 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 preferential pairing that occurs in the corresponding wild-type system.

[0313] The degree of preferential pairing, or design strength, is a measure of the ability of amino acid modifications to promote preferential pairing. The degree of preferential pairing can be assessed as described elsewhere in this specification and in the examples, and is based on the measurement of correctly paired heterodimers (i.e., H1L1 and H2L2) compared to mispaired heterodimers (i.e., H1L2 and H2L1). The degree of preferential pairing can be assessed in the context of LCCA design sets (H1L1L2, or H2L1L2) in which one heavy chain is co-expressed with two intrinsic light chains, or Mab design sets (H1L1H2L2) in which the heavy and light chains of the parent antibody are co-expressed.

[0314] The following embodiments are relevant in the context of the LCCA design set. In all embodiments in this section, the term “about” means ±5% of the specified ratio, and unless otherwise specified, preferred pairings are compared to the wild type. In one embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferred pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferred pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 70:30. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 75:25. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 80:20. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 85:15. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 90:10. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 95:5. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where the ratio of H1L1:H1L2 is at least about 40:60 and the ratio of H2L2:H2L1 is at least about 99:1. In another embodiment, one or more of H1, H2, L1, and L2 include an amino acid modification that promotes preferential pairing of L1 and H1 compared to L2 to form H1L1, or promotes preferential pairing of L2 and H2 compared to L1 to form H2L2, where 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 include amino acid modifications that promote preferential pairing of L1 and H1 compared to L2 to form H1L1, or promote preferential pairing of L2 and H2 compared 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, preferred pairing is measured by LCCA as described in the example. The LCCA results generally predict the outcome in the context of preferred pairing in a Mab design set (described below) in which H1, L1, H2, and L2 are simultaneously expressed.

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

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

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

[0328] Prioritized pairings in the Mab design set Preferential pairing can also be evaluated in the context of a Mab design set that includes amino acid modifications to co-express H1, L1, H2, and L2, where one or more of H1, L1, H2, and L2 promote preferential pairing of L1 and H1, and preferential pairing of L2 and H2, forming a bispecific antigen-binding polypeptide construct containing a correctly paired first heterodimer (H1L1) and a correctly paired second heterodimer (H2L2). In this type of embodiment, when two different immunoglobulin heavy chain polypeptide sequences are co-expressed with two different immunoglobulin light chain polypeptide sequences, as shown in Figure 8, 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 evaluating correct pairing between heavy and light chains based on the Mab design set, some of the additional products may also be considered to exhibit correct pairing in the context of the Fab region, as they contain correctly paired heterodimers at the Fab level (see, for example, antibody species E, H, K, and M in Figure 8). In some embodiments, the Fc portion of the antigen-binding polypeptide construct contains asymmetric amino acid modifications that facilitate the formation of heterodimer Fc. In these embodiments, the number and amount of species E-J are expected to be reduced.

[0329] In the context of LCCA design sets, specifically Mab design sets that co-express all four immunoglobulin polypeptide sequences, H1, L1, H2, and L2, there may be an inherent bias in the pairing resulting from one of the light chains (either L1 or L2) preferentially pairing with both H1 and H2. Therefore, when determining the strength of a Mab design in the context of bispecific antigen-binding polypeptide constructs, it may be necessary to evaluate the degree of pairing of the Mab design with amino acid modifications by comparing it to the amount of correct pairing observed in the corresponding wild-type parent line (H1, L1, H2, and L2 polypeptide sequences without the amino acid modifications of the Mab design). Thus, in one embodiment, 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 parent line, the Mab design is considered to exhibit preferential pairing. Alternatively, 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 antibodies obtained in the total expression product of the corresponding wild-type parent line, the Mab design is considered to exhibit preferential pairing. In one embodiment, the total expression product may include antibody species A-N in Figure 8. In another embodiment, the total expression product may consist only of antibody species having two heavy chains and two light chains (antibody species A-J in Figure 8). In the latter embodiment, preferential pairing is measured as the percentage of total bispecific antibodies excluding half-antibodies such as species K-N in Figure 8.

[0330] In another embodiment, if the amount of correct pairing increases in the heterodimer of a bispecific antigen-binding polypeptide construct that exhibits a high degree of mispairing in the corresponding wild-type parent line, the Mab design is considered to exhibit preferential pairing. In another embodiment, 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 parent line, the Mab design is considered to exhibit preferential pairing. For example, referring to Figure 8, species A, B, H, I, and M are considered to have paired correctly with H1L1, and species A, C, E, F, and K are considered to have paired correctly with H2L2. In this embodiment, preferential pairing is measured as a percentage of total pairings.

[0331] In one embodiment, the preferred match is measured by SMCA as described herein.

[0332] In some embodiments, if the change in the total amount of correct pairings, as measured by the sum of H1L1 and H2L2 pairings, is greater than approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% compared to the pairings of the corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab region that promotes preferential pairing, then the Mab design is considered to promote preferential pairing.

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

[0334] In one embodiment, if the change in the total amount of correct pairings, as measured by the amount of bispecific antibodies produced as a percentage of all species produced, is greater than approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to pairings of the corresponding H1, L1, H2, and L2 polypeptide chains that do not have amino acid substitutions in the Fab region that promotes preferential pairing, then the Mab design is considered to 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 and H1 compared to L2, and preferential pairing of L2 and H2 compared 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 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 sequence (the first wild-type Fab region) or the Fab region formed by the corresponding wild-type H2 and L2 polypeptide sequence (the second wild-type Fab region). The terms "corresponding wild-type H1 and L1 polypeptide sequence" and "corresponding wild-type H2 and L2 polypeptide sequence" mean describing the corresponding H1, L1, H2, and L2 polypeptide sequence that does not have the amino acid modifications that promote the preferential pairing described herein.

[0336] Thermal stability can be measured by various methods known in the art and described herein, including differential scanning calorimetry (DSC) or differential scanning fluorescence quantification (DSF). The latter method provides a measure of thermal stability in terms of "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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a first Fab region having a Tm within approximately 6°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm within approximately 5°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm within approximately 4°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm within approximately 3°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm within approximately 2°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm within approximately 1°C of the Tm of the corresponding wild-type first Fab region. In one embodiment, the antigen-binding polypeptide construct includes a first Fab region having a Tm substantially the same as the Tm of the corresponding wild-type first Fab region.

[0338] In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 20°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 15°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 10°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 9°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 8°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm of approximately 7°C or less than the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 6°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 5°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 4°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 3°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 2°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm within approximately 1°C of the Tm of the corresponding wild-type second Fab region. In one embodiment, the antigen-binding polypeptide construct includes a second Fab region having a Tm substantially 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 approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Fab region formed by wild-type H1 and L1 polypeptide sequences corresponding to the first antigen (wild-type first Fab region), and / or the melting temperature (Tm) of the second Fab region is within approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20°C of the Fab region formed by wild-type H2 and L2 polypeptide sequences corresponding to 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 rises to 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, or 5.0°C or higher 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 includes amino acid substitutions corresponding to KL design numbers 2979, 3018, 3041, 3102, 3898, and / or 3947. In one embodiment, the antigen-binding polypeptide construct includes amino acid substitutions corresponding to KL design numbers 3025, 3109, 3113, 3878, 3890, 3910, 3931, 3954, 3967, 4010, and / or 4040.

[0342] The ability of the Fab region to bind to the antigen. Amino acid modifications in one or more of the H1, L1, H2, and L2 polypeptide sequences promote preferential pairing of L1 and H1 compared to L2, and preferential pairing of L2 and H2 compared to L1, minimizing the impact on the ability of each heterodimer of the antigen-binding polypeptide construct to bind to 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 by H2 and L2 to bind to their respective antigens, and comparing this to the ability of the corresponding wild-type first Fab region or the corresponding wild-type second Fab region to bind to their respective antigens.

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

[0344] In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 100 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 50 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 40 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 30 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 20 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 10 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 9 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 8 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 7 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 6 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately 5 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within 4 times the affinity of the wild-type first Fab region to the first antigen. In one embodiment, the affinity of the first Fab region to the first antigen is within approximately three times the affinity of the wild-type first Fab region to the first antigen. In another embodiment, the affinity of the first Fab region to the first antigen is within approximately two times the affinity of the wild-type first Fab region to the first antigen.In one embodiment, the affinity of the first Fab region to the first antigen is approximately the same as the affinity of the wild-type first Fab region to the first antigen.

[0345] In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 100 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 50 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 40 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 30 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 20 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 10 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 9 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 8 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 7 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 6 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately 5 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within 4 times the affinity of the wild-type second Fab region to the second antigen. In one embodiment, the affinity of the second Fab region to the second antigen is within approximately three times the affinity of the wild-type second Fab region to the second antigen. In another embodiment, the affinity of the second Fab region to the second antigen is within approximately two times the affinity of the wild-type second Fab region to the second antigen.In one embodiment, the affinity of the second Fab region to the second antigen is approximately the same as the affinity of the wild-type second Fab region to the second antigen.

[0346] Amino acid modification or mobility of the design set Using the amino acid modifications or design sets described herein, each heterodimer immunoglobulin heavy chain polypeptide sequence and immunoglobulin light chain polypeptide can be used to prepare bispecific antigen-binding polypeptide constructs, which can be obtained from one or more parental antibodies, each containing at least one kappa light chain and at least one other lambda light chain. Based on the following discussion, the Mab design sets can be applied to the vast majority of such bispecific antigen-binding polypeptide constructs.

[0347] The VH:VL and CH1:CL junction residues in the junction 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 be formed upon pairing of the light-heavy chain combination. As a result of this sequence conservation, the Mab design sets described herein can be moved to kappa Fab and lambda of other parental antibodies to produce preferential pairing, based on the structural modeling of D3H44 kappa Fab and CAT-2200 lambda Fab that produce preferential pairing, because this region exhibits high sequence conservation across antibodies. Furthermore, when sequence differences occur, they are usually distal to the CH1:CL junction. This is particularly true with respect to the CH1 and CL domains. In one embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which kappaFab has one or more amino acid modifications in the CL and / or CH1 domains that promote preferential pairing. In one embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which lambdaFab has one or more amino acid modifications in the CL and / or CH1 domains that promote preferential pairing.

[0348] However, with respect to the CDR (complementarity-determining region) loop residue (and length), specifically with respect to CDR-H3, there are several sequence changes at the antigen-binding site. Therefore, in one embodiment, when the amino acid sequence of the antigen-binding site differs significantly from that of the D3H44 antibody, the antigen-binding polypeptide construct described herein includes a heterodimer in which kappaFab has one or more amino acid modifications in the VH and / or VL domain distal to the CDR loop. In another embodiment, when the amino acid sequence of the antigen-binding site differs significantly from that of the CAT-2200 antibody, the antigen-binding polypeptide construct described herein includes a heterodimer in which lambdaFab promotes preferential pairing and has one or more amino acid modifications in the VH and / or VL domain distal to the CDR loop. In another embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which kappaFab includes one or more amino acid modifications in the VH and / or VL domains that promote preferential pairing and are proximal or distal to the CDR loop, when the amino acid sequence of the antigen-binding site is substantially similar to that of the D3H44 antibody. In another embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which lambdaFab includes one or more amino acid modifications in the VH and / or VL domains that promote preferential pairing and are proximal or distal to the CDR loop, when the amino acid sequence of the antigen-binding site is substantially similar to that of the CAT-2200 antibody. In a particular embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which kappaFab includes one or more amino acid modifications in the CL and / or CH1 domain, as well as modifications in the VH and / or VL domains that promote preferential pairing. In a particular embodiment, the antigen-binding polypeptide construct described herein comprises a heterodimer in which lambdaFab comprises one or more amino acid modifications in the CL and / or CH1 domain, as well as modifications in the VH and / or VL domain that promote preferential pairing.

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

[0350] In another embodiment, the amino acid modifications described herein are transferable to the immunoglobulin heavy and light chains of antibodies using commonly used VH and VL subgroups.

[0351] In one embodiment, the amino acid modifications described herein are transferable to the immunoglobulin heavy and light chains of antibodies having a germline-like framework. An example of such an antibody is obinutuzumab.

[0352] In one embodiment, the amino acid modifications described herein are transferable to the immunoglobulin heavy and light chains of an antibody having a VH:VL domain-to-domain angle close to the average observed for the heavy and light chain pair. Examples of this type of antibody include, but are not limited to, pertuzumab. In another embodiment, the amino acid modifications described herein are transferable to the immunoglobulin heavy and light chains of an antibody having standard CL and CH1 domains. Preferred examples of such antibodies include, but are not limited to, trastuzumab.

[0353] Examples, figures, and tables demonstrate that amino acid modifications (e.g., within one or more Fab fragments including variable and constant regions) that can promote preferential pairing are migrateable to other immunoglobulin heavy and light chains, resulting in similar patterns 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 may be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance. The heterodimer of the antigen-binding polypeptide construct may 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 immunoglobulin Fc(Fc) or a portion thereof. In some embodiments, Fc comprises at least one or two CH3 domain sequences. In some embodiments, Fc further comprises at least one or two CH2 domain sequences. In some embodiments, the antigen-binding polypeptide construct comprises Fc linked to a first heterodimer and / or a second heterodimer with or without one or more linkers. In some embodiments, Fc is human Fc. In some embodiments, Fc is human IgG or IgG1 Fc. In some embodiments, Fc is a heterodimer Fc. In some embodiments, Fc is a single-chain polypeptide. In some embodiments, Fc is a plurality of peptides, for example, two polypeptides.

[0356] In some embodiments, Fc includes one or more amino acid modifications in at least one of the CH3 domain sequences. Amino acid modifications may be made to immunoglobulin heavy chain Fc to result in preferential pairing between heterodimeric CH3 domain sequences and homodimeric CH3 domain sequences. Such amino acid modifications are known in the art and include, for example, those described in U.S. Patent Publication 2012 / 0149876. Alternative strategies for resulting in preferential pairing between heterodimeric CH3 domain sequences and homodimeric CH3 sequences include, for example, "knob-into-hole" techniques, charged residues via ionic interactions, and strand-exchange gene manipulation domain (SEED) techniques may also be used. The latter strategies are described in the art and outlined above by Klein et al. Further consideration of the Fc domain follows below.

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

[0358] In some embodiments, the antigen-binding polypeptide constructs described herein include a heterodimer Fc containing an asymmetrically modified modified CH3 domain. The heterodimer Fc may contain two heavy chain constant-domain polypeptides, namely a first Fc polypeptide and a second Fc polypeptide, which can be used interchangeably, provided that Fc contains one first heavy chain polypeptide and one second heavy chain polypeptide. Generally, the first heavy chain polypeptide contains a first CH3 sequence, and the second heavy chain polypeptide contains a second CH3 sequence.

[0359] Two CH3 sequences containing one or more asymmetrically introduced amino acid modifications generally result in a heterodimer 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 the first CH3 sequence differs from an amino acid at the same position on the second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer rather than a homodimer. This heterodimerization may result from modification of only one of two amino acids at the same individual amino acid positions on each sequence; or from modification of both amino acids on each sequence at the same individual positions on each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimer Fc may contain one or more asymmetric amino acid modifications.

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

[0361] Typically, Fc may contain two dimerizable consecutive heavy chain sequences (A and B). In some embodiments, one or both sequences of Fc contain 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, Fc contains the variant sequences shown in Table X. In some embodiments, Fc contains the mutations of variants 1A-B. In some embodiments, Fc contains the mutations of variants 2A-B. In some embodiments, Fc contains the mutations of variants 3A-B. In some embodiments, Fc contains the mutations of variants 4A-B. In some embodiments, Fc contains the mutations of variants 5A-B.

[0362] TIFF0007901718000051.tif97170

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

[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 antigen-binding polypeptide construct Fc to bind to the 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 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 its binding to the Fc-γ receptor.

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

[0366] TIFF0007901718000052.tif94170

[0367] Accordingly, in one embodiment, the antigen-binding polypeptide construct described herein may include a dimer Fc having one or more amino acid modifications as described in the table above, which result in improved effector function. In another embodiment, the antigen-binding polypeptide construct may be unfucosylated to improve effector function.

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

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

[0370] In some embodiments, one or more linkers are one or more polypeptide linkers. In some embodiments, one or more linkers include one or more antibody hinge regions. In some embodiments, one or more linkers include 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 may be further modified (i.e., by covalent bonding of various types of molecules) such that the covalent bond does not interfere with the preferential pairing between the heavy and light chains, or affects the ability of the heterodimer to bind to the 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, and binding to intracellular ligands or other proteins. Any of the many chemical modifications may be carried out by known techniques, including, but are not limited to, specific chemical degradation, acetylation, formylation, and metabolic synthesis of tunicamycin.

[0372] In another embodiment, the immunoglobulin heavy and light chains of the antigen-binding polypeptide constructs described herein may be conjugated (directly or indirectly) to a therapeutic or drug moiety that modifies a given biological reaction. In certain embodiments, the antigen-binding polypeptide construct is conjugated to a drug, such as a toxin, chemotherapeutic agent, immunomodulator, or 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 No. 8,624,003 (Pot Method), No. 8,163,888 (One-Step Method), and No. 5,208,020 (Two-Step Method). In some embodiments, the drug is selected from maytansine, auristatin, calicheamicin, or derivatives thereof. In other embodiments, the drug is maytansine selected from DM1 and DM4.

[0373] In some embodiments, antigen-binding polypeptide constructs are conjugated to cytotoxic agents. The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or blocks cellular function and / or causes cellular destruction. This term is intended to include radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu177), chemotherapeutic agents, and toxins such as small molecule toxins, or enzyme-active toxins (including fragments and / or variants thereof) derived from bacteria, fungi, plants, or animals.

[0374] The therapeutic or drug portion should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug portion may be a protein or polypeptide with the desired biological activity. Such proteins include toxins such as abrin, lysine A, onconase (or another cytotoxic RNase), Pseudomonas aeruginosa 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; and apoptotic agents such as TNF-alpha, TNF-beta, AIMI (see International Publication WO97 / 33899), AIM II (see International Publication WO97 / 34911), Fas ligand (Takahashi et al.). These may include biological response modifiers such as (al., 1994, J.Immunol., 6:1567), and VEGI (see International Publication WO99 / 23105); thrombotic agents or anti-angiogenic agents, e.g., angiostatin or endostatin; or, 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, antigen-binding polypeptide constructs may be conjugated with therapeutic moieties such as radioactive materials or macrocyclic chelating agents useful for conjugating with radioactive metal ions (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA), which can be bound to the antibody via a linker molecule. Such linker molecules are commonly 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 an antigen-binding polypeptide construct are represented as a fusion protein containing a tag to facilitate purification and / or testing, etc. As described herein, a “tag” is any further set of amino acids provided to a protein, either at the C-terminus, N-terminus, or internally, that contribute 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 domains (ABDs), His tags, FLAG tags, glutathione-s-transferases, hemagglutinin (HA), and maltose-binding proteins. Such tagged proteins may also be genetically engineered to include cleavage sites, such as thrombin, enterokinase, or factor X cleavage sites, to simplify tag removal before, during, or after purification.

[0376] Method for preparing antigen-binding polypeptide constructs As described above, the antigen-binding polypeptide constructs described herein may comprise a first heterodimer and a second heterodimer. The first heterodimer comprises an immunoglobulin heavy chain or fragment thereof having at least VH and CH1 domains, and an immunoglobulin lambda light chain having VL and CL domains, and the second heterodimer comprises an immunoglobulin heavy chain or fragment thereof having at least VH and CH1 domains, and an immunoglobulin kappa light chain having VL and CL domains. The immunoglobulin polypeptide sequences are genetically engineered to incorporate amino acid modifications that promote the preferential pairing described herein. Thus, in the case of a bispecific antigen-binding polypeptide construct, there are typically four different polypeptide sequences: two immunoglobulin heavy chain polypeptide sequences or fragments thereof, and two immunoglobulin light chain polypeptide sequences that constitute 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.For example, standard techniques 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 (ASMPress, 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 parental antibody constituting the antigen-binding polypeptide construct are either publicly known in the art or can be readily determined using nucleic acid and / or protein sequencing methods.

[0378] Accordingly, polynucleotides or sets of polynucleotides encoding the immunoglobulin heavy and light chains of antigen-binding polypeptide constructs are also provided. Such polynucleotides include DNA and RNA in both single-stranded and double-stranded forms, as well as their corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. Polynucleotides include full-length gene or cDNA molecules, as well as combinations of their fragments.

[0379] The polynucleotides encoding genetically modified immunoglobulin heavy and light chain polypeptides described herein can be prepared by site-specific mutation of nucleotides within the polypeptide-encoding DNA using cassette mutagenesis, PCR mutagenesis, or other techniques known in the art to generate DNA encoding the genetically modified immunoglobulin heavy and light chain polypeptides, and then by expressing the recombinant DNA in a cell culture medium as outlined herein. However, the polynucleotides encoding genetically modified immunoglobulin heavy and light chain polypeptides may also be prepared by in vitro gene synthesis using established techniques.

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

[0381] Further provided are expression systems and constructs in the form of plasmids, expression vectors, transcriptions, or expression cassettes containing at least one of the polynucleotides mentioned above. Further provided are host cells containing such expression systems or constructs.

[0382] Typically, expression vectors used in host cells contain sequences for plasmid retention, as well as sequences for the cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” in certain embodiments, typically include the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication start site, 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 inserting a polynucleotide encoding the polypeptide to be expressed, and one or more selection marker elements. The vector may be multicistronic, meaning it can express two or more polynucleotides encoding the immunoglobulin heavy and light chains of an antigen-binding polypeptide construct, or the antigen-binding polypeptide construct may be expressed by a vector set, each vector expressing one or more polynucleotides. The antigen-binding polypeptide construct may also be expressed using a vector set, which may include a multicistronic vector and a vector containing 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 codes for another “tag,” such as polyHis (e.g., hexaHis), or FLAG, HA (hemagglutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide during polypeptide expression and can function 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 an antibody against the tag as an affinity matrix. If necessary, the tag can then be removed from the purified polypeptide by various means, such as peptidase cleavage.

[0384] Vectors typically contain a promoter that is recognized by a host organism and functionally linked to a polynucleotide encoding a polypeptide. A promoter is a non-transcriptional sequence located upstream (i.e., at the 5' end) of the start codon of a structural gene (generally about 100–1000 bp) that controls the transcription of that structural gene. Promoters are traditionally classified into two classes: inductive promoters and constitutive promoters. Inductive promoters initiate transcription by increasing the level of transcription from the DNA they control in response to several changes in culture conditions, such as the presence or absence of nutrients or temperature changes. Constitutive promoters, on the other hand, uniformly transcribe the gene they functionally link to, meaning they have little to no control over gene expression. Many promoters recognized by various promising host cells are well known.

[0385] Promoterians suitable for use with yeast hosts, bacterial hosts, and insect hosts are also well known in the art. Yeast enhancers are advantageously used in conjunction with yeast promoters. Promoterians suitable for use with mammalian host cells are well known and not limited to, but include promoters obtained from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (e.g., adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably Simian virus 40 (SV40). Other suitable mammalian promoters include heteromammalian 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 the EASE element (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and a matrix-binding region (MAR). MARs can mediate chromatin structure construction and protect the integrated vector from "position" effects. Therefore, MARs are particularly useful when vectors are used to create stable transmitter organisms. Many natural and synthetic MAR-containing nucleic acids are known in the art, for example, 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 polynucleotides are inserted into the appropriate sites in the vector, the completed vector may be inserted into host cells suitable for amplification and / or polypeptide expression. Transformation of selected host cells with the expression vector can be achieved by well-known methods, e.g., transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method of selection is, in part, functional to the type of host cell used. The host cell can be transiently transfected, or it can be stably transfected. These methods and other suitable methods are well-known to those skilled in the art and are described, for example, in Sambrook et al., 2001.

[0388] For long-term high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express genetically modified heavy and light chains of antigen-binding polypeptide constructs can be created. Rather than using expression vectors containing viral replication origins, host cells can be transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription termination factors, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA or polynucleotides, the genetically modified cells can be grown in enriched medium for 1-2 days and then transferred to selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing the plasmid to be stably incorporated into the cell's chromosomes, proliferate, and form lesions, which can then be cloned and expanded into cell lines.

[0389] Many selective systems may 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), which may be used in tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr (which gives 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 gives resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072)); neo (which gives resistance to aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1)); and hygro (which gives resistance to hygromycin (Santerre et al., 1984, Gene 30:147)).

[0390] Host cells, when cultured under appropriate conditions, produce antigen-binding polypeptide constructs that can later be recovered from the culture medium (if the host cells secrete the antigen-binding polypeptide constructs into the culture medium) or directly from the host cells producing the antigen-binding polypeptide constructs (if the antigen-binding polypeptide constructs are not secreted). The selection of appropriate host cells depends on various factors, such as the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and the ease of folding into biologically active molecules. The host cells may be eukaryotes or prokaryotes. For example, expression in bacterial systems produces non-glycosylated products, while expression in yeast produces glycosylated products. Eukaryotic host cells with cellular mechanisms for appropriately handling 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, and include, for example, immortalized cell lines available from the United States Cell Culture and Cell Line Preservation Center (ATCC), and any cell line used in expression systems known in the art can be used to produce the recombinant polypeptides described herein. Generally, the host cell is transformed with a recombinant expression vector containing DNA encoding the antigen-binding polypeptide construct. Host cells that can be used include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms (e.g., Escherichia coli or bacilli). Higher eukaryotic cells include insect cells and mammalian cell lines. Examples of suitable mammalian host cell lines include the COS-7 (ATCC CRL 1651) monkey kidney cell line (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, BHK (ATCC CRL 10) cell line, and the African green monkey kidney cell line CVI (ATCC CCL 1651) described by McMahan et al., 1991, EMBO J.10:2821. Examples include CV1 / EBNA cell lines derived from 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 cultures of primary tissues, primary grafts, 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 strain, Candida, or any yeast strain capable of expressing heterologous polypeptides.Suitable bacterial species include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial species capable of expressing heterologous polypeptides.

[0392] When antigen-binding polypeptides are produced in yeast or bacteria, it may be desirable to modify the products produced within them, for example, by phosphorylation or glycosylation at appropriate sites, in order to obtain functional products. Such covalent bonding can be achieved using known chemical or enzymatic methods. Antigen-binding polypeptide constructs can also be generated by using insect expression systems, by functionally linking a set of polynucleotides to appropriate regulatory sequences in one or more insect expression vectors. Materials and methods for baculovirus / insect cell expression systems are available, for example, in kit form from Invitrogen (San Diego, California, USA) (MaxBac® kits), and 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, a cell-free protein expression system can be used to co-express polypeptides (e.g., heavy and light chain polypeptides) from a polynucleotide set without the use of living cells. Instead, all the components required to transcribe DNA to RNA and translate RNA to protein (e.g., ribosomes, tRNA, 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 gene template (mRNA or DNA) encoding heavy and light chain polypeptides, and (2) the essential molecular mechanisms of transcription and translation. In certain embodiments, a cell extract substantially supplies the components of the reaction solution, e.g., RNA polymerase for mRNA transcription, ribosomes, tRNA, amino acids, enzyme cofactors, energy sources, and cellular components essential for proper protein folding. The cell-free protein expression system 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 necessary 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 outlined in Carlson et al. (2012) Biotechnol. Adv. 30:1185-1194. For example, cell-free protein systems are available based on prokaryotes or eukaryotic cells. An example of a prokaryotic cell-free expression system is one derived from E. coli. Eukaryotic cell-free protein expression systems are available, for example, 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 a suitable cell-free protein expression system capable of producing polypeptides (e.g., heavy-chain 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 effectiveness of IgG folding.

[0395] Simultaneous expression of heavy and light chains The genetically 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 host cells. Thus, in the case of a bispecific antigen-binding polypeptide construct, two immunoglobulin heavy chains and two immunoglobulin light chains are co-expressed in host cells. However, other methods for generating bispecific antigen-binding polypeptide constructs that do not rely solely on the use of a single clonal or transient cell line expressing all four chains are also known in the art (Gramer, et al. (2013) mAbs 5, 962, Strop et al. (2012) J Mol Biol 420, 204). These methods rely on post-production arm exchange of the two pairs of light and heavy chains involved in the formation of bispecific antibodies under redox conditions (redox generation). In this approach, H1L1 and H2L2 heterodimers are expressed in two different cell lines to independently generate the two heterodimers. Subsequently, the two heterodimers are mixed under selective redox conditions to achieve the reassociation of the two intrinsic heavy chains H1 and H2, forming a bispecific antigen-binding polypeptide construct containing H1L1H2L2.

[0396] Preferential pairing is primarily driven by incorporating Mab design set amino acid modifications into immunoglobulin heavy and light chain polypeptides, but the amount of correctly paired heterodimers may be further optimized by varying the ratio of polynucleotides encoding each polypeptide to each other, as shown in the examples.

[0397] Testing of antigen-binding polypeptide constructs As described above, the antigen-binding polypeptide construct comprises a first heterodimer containing H1 and L1, and a second heterodimer containing H2 and L2, where L1 is a lambda light chain, L2 is a kappa light chain, and H1 and H2 are distinct from each other. One or more of H1, L1, H2, and L2 contain amino acid modifications that promote preferential pairing of L1 and H1 compared to L2, and preferential pairing of L2 and H2 compared to L1. The first Fab region of the H1L1 heterodimer and the second Fab region of the H2L2 heterodimer can bind to antigens having similar affinity to 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 in a heterodimer pair for each antigen can be tested as described below. The thermal stability of each heterodimer in 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 the LCCA design set 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] Method for measuring preferential pairings The degree of preferential matching can be evaluated, for example, by using the methods described below and in the examples. Preferential matching can be evaluated in the context of an LCCA design set (H1L1L2, or H2L1L2) or a Mab design set (H1L1H2L2).

[0401] In one embodiment, a light chain competition assay (LCCA) can be used to evaluate preferential pairing in the context of an LCCA design set. Joint patent application PCT / US2013 / 063306, filed on 3 October 2013, describes various embodiments of the LCCA and is incorporated herein by reference in whole. The method allows for the quantitative analysis of pairing between heavy chains and specific light chains in a mixture of co-expressed proteins and can be used to determine whether one particular immunoglobulin heavy chain selectively associates with one of two immunoglobulin light chains when heavy and light chains are co-expressed. The method is concisely described as follows: at least one heavy chain and two different light chains are co-expressed in a cell in a ratio such that the heavy chain is the reactant that constrains pairing. The heavy and light chains may be tagged to facilitate detection. Secretory proteins may be isolated from the cell, and the immunoglobulin light chain polypeptide that binds to the heavy chain is isolated from other secretory proteins to produce a fraction paired with the isolated heavy chain. Subsequently, the amounts of each different light chain are detected in the isolated heavy chain fraction, and the relative amounts of each different light chain in the isolated heavy chain fraction are 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, preferred pairings are evaluated in the context of a Mab design set, and 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 paired heavy and light chains of the resulting species are evaluated using LCMS (liquid chromatography-mass spectrometry) based on the molecular weight difference of each species. Alternatively, antigen activity assays can be used to quantify the relative heterodimer populations containing each light chain, thereby allowing estimation of each relative heterodimer population using the measured degree of binding (compared to a control).

[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 a heterodimer 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 a heterodimer can also be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

[0404] affinity for antigen The binding affinity of each heterodimer to the respective antigen, and the interaction off-rate, can be determined by competitive binding assays according to methods well known in the art. An example of a competitive binding assay is a radioimmunoassay comprising incubation of a labeled antigen (a heterodimer described herein (e.g., 3H or 125I) and the target molecule) in the presence of an increased amount of unlabeled antibody, and detection of the molecule bound to the labeled ligand. The affinity of the heterodimer to the antigen and the binding off-rate can be determined from saturated data obtained by scatchard plot analysis.

[0405] Furthermore, the kinetic parameters of the heterodimers described herein can be determined using surface plasmon resonance (SPR)-based assays known in the art (BIAcore kinetic analysis). For an overview of SPR-based techniques, 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. Furthermore, any of the SPR apparatuses and SPR-based methods for measuring protein-protein interactions described in U.S. Patents No. 6,373,577; 6,289,286; 5,322,798; 5,341,215; and 6,268,125 are conceivable in the methods of the present invention. As is well known in the art, affinity can also be measured using FACS.

[0406] Pharmaceutical composition Also provided herein are pharmaceutical compositions comprising antigen-binding polypeptide constructs as described herein. Such compositions comprise a therapeutically effective amount of antigen-binding polypeptide construct and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” means approval by a federal or state government supervisory authority, or approval as described in the United States Pharmacopeia, or any other generally recognized pharmacopoeia for use in animals, or more specifically, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle, with which the therapeutic agent may be administered. Such pharmaceutical carriers may be sterile liquids (e.g., water and oil), including, but not limited to, oils derived from petroleum, animal oils, vegetable oils, or synthetic oils (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Furthermore, saline solutions, aqueous dextrose solutions, and glycerol solutions may 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, and ethanol. If desired, the compositions may also contain trace amounts of wetting agents or emulsifiers, or pH buffers. These compositions may take the form of solutions, suspensions, emulsifiers, tablets, pills, capsules, powders, or sustained-release formulations. The composition may be formulated as a suppository with traditional binders and carriers (e.g., triglycerides). Oral formulations may contain standard carriers (e.g., pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.). Examples of suitable pharmaceutical carriers are listed in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of a compound, preferably in a purified form, together with an appropriate amount of carrier to provide a form for appropriate administration to a patient.This formulation should be adapted to the prescribed mode of administration.

[0407] In certain embodiments, compositions comprising antigen-binding polypeptide constructs are formulated according to routine procedures as pharmaceutical compositions suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers and local anesthetics such as lignocaines to relieve pain at the injection site. Generally, these components are supplied either separately or mixed together in unit dosage forms, as lyophilized powders or anhydrous concentrates in sealed containers, for example, ampoules or pouches indicating the amount of the activator. If the composition is to be administered by injection, it may be dispensed from injection bottles containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed before administration.

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

[0409] The amount of the compositions described herein that would be effective in treating, inhibiting, and preventing diseases or disorders associated with the abnormal expression and / or activity of therapeutic proteins can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify the optimal dosage range. The appropriate dose used in the formulation should be determined according to the operator's judgment and the individual patient's condition, depending on the route of administration and the severity of the disease or disorder. The effective dose is estimated from dose-response curves derived from in vitro or animal model test systems.

[0410] Use of antigen-binding polypeptide constructs As described above, the antigen-binding polypeptide constructs described herein are obtained from parental antibodies, where each heterodimer of the antigen-binding polypeptide construct corresponds to one of the parental antibodies and is genetically engineered to incorporate amino acid modifications that promote preferential pairing of the immunoglobulin heavy and light chains constituting the heterodimer. Accordingly, the antigen-binding polypeptide constructs described herein may be used for the treatment or prevention of the same disease, disorder, or infection using the parental antibody or combination of parental antibodies.

[0411] In another embodiment, the antigen-binding polypeptide constructs described herein may also be used 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 monoclonal or chimeric antibodies, lymphokines, or hematopoietic growth factors (e.g., IL-2, IL-3, and IL-7) that interact with molecules and work to increase the number or activity of effector cells that increase the immune response. The antigen-binding polypeptide constructs described herein may also be used in combination with one or more drugs used to treat diseases, disorders, or infections, such as anticancer agents, anti-inflammatory agents, or antiviral agents.

[0412] Generation of bispecific antibodies using a Mab design set library In one embodiment, the Mab design sets described herein can be used to produce bispecific antigen-binding polypeptide constructs. The Mab design sets described herein may be available in the form of a Mab design set library, which comprises Mab design sets that demonstrate usefulness 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 to 10-A12 and 10-B1 to 10-B10. In another embodiment, the Mab design set library is represented by one or more of Tables 10-A1 to 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 produce antigen-binding polypeptide constructs starting from two parent antibodies (i.e., Mab1 and Mab2), as follows: For illustrative purposes, Mab1 contains lambda Fab and immunoglobulin heavy chain polypeptide H1 and immunoglobulin light chain polypeptide L1, while Mab2 contains kappa Fab and immunoglobulin heavy chain polypeptide H2 and immunoglobulin light chain polypeptide L2.

[0413] The Mab design set amino acid modification (H1L1H2L2) of the Mab design set library is introduced into the immunoglobulin heavy and light chains (H1 and L2) of Mab1 and Mab2 (H2 and L2). Subsequently, H1, L1, H2, and L2 are co-expressed to determine the amount of correctly paired bispecific antigen-binding polypeptide construct. One or more Mab design sets from the Mab design set library may be individually tested or screened to determine which provides the desired amount of bispecific antigen-binding polypeptide construct. Each heterodimer of the bispecific antigen-binding polypeptide construct can be further tested to evaluate the ability of each heterodimer to bind to an antigen or to evaluate its thermal stability as described herein. Further properties that can be evaluated include the solubility, aggregation, kon and koff rates, and resistance to exposure to acids, bases, oxidation, freeze / thaw cycles, shaking, and pressure of the bispecific antigen-binding polypeptide construct compared to the parent antibody or the Fab region of the parent antibody. The latter characteristic can be influenced by the complementarity-determining region (CDR) of the antibody in question, and therefore may be tested for each bispecific antigen-binding polypeptide construct produced.

[0414] In some embodiments, the amount of correctly paired bispecific antigen-binding polypeptide constructs is assessed by LC-MS. 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 chromatography. A schematic procedure for preparing bispecific antigen-binding polypeptide constructs from Mab1 and Mab2 using the Mab design set library is shown in Figure 9.

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

[0416] Computer implementation In one embodiment, the computer includes at least one processor coupled to a chipset. The chipset is also coupled to memory, storage devices, a keyboard, a graphics adapter, a pointing device, and a network adapter. The display is coupled to the graphics adapter. In one embodiment, the functionality of the chipset is provided by a memory controller hub and an I / O controller hub. In another embodiment, the 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 can be a mouse, trackball, or other type of pointing device, used in conjunction with a keyboard to input data into the computer system. A graphics adapter displays images and other information on a display. A network adapter connects the computer system to a local or wide area network.

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

[0419] As is known in the art, a computer is adapted to run computer program modules to provide the functionality described herein. As used herein, the term “module” refers to the computer program logic used to provide the specified functionality. Thus, modules may be implemented in hardware, firmware, and / or software. Program modules are recorded on storage devices, loaded into memory, and executed by a processor.

[0420] The examples and embodiments described herein are for illustrative purposes only, and it will be understood that various modifications or changes in consideration therein are suggested to those skilled in the art and are included in the spirit and scope of this application and the appended claims. [Examples]

[0421] The following are examples of specific embodiments for preparing and using the antigen-binding polypeptide constructs described herein. These examples are provided solely for illustrative purposes and are not intended to limit the scope of this disclosure. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), a certain degree of experimental error and deviation should naturally be acceptable.

[0422] The constructs and methods described herein may be prepared and carried out by conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the scope of the art, unless otherwise specified. Such techniques are well described 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). Examples

[0423] Example 1: Molecular modeling of Fab connection region and computer-guided gene manipulation Using a structure- and computer molecular modeling-guided approach, a kappa-lambda (KL) design library was generated for preparing bispecific antibodies (i.e., kappa-lambda systems, or KL systems) in which one Fab has a kappa light chain and the other Fab has a lambda light chain. The KL design library includes designs with amino acid modifications in the heavy and light chains of Fabs that promote the preferential formation of the desired bispecific antibody when these heavy and light chains are co-expressed. The KL design library is optimized for the kappa-lambda system because it takes 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), but the library can be used in the context of other bispecific KL system antibodies or fragments to identify designs that exhibit the desired pairing specificity in the antibody of interest.

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

[0425] First, a global analysis of sequence conservation across Fab variable and constant connection regions was performed via sequence and structural alignment of known antibodies. Figure 1 shows alignments of constant and variable domain sequences from various antibody subgroups compared to the sequences of D3H44 and the anti-HER2 antibody pertuzumab (both containing kappa light chains) and CAT-2200 (containing lambda light chains). Figures 1A and 1E show alignments of representative human VH germline subgroups compared to those of D3H44 / pertuzumab and CAT-2200, respectively. Figure 1B shows alignments of representative human kappa VL germline subgroups compared to D3H44 / pertuzumab. Figures 1C and 1G show alignments of human CH1 allele sequences compared to those of D3H44 / pertuzumab and CAT-2200, respectively. Figure 1D shows alignments of D3H44 / pertuzumab compared to human kappa allele sequences. Figure 1F shows the alignment of CAT-2200 compared to a representative human lambda VL germline subgroup. Figure 1H shows the alignment of CAT-2200 compared to a human lambda allele sequence. This analysis revealed that pertuzumab and D3H44 exhibit high sequence conservation by kappa constant and variable domain germlines, while CAT-2200 exhibits high sequence conservation by lambda constant and variable domain germlines. Furthermore, these alignments exemplify high conservation in both heavy and light chain constant domains, suggesting that designing the constant domain connection region increases the potential for transfer to other antibodies.

[0426] The second approach involved analysis of the D3H44 and CAT-2200 crystal structure connection regions using a variety of molecular modeling tools, as shown in Figure 2 (e.g., ResidueContacts® and AffinityDecomposition®). To improve the possibility of migration to other antibodies or fragments, the analysis focused on constant domains with higher sequence conservation (see Figure 1). Using these analyses, we identified differences in hotspot locations (critical connection region residues) between representative kappa Fab (D3H44) and lambda Fab (CAT-2200) structures, as shown in Table 2. Significant conformational differences exist in the constant domains between the CH1-CL (kappa) and CH1-CL (lambda) structures. By superimposing available Fab structures onto the CH1 domain, it was shown that the CL (kappa) structure adopts a very similar conformation and forms a tight cluster. However, the CL (lambda) conformation tends to exist in two distinct clusters: one is close to the kappa orientation (kappa-like cluster), and the other is a more contradictory orientation (lambda cluster). This analysis shows that D3H44 represents the general kappa structure, and CAT-2200 represents the general lambda structure (lambda cluster). Figure 3 illustrates the difference in general constant-domain kappa-lambda conformations using D3H44 and CAT-2200 as representative structures. These differences are thought to mainly stem from the rigid motion of the light chain constant domain relative to the CH1 domain, altering the properties of the HL connection portion in the kappa constant domain compared to the lambda system. The identified hotspot mismatches (Table 2) and the conformational differences described above (Figure 3) served as a starting point for genetic engineering of HL pair design for bispecific systems containing kappa Fab and lambda Fab. Amino acid numbering in the parental D3H44 and CAT-2200 sequences according to Kabat is provided in Tables 3A and 3B.

[0427] Next, the hotspot locations in the 3D crystal structure, as well as potential mutations adjacent to the target hotspots, were simulated and identified via in silico mutagenesis and packing / modeling using Zymepack®. Zymepack® is a software suite that, given an input structure and a set of mutations, modifies the residue types in the input structure according to the supplied mutations to generate a new structure that approximates the physical structure of the mutant protein. In addition, Zymepack® evaluates the properties of the mutant protein by calculating various quantitative metrics. These metrics include measurements of stereochemistry and electrostatic complementarity that can correlate with the stability, binding affinity, or heterodimer specificity of the mutant protein.

[0428] By utilizing mismatches in the conjugate region, mutations can be introduced to promote the selective pairing of desired or preferred polypeptide chains or heterodimers, while avoiding the formation of mispaired or mismatched polypeptide chains or heterodimers. For example, four polypeptide chains are required to prepare a bispecific antibody having one kappa Fab and one lambda Fab. Kappa Fab contains heavy chain 1 (H1) and kappa light chain 1 (L1), while lambda Fab contains heavy chain 2 (H2) and lambda light chain 2 (L2). In this case, the desired HL pairings are H1L1 and H2L2, while the mispairings are H1L2 and H2L1. Note that the designation / numbering of polypeptide chains is arbitrary. Therefore, mutations preferring paired CH1-CL (kappa) junctions (H1L1) over mispaired CH1-CL (lambda) junctions (H1L2), and mutations preferring paired CH1-CL (lambda) junctions (H2L2) over mispaired CH1-CL (kappa) junctions (H2L1) were identified, and kappaFab-lambdaFab-tuned designs were generated 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 evaluated based on Coulomb interactions between charge, hydrogen bonding, and desolvation effects. Relative steric and electrostatic scores were calculated by simulating both preferred heavy-and-light-chain pairing models (H1L1 and H2L2) and mispairing models (H1L2 and H2L1) obtained by introducing the target mutations. This allowed us to determine whether a particular set of mutations results in preferred energies, i.e., greater steric or electrostatic complementarity for preferred heavy-and-light-chain pairs compared to inappropriate pairs. The calculated steric and electrostatic energies are components of the free energy associated with light-and-heavy-chain pairing. Therefore, greater steric and electrostatic complementarity indicates a greater change in free energy associated with the desired pairing compared to the pairing of inappropriate pairs.Greater steric and electrostatic complementarity results in preferred (selective) pairing of the desired heavy and light chains compared to the steric penalty and / or electrostatic repulsion of improper pairings.

[0429] Example 2: Design Selection and Description Using the approach described in Example 1, we design heavy-light heterodimer pairs (i.e., H1L1 and H2L2) that exhibit selective or preferential pairing when one of the HL heterodimer pairs contains a kappa light chain and the other contains a lambda light chain. The heterodimers are designed in pairs called “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. The Mab design sets were initially tested as “LCCA designs” in which one heavy chain of the Mab design set is co-expressed with the two light chains of the Mab design set, one kappa and one lambda, to evaluate relative pairing. The amino acid substitutions described throughout the examples were identified using the Kabat numbering system, as described 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), with reference to Tables 3A and 3B (for pertuzumab and CAT-2200 Fab), unless otherwise specified. However, for reference, Tables 22A, 22B, and 22C provide identification of selected amino acid positions in the heavy chain, kappa light chain, and lambda light chain using the applicable IMGT, 1JPT, and EU numbering systems.

[0430] Mab designs were placed on molecular models of D3H44 and CAT-2200, and metrics were calculated as described in Example 1. Then, the top designs were selected based on risk (minimizing the impact on stability and immunogenicity) and impact (considering the proposing power of drive pairing specificity). Next, the top designs were tested by light chain competition assay (LCCA) to experimentally determine their pairing specificity (see Example 4). While Mab designs were identified using D3H44 and CAT-2200 as representative Fabs, the Mab designs 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 their constant domains and can be seamlessly interconverted between the two systems. These Mab designs are referred to as KL designs.

[0431] A second set of designs was also tested with the pertuzumab / CAT-2200 KL system. These designs were proposed using representative designs that reflect the diversity of designs from the kappa-kappa (KK) design library listed in Table 30 of International Patent Application PCT / CA2013 / 050914 (International Patent Publication WO2014 / 082179) as a starting point. These representative KK-derived designs included a subset of designs that, where possible, were either transplanted into the KL system without modification, or adapted to the kappa-lambda system by modifying amino acid residues as necessary to account for differences in the sequence and structure of the kappa and lambda light chains. Representative KK-derived designs of both types 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. 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 back-translated into DNA, codon-optimized for mammalian expression, and the 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 extracted from PDB entry 2VXS, back-translated into DNA, codon-optimized for mammalian expression, and the 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] A light chain vector consisting of a 5'-EcoRI cleavage site-HLA-A signal peptide-HA or FLAG tag-light chain Ig clone-"TGA or TAA stop"-BamH1 cleavage site-3' was inserted into a pTT5 vector (Durocher Y et al., Nucl. Acids Res. 2002;30, No.2 e9) to produce a light chain expression vector. The resulting light chain expression vector was sequenced to confirm the correct reading frame and sequence of the coding DNA. Similarly, a heavy chain vector consisting of a 5'-EcoR1 restriction site-HLA-A signal peptide-heavy chain clone (terminating at T238, see Table 3A)-ABD2-His6 tag-TGA stop-BamH1 cleavage site-3' was inserted into a pTT5 vector (ABD2 = two copies of tandem-linked albumin-binding domain protein) to produce a heavy chain expression vector. The resulting heavy chain expression vectors were also sequenced to confirm the correct reading frame and sequence of the coding DNA. Various pertuzumab or CAT-2200 Fab constructs containing amino acid substitutions from the Mab design set were generated by either 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 the assessment of preferential pairing by competitive assay-SPR screening (LCCA). The ABD2-His6 heavy chain tag trapped the HL complex on the surface of the anti-His tag SPR chip, while the FLAG and HA light chain tags quantified the relative L1 and L2 populations.

[0436] Example 4: Evaluation of preferential pairing of designed Fab heterodimers using light chain competition assay (LCCA). Constructs encoding CAT-2200 and pertuzumab IgG heavy and light chains in Fab form, including amino acid modifications, ...

Claims

1. An antigen-binding polypeptide construct comprising a first heterodimer and a second heterodimer, The first heterodimer (H1L1) comprises a first immunoglobulin G (IgG) heavy chain polypeptide sequence (H1) and an immunoglobulin lambda light chain polypeptide sequence (L1) that forms a first Fab region that specifically binds to the first antigen; the second heterodimer (H2L2) comprises a second immunoglobulin G (IgG) heavy chain polypeptide sequence (H2) and an immunoglobulin kappa light chain polypeptide sequence (L2) that forms a second Fab region that specifically binds to the second antigen. Unlike H2, H1 contains a heavy chain variable domain (VH domain) and a heavy chain constant domain 1 (CH1 domain); L1 comprises a variable lambda light chain (VL-lambda) domain and a steady-state lambda light chain (CL-lambda) domain, and L2 comprises a variable kappa light chain (VL-kappa) domain and a steady-state kappa light chain (CL-kappa) domain; H1, H2, L1, and L2 are subjected to the following amino acid substitutions: a) H1 contains amino acid substitutions 125R, 143D, and 145T; L1 contains amino acid substitutions 122D, 124Q, 131K, and 133S; H2 contains amino acid substitutions 143I, 186K, and 228D; and L2 contains amino acid substitutions 121K, 124E, and 133D; (13173-13477) b) H1 comprises amino acid substitutions 125R, 143D, and 145T; L1 comprises amino acid substitutions 122D, 124Q, 131K, and 133S; H2 comprises amino acid substitutions 186K and 228D; and L2 comprises amino acid substitutions 121K, 124E, and 133D; (13173-13506) c) H1 comprises amino acid substitutions 125R, 143D, 145T and 179E; L1 comprises amino acid substitutions 122D, 124Q and 131R; H2 comprises amino acid substitutions 188K and 228D; and L2 comprises amino acid substitutions 121K, 133I, 176D and 178E; (13180-13515) d) H1 contains amino acid substitutions 125R, 143D and 145T; L1 contains amino acid substitutions 122D, 124Q and 131R; H2 contains amino acid substitutions 188K and 228D; and L2 contains amino acid substitutions 121K, 131D, 133G and 178F; (13177-13513) e) H1 contains amino acid substitutions 125R, 143D, and 145T; L1 contains amino acid substitutions 122D, 124Q, and 131K; H2 contains amino acid substitutions 124R, 186R, and 228D; and L2 contains amino acid substitutions 121K, 133G, 176D, and 180E; (13178-13417) f) H1 ​​contains amino acid substitutions 125R, 143D, and 145T; L1 contains amino acid substitutions 122D, 124Q, and 131R; H2 contains amino acid substitutions 124R, 186R, and 228D; and L2 contains amino acid substitutions 121K, 133G, 176D, and 180E; (13171-13416) g) H1 contains amino acid substitutions 125R, 143D, and 145T; L1 contains amino acid substitutions 122D, 124Q, and 131R; H2 contains amino acid substitutions 143R and 228D; and L2 contains amino acid substitutions 121K, 124E, 133D, and 180D; (13176-13487) h) H1 contains amino acid substitutions 125R, 143D, and 145T; L1 contains amino acid substitutions 122D, 124Q, and 131R; H2 contains amino acid substitutions 143R and 228D; and L2 contains amino acid substitutions 121K, 124E, and 133D; (13175-13486) i) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q, 131K, and 133S, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions Q124E and V133D; (12906-13152) j) H1 contains amino acid substitutions 143D, 145T, and 179E, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions 133D and 160E; (10970-11348) k) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q and 131K; H2 contains amino acid substitution 186K; and L2 contains amino acid substitution 133D; (10969-11344) l) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q and 131K; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitution 133D; (10969-11290) m) H1 contains amino acid substitutions 143D and 145T; L1 contains amino acid substitutions 131K and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitutions 133D and 160E; (10966-11351) n) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 131K and 133S, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitutions 133D and 160E; (10966-11297) o) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 131K and 133S, H2 contains amino acid substitution 186K, and L2 contains amino acid substitution 133D; (10965-11347) p) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 131K and 133S, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitution 133D; (10965-11293) q) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitution 131K, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions 133D and 160E; (10962-11350) r) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitution 131K, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitutions 133D and 160E; (10962-11296) s) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 188K, and L2 contains amino acid substitutions 131E, 133G, and 178F; (10946-11382) t) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 188K, and L2 contains amino acid substitutions 131D, 133G, and 178F; (10945-11377) u) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 143R, and L2 contains amino acid substitutions 124E and 133D; (10944-11314) v) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 143K, and L2 contains amino acid substitutions 124E and 133D; (10944-11307) w) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q, 131K, and 133S, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions 133D and 160E; (10943-11349) x) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q, 131K, and 133S, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitutions 133D and 160E; (10943-11295) y) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q, 131K, and 133S, H2 contains amino acid substitution 186K, and L2 contains amino acid substitution 133D; (10942-11345) z) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q, 131K, and 133S, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitution 133D; (10942-11291) aa) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions 133D and 160E; (10928-11348) bb) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitutions 133D and 160E; (10928-11294) cc) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitution 186K, and L2 contains amino acid substitution 133D; (10927-11344) dd) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitution 133D; (10927-11290) ee) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitution 188K, and L2 contains amino acid substitutions 131D, 133G, and 178F; (10924-11376) ff) H1 ​​contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q and 131K; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitutions 133D and 160E; (10970-11294) gg) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q, 131K, and 133S; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitution 133D; (10973-11291) hh) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitutions 39E and 143R, and L2 contains amino acid substitutions 38R, 124E, and 133D; (13279-13501) ii) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q, 131K, and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitution 133D; (10973-11345) jj) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitutions 45P and 143R, and L2 contains amino acid substitutions 44F, 124E, and 133D; (13278-13372) kk) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q, 131K, and 133S; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitutions 124E, 133D, and 135W; (13216-13476) ll) H1 contains amino acid substitutions 143D, 145T, and 174G; L1 contains amino acid substitutions 124Q, 131K, 133S, and 176F; H2 contains amino acid substitutions 143I, 186K, and 190F; and L2 contains amino acid substitutions 124E, 133D, and 135A; (13283-13478) (mm) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q, 131K, and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitutions 124E, 133D, and 135W; (13216-13505) nn) H1 contains amino acid substitutions 143D, 145T, and 174G; L1 contains amino acid substitutions 124Q, 131K, 133S, and 176F; H2 contains amino acid substitutions 186K and 190F; and L2 contains amino acid substitutions 124E, 133D, and 135A; (13283-13507) oo) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q and 131R; H2 contains amino acid substitution 188K; and L2 contains amino acid substitutions 131D, 133G, 135W, and 178F; (13220-13511) pp) H1 contains amino acid substitutions 143D, 145T, and 174G; L1 contains amino acid substitutions 116F, 124Q, 131K, and 176F; H2 contains amino acid substitutions 124R, 186K, and 190F; and L2 contains amino acid substitutions 133G, 135A, 176D, and 180E; (13285-13414) qq) H1 contains amino acid substitutions 143D, 145T, and 174G; L1 contains amino acid substitutions 116F, 124Q, 131R, and 176F; H2 contains amino acid substitutions 143R and 190F; and L2 contains amino acid substitutions 124E, 133D, and 135A; (13287-13494) rr) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q and 131R; H2 contains amino acid substitution 143R; and L2 contains amino acid substitutions 124E, 133D, 135W, and 180D; (13219-13483) ss) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q and 131R; H2 contains amino acid substitution 143R; and L2 contains amino acid substitutions 124E, 133D, and 135W; (13218-13482) tt) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 143R, and L2 contains amino acid substitutions 124E, 133D, and 180D; (13282-13484) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 131K and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitutions 133D and 160E; (10982-11351) vv) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 131K and 133S; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitutions 133D and 160E; (10982-11297) (ww) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 131K and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitution 133D; (10981-11347) xx) H1 contains amino acid substitutions 143D, 145T, and 179E, L1 contains amino acid substitutions 131K and 133S, H2 contains amino acid substitutions 143I and 186K, and L2 contains amino acid substitution 133D; (10981-11293) yy) H1 contains amino acid substitutions 143D, 145T, and 179E, L1 contains amino acid substitution 131K, H2 contains amino acid substitution 186K, and L2 contains amino acid substitutions 133D and 160E; (10978-11350) zz) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitution 131K; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitutions 133D and 160E; (10978-11296) aaa) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitution 131K; H2 contains amino acid substitution 186K; and L2 contains amino acid substitution 133D; (10977-11346) bbb) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitution 131K; H2 contains amino acid substitutions 143I and 186K; and L2 contains amino acid substitution 133D; (10977-11292) (ccc) H1 contains amino acid substitutions 143D, 145T, and 179E; L1 contains amino acid substitutions 124Q, 131K, and 133S; H2 contains amino acid substitution 186K; and L2 contains amino acid substitutions 133D and 160E; (10974-11349) ddd) H1 comprises amino acid substitutions 143D, 145T, and 179E; L1 comprises amino acid substitutions 124Q, 131K, and 133S; H2 comprises amino acid substitutions 143I and 186K; and L2 comprises amino acid substitutions 133D and 160E; (10974-11295) or eee) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131K, H2 contains amino acid substitution 143K, and L2 contains amino acid substitutions 124E and 133D; (10923-11306) fff) H1 ​​contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 39E, 124E, 145T and 179E, and L2 contains amino acid substitutions 38R, 131R, 133G and 176R; (13303-13496) ggg) H1 contains the amino acid substitution 143K, L1 contains the amino acid substitutions 129T and 133D, H2 contains the amino acid substitutions 45P, 124E, 145T and 179E, and L2 contains the amino acid substitutions 44F, 131R, 133G and 176R; (13302-13367) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T, 179E, 186I and 188W, and L2 contains amino acid substitutions 131K, 133G, 176R and 178A; (11033-11209) iii) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T, 179E and 188W, and L2 contains amino acid substitutions 131K, 133G, 176R and 178A; (11033-11215) jjj) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G and 176R; (11034-11204) kkk) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T, 179E, 186I and 188W, and L2 contains amino acid substitutions 131R, 133G, 176R and 178A; (11035-11212) lll) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T, 179E and 188W, and L2 contains amino acid substitutions 131R, 133G, 176R and 178A; (11035-11218) (mmm) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T and 186E, and L2 contains amino acid substitutions 131R, 133S and 135K; (11036-11221) nnn) H1 contains the amino acid substitution 143K, L1 contains the amino acid substitutions 129T and 133D, H2 contains the amino acid substitutions 124E, 143E and 145T, and L2 contains the amino acid substitutions 131R, 133T, 135K and 178S; (11039-11224) ooo) H1 contains amino acid substitutions 186K and 188T, L1 contains amino acid substitutions 133D and 178T, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131K, 133G and 176R; (11064-11201) ppp) H1 contains amino acid substitutions 186K and 188T, L1 contains amino acid substitutions 133D and 178T, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G and 176R; (11065-11206) (qqq) H1 contains amino acid substitutions 139W and 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G, 135W and 176R; (13227-13383) (rrr) H1 contains amino acid substitutions 125R and 143K, L1 contains amino acid substitutions 122D, 129T and 133D, H2 contains amino acid substitutions 124E, 145T, 179E and 228D, and L2 contains amino acid substitutions 121K, 131R, 133G and 176R; (13184-13388) sss) H1 contains amino acid substitutions 139W, 186K, and 188T; L1 contains amino acid substitutions 133D and 178T; H2 contains amino acid substitutions 124E, 145T, and 179E; and L2 contains amino acid substitutions 131R, 133G, 135W, and 176R; (13231-13386) (ttt) H1 contains amino acid substitutions 125R, 186K and 188T, L1 contains amino acid substitutions 122D, 133D and 178T, H2 contains amino acid substitutions 124E, 145T, 179E and 228D, and L2 contains amino acid substitutions 121K, 131R, 133G and 176R; (13199-13391) H1 contains amino acid substitutions 139W and 186K, L1 contains amino acid substitutions 129T, 133D and 178T, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G, 135W and 176R; (13230-13384) vvv) H1 contains amino acid substitutions 125R and 186K, L1 contains amino acid substitutions 122D, 129T, 133D and 178T, H2 contains amino acid substitutions 124E, 145T, 179E and 228D, and L2 contains amino acid substitutions 121K, 131R, 133G and 176R; (13198-13389) (lol) H1 contains amino acid substitutions 139W, 143I and 186K; L1 contains amino acid substitutions 129T, 133D and 178T; H2 contains amino acid substitutions 124E, 145T and 179E; and L2 contains amino acid substitutions 131R, 133G, 135W and 176R; (13225-13384) xxx) H1 comprises amino acid substitutions 125R, 143I and 186K, L1 comprises amino acid substitutions 122D, 129T, 133D and 178T, H2 comprises amino acid substitutions 124E, 145T, 179E and 228D, and L2 comprises amino acid substitutions 121K, 131R, 133G and 176R; (13181-13389) or yyy) H1 contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131K, 133G and 176R; (11032-11199) The antigen-binding polypeptide construct comprises, where the amino acid substitution preferentially promotes the pairing of H1 to L1 compared to the pairing of H1 to L2, and / or preferentially promotes the pairing of H2 to L2 compared to the pairing of H2 to L1, where the amino acid residue numbering is by Kabat.

2. a) H1 contains amino acid substitutions 125R, 143D, 145T and 179E; L1 contains amino acid substitutions 122D, 124Q and 131R; H2 contains amino acid substitutions 188K and 228D; and L2 contains amino acid substitutions 121K, 133I, 176D and 178E; (13180-13515) b) H1 contains amino acid substitutions 139W, 143D, and 145T; L1 contains amino acid substitutions 124Q and 131R; H2 contains amino acid substitution 143R; and L2 contains amino acid substitutions 124E, 133D, and 135W; (13218-13482) c) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 143R, and L2 contains amino acid substitutions 124E, 133D, and 180D; (13282-13484); d) H1 contains amino acid substitutions 143D, 145T, and 174G; L1 contains amino acid substitutions 116F, 124Q, 131R, and 176F; H2 contains amino acid substitutions 143R and 190F; and L2 contains amino acid substitutions 124E, 133D, and 135A; (13287-13494) e) H1 contains amino acid substitutions 143D and 145T, L1 contains amino acid substitutions 124Q and 131R, H2 contains amino acid substitution 188K, and L2 contains amino acid substitutions 131D, 133G, and 178F; (10945-11377) f) H1 ​​contains amino acid substitution 143K, L1 contains amino acid substitutions 129T and 133D, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G and 176R; (11034-11204) or g) H1 contains amino acid substitutions 186K and 188T, L1 contains amino acid substitutions 133D and 178T, H2 contains amino acid substitutions 124E, 145T and 179E, and L2 contains amino acid substitutions 131R, 133G and 176R; (11065-11206), The structure according to claim 1.

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

4. The construct according to any one of claims 1 to 3, wherein each of the sequences H1, H2, L1, and L2 is derived from a human sequence or a humanized sequence.

5. The construct according to any one of claims 1 to 4, further comprising a dimer Fc having two Fc polypeptides, each Fc polypeptide comprising a CH2 domain sequence and a CH3 domain sequence, and bonded to one of the first Fab region and the second Fab region with or without a linker.

6. The structure according to claim 5, wherein Fc is 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.

7. The construct according to claim 5, wherein the Fc comprises, relative to the wild type, one or more substitutions in at least one of the CH3 domain sequences that promote preferential pairing between heterodimeric CH3 domain sequences compared to homodimeric CH3 domain sequences.

8. The aforementioned Fc is, i) A heterodimer IgG1 Fc having the substitution L351Y_F405A_Y407V in the first Fc polypeptide and the substitution T366L_K392M_T394W in the second Fc polypeptide; ii) A heterodimer IgG1 Fc having the substitution L351Y_F405A_Y407V in the first Fc polypeptide and the substitution T366L_K392L_T394W in the second Fc polypeptide; iii) Heterodimer IgG1 Fc having the substitution T350V_L351Y_F405A_Y407V in the first Fc polypeptide and the substitution T350V_T366L_K392L_T394W in the second Fc polypeptide; iv) A heterodimer IgG1 Fc having the substitution T350V_L351Y_F405A_Y407V in the first Fc polypeptide and the substitution T350V_T366L_K392M_T394W in the second Fc polypeptide; or v) The construct according to claim 7, comprising a heterodimer IgG1Fc having substituted T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide and substituted T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide, wherein the numbering of amino acid residues of Fc follows EU numbering.

9. The construct according to any one of claims 5 to 8, wherein the Fc comprises one or more modifications to promote selective binding to the Fc-γ receptor, reduce or eliminate binding to the Fc-γ receptor, or promote binding to FcRn.

10. The structure according to any one of claims 5 to 9, wherein the linker is one or more polypeptide linkers.

11. The construct according to claim 10, wherein the linker includes one or more antibody hinge regions.

12. The structure according to claim 11, wherein the linker includes one or more IgG1 hinge regions.

13. A structure according to any one of claims 1 to 12, conjugated with a therapeutic agent or drug.

14. A polynucleotide or polynucleotide set encoding a construct according to any one of claims 1 to 12.

15. A vector or vector set comprising one or more polynucleotides or polynucleotide sets as described in claim 14.

16. Isolated cells comprising a polynucleotide or polynucleotide set according to claim 14, or a vector or vector set according to claim 15.

17. A pharmaceutical composition comprising an antigen-binding polypeptide construct according to any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, further comprising one or more substances selected from the group consisting of buffers, antioxidants, low molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelating agents, stabilizers, and excipients.

19. A method for preparing a structure according to any one of claims 1 to 12, (a) A step of obtaining a host cell containing a polynucleotide or set of polynucleotides encoding the antigen-binding polypeptide construct; (b) The step of culturing the host cells in host cell culture under conditions that express the antigen-binding polypeptide construct, and (c) Step of collecting the antigen-binding polypeptide construct from the host cell culture. The method, including the method described above.

20. The method according to claim 19, wherein the host cell is transiently transfected or stably transfected with the polynucleotide or polynucleotide set.

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