Multispecific antigen-binding proteins

Abstract

To provide a method or the like for reducing mispaired heavy chain / light chain by-products and increasing bispecific antibody yield.SOLUTION: Provided are, inter alia, multispecific antigen binding proteins, or antigen-binding fragments thereof, comprising one or more mutations in the VH / VL domains and / or CH1 / CL domains, pharmaceutical compositions comprising the same, isolated nucleic acids, vectors, and host cells encoding / expressing the same, method of making the multispecific antigen binding proteins, computer readable media for evaluating multispecific antigen binding proteins, and libraries.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 152,735, filed April 24, 2015, 62 / 264,291, filed December 7, 2015, and 62 / 310,555, filed March 18, 2016, all of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 22, 2016, is named P05841WO_PCTSequenceListing.txt and is 131,134 bytes in size. [Background technology]

[0003] The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential. However, producing bispecific antibodies in the IgG format has been difficult because antibody heavy chains have evolved to bind to antibody light chains in a relatively promiscuous manner. As a result of this promiscuous pairing, for example, co-expression of two antibody heavy chains and two antibody light chains naturally leads to heavy chain homodimerization and scrambling of heavy chain / light chain pairing.

[0004] One approach to circumventing the problem of heavy chain homodimerization, known as "knobs-into-holes," aims to force pairing of two different antibody heavy chains by modifying the contact interface through mutations in the CH3 domain. On one heavy chain, an amino acid with a short side chain was substituted for the original amino acid, creating a "hole." Conversely, an amino acid with a long side chain was introduced into the CH3 domain of the other heavy chain, creating a "knob." By coexpressing these two heavy chains (and two identical light chains that must be compatible with both heavy chains), a higher yield of heterodimerization ("knobs-holes") was observed relative to homodimerization ("hole-holes" or "knobs-knobs") (Ridgway, JB, Protein Eng. 9 (1996) 617-621, and WO 96 / 027011).

[0005] Minimizing heavy / light chain scrambling has been more challenging due to the complex multi-domain heterodimeric interactions within antibody Fabs. Bispecific antibody formats aimed at addressing heavy chain / light chain scrambling include DVD-Ig (dual variable domain Ig) (Nature Biotechnology 25, 1290-1297 (2007)), crossover Ig (Schaefer W et al (2011) PNAS 108 (27): 11187-11192), two-in-one Ig (Science 2009, 323, 1610), BiTE® antibody (PNAS 92 (15): 7021-7025; 1995), and Lewis et al. (2014) "Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface." Nat Biotechnol 32, 191-8, Liu et al. (2015) "A Novel Antibody Engineering Strategy for These include strategies described in “Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism,” J Biol Chem. (Published online January 12, 2015, doi:10.1074 / jbc.M114.620260), Mazor et al. 2015. “Improving target cell specificity using a novel monovalent bispecific IgG design,” Mabs. (Published online January 26, 2015, doi:10.1080 / 19420862.2015.1007816), WO 2014 / 081955, WO 2014 / 082179, and WO 2014 / 150973.

[0006] There is a continuing need to reduce mispaired heavy / light chain by-products and increase the yield of bispecific antibodies. Summary of the Invention

[0007] As described in more detail below, multispecific antigen-binding proteins (such as bispecific antibodies) that have been engineered to contain such asymmetric mutations are produced in a single cell with, among other things, improved and correct heavy / light chain pairing and / or improved yields of multispecific antigen-binding protein when compared to multispecific antigen-binding proteins having sequences that lack the modifications. In certain embodiments, the multispecific antigen-binding protein comprises modification(s) in the VH / VL and / or CH1 / CL regions that promote correct heavy / light chain pairing. In certain other embodiments, the multispecific antigen-binding protein further comprises modification(s) in the Fc region that promote heterodimerization of the two arms of the multispecific antigen-binding protein.

[0008] Provided herein is a multispecific antigen-binding protein, or antigen-binding fragment thereof, comprising: a) a first heavy chain / light chain pair that binds to a first antigen, the first heavy chain polypeptide (H1) and the first light chain polypeptide (L1); and b) a second heavy chain / light chain pair that binds to a second antigen, the first heavy chain polypeptide (H2) and the second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises an amino acid substitution at S183 (EU numbering) and the CL domain of L1 comprises an amino acid substitution at V133 (EU numbering). In certain embodiments according to (or as applied to) any of the above embodiments, L1 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L2 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L1 and L2 are each a kappa chain.

[0009] In some embodiments according to (or as applied to) any of the above embodiments, the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K, and the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H1 is replaced with a positively charged residue, and the amino acid at V133 on the CL domain of L1 is replaced with a negatively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H1 is replaced with a negatively charged residue, and the amino acid at V133 on the CL domain of L1 is replaced with a positively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the positively charged residue is selected from the group consisting of R and K. In some embodiments according to (or as applied to) any of the above embodiments, the negatively charged residue is selected from the group consisting of D and E.

[0010] In particular embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 consists of an amino acid substitution at S183 (EU numbering) and the CL domain of L1 consists of an amino acid substitution at V133 (EU numbering).

[0011] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises a S183D mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183E mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133E mutation, or the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133S mutation, or the CH1 domain of H1 comprises a S183A mutation; The CL domain of L1 contains a V133L mutation; the CH1 domain of H1 contains a S183A mutation; the CL domain of L1 contains a V133W mutation; the CH1 domain of H1 contains a S183A mutation; the CL domain of L1 contains a V133K mutation; the CH1 domain of H1 contains a S183A mutation; the CL domain of L1 contains a V133R mutation; the CH1 domain of H1 contains a S183A mutation; the CL domain of L1 contains a V133D mutation; the CH1 domain of H1 contains a S183T mutation; and the CL domain of L1 contains a V133E mutation. or the CH1 domain of H1 contains a S183T mutation and the CL domain of L1 contains a V133S mutation; or the CH1 domain of H1 contains a S183T mutation and the CL domain of L1 contains a V133L mutation; or the CH1 domain of H1 contains a S183T mutation and the CL domain of L1 contains a V133W mutation; or the CH1 domain of H1 contains a S183T mutation and the CL domain of L1 contains a V133K mutation; or the CH1 domain of H1 contains a S183T mutation and the CL domain of L1 contains a V133R mutation; or the CH1 domain of H1 contains a S18 The CL domain of L1 contains a V133D mutation, the CH1 domain of H1 contains a S183V mutation, and the CL domain of L1 contains a V133E mutation, or the CH1 domain of H1 contains a S183V mutation, and the CL domain of L1 contains a V133S mutation, or the CH1 domain of H1 contains a S183V mutation, and the CL domain of L1 contains a V133L mutation, or the CH1 domain of H1 contains a S183V mutation, and the CL domain of L1 contains a V133W mutation, or the CH1 domain of H1 contains a S183V mutation, and the CL domain of L1 containsThe H1 CH1 domain contains a V133K mutation, or the H1 CH1 domain contains a S183V mutation and the L1 CL domain contains a V133R mutation, or the H1 CH1 domain contains a S183V mutation and the L1 CL domain contains a V133D mutation, or the H1 CH1 domain contains a S183Y mutation and the L1 CL domain contains a V133E mutation, or the H1 CH1 domain contains a S183Y mutation and the L1 CL domain contains a V133S mutation, or the H1 CH1 domain contains a S183Y mutation and the L1 CL domain contains a V133L mutation, or the H1 The CH1 domain contains a S183Y mutation and the CL of the L1 domain contains a V133W mutation; the CH1 domain of H1 contains a S183Y mutation and the CL of the L1 domain contains a V133K mutation; the CH1 domain of H1 contains a S183Y mutation and the CL domain of L1 contains a V133R mutation; the CH1 domain of H1 contains a S183Y mutation and the CL domain of L1 contains a V133D mutation; the CH1 domain of H1 contains a S183F mutation and the CL domain of L1 contains a V133E mutation; or the CH1 domain of H1 contains a S183F mutation. The CL domain of L1 contains a V133S mutation, the CH1 domain of H1 contains a S183F mutation, the CL domain of L1 contains a V133L mutation, the CH1 domain of H1 contains a S183F mutation, and the CL domain of L1 contains a V133W mutation, the CH1 domain of H1 contains a S183F mutation, and the CL domain of L1 contains a V133K mutation, the CH1 domain of H1 contains a S183F mutation, and the CL domain of L1 contains a V133R mutation, or the CH1 domain of H1 contains a S183F mutation, and the CL of the L1 domain contains a V133R mutation. , or the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133S mutation; or the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133L mutation; or the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133W mutation; or the CH1 domain of H1 comprises a S183N mutation and the CL domain of L1 comprises a V133L mutation; or the CH1 domain of H1 comprises a S183E mutation and the CL domain of L1 comprises a V133L mutation.

[0012] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises a S183D mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183E mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183V mutation and the CL domain of L1 comprises a V133E mutation.

[0013] In certain embodiments, the CH1 domain of H1 comprises a S183K mutation and the CL domain of L1 comprises a V133E mutation.

[0014] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises a S183D mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183E mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133K mutation, or the CH1 domain of H1 comprises a S183V mutation and the CL domain of L1 comprises a V133E mutation. In certain embodiments, the CH1 domain of H1 comprises a S183K mutation and the CL domain of L1 comprises a V133E mutation.

[0015] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 and / or the CL domain of L2 do not comprise an amino acid substitution. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 does not comprise a substitution at S183, and the CL domain of L2 does not comprise a substitution at V133.

[0016] In some embodiments according to (or as applied to) any of the above embodiments, the H1 and / or H2 VH domain comprises an amino acid substitution at position Q39 (Kabat numbering), and the L1 and / or L2 VL domain comprises an amino acid substitution at position Q38 (Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at Q39 in the VH domain is replaced with a positively charged residue, and the amino acid at Q38 in the VL domain is replaced with a negatively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at Q39 in the VH domain is replaced with a negatively charged residue, and the amino acid at Q38 in the VL domain is replaced with a positively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the positively charged residue is selected from the group consisting of R and K. In some embodiments according to (or as applied to) any of the above embodiments, the negatively charged residue is selected from the group consisting of D and E.

[0017] In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H1 is replaced with a negatively charged residue, the amino acid at position Q39 in the VH domain is replaced with a positively charged residue, the amino acid at position V133 on the CL domain of L1 is replaced with a positively charged residue, and the amino acid at Q38 in the VL domain is replaced with a negatively charged residue (Kabat numbering).

[0018] In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 in the VH domain is replaced by a negatively charged residue, the amino acid at V133 on the CL domain of L1 is replaced by a negatively charged residue, and the amino acid at Q38 in the VL domain is replaced by a positively charged residue (Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 in the VH domain is replaced by a negatively charged residue, the amino acid at V133 on the CL domain of L1 is replaced by a negatively charged residue, and the amino acid at Q38 in the VL domain of L1 is replaced by a positively charged residue (Kabat numbering). In a further embodiment, the amino acid at EU position S183 on the CH1 domain of H2 is replaced with a negatively charged residue, the amino acid at Q39 in the VH domain of H2 is replaced with a positively charged residue, the amino acid at V133 on the CL domain of L2 is replaced with a positively charged residue, and the amino acid at Q38 in the VL domain is replaced with a negatively charged residue (Kabat numbering).

[0019] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation (Kabat numbering), and the VL domain of L1 comprises a Q38K substitution mutation (Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, and the VH domain of H2 comprises a Q39K substitution mutation. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, and the VL domain of L2 comprises a Q38E substitution mutation.

[0020] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation (Kabat numbering), and the VL domain of L1 comprises a Q38E substitution mutation (Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, and the VH domain of H2 comprises a Q39E substitution mutation (all Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation (all Kabat numbering). In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation (all Kabat numbering).

[0021] In some embodiments according to (or as applied to) any of the above embodiments, the interaction between the two substituted amino acids is by hydrogen bonding. In some embodiments according to (or as applied to) any of the above embodiments, the interaction between the two substituted amino acids is by electrostatic interaction.

[0022] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133K mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation.

[0023] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183T substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133K mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation.

[0024] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183Y substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133K mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation.

[0025] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183F substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133K mutation, the VH domain of H2 comprises a Q39E substitution mutation, and the VL domain of L2 comprises a Q38K substitution mutation.

[0026] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, the CL domain of L1 comprises a V133K mutation, the VH domain of H2 comprises a Q39K substitution mutation, and the VL domain of L2 comprises a Q38E substitution mutation.

[0027] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L1 comprises a V133K mutation.

[0028] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183K substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, and the CL domain of L1 comprises a V133E substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38E substitution mutation, and the CL domain of L2 comprises a V133K mutation.

[0029] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, and the CL domain of L1 comprises a V133K substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, the CH1 domain of H2 comprises a S183K substitution mutation, the VL domain of L2 comprises a Q38E substitution mutation, and the CL domain of L2 comprises a V133E mutation.

[0030] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, and the CL domain of L1 comprises a V133K substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, the CH1 domain of H2 comprises a S183K substitution mutation, the VL domain of L2 comprises a Q38E substitution mutation, and the CL domain of L2 comprises a V133E mutation.

[0031] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, and the CL domain of L1 comprises a V133E substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L2 comprises a V133K mutation.

[0032] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, and the CL domain of L1 comprises a V133K substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183K substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L2 comprises a V133E mutation.

[0033] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183K substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, and the CL domain of L1 comprises a V133E substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L2 comprises a V133K mutation.

[0034] In some embodiments according to (or as applied to) any of the above embodiments, H1 and / or H2 each comprise an Fc region comprising a CH2 and a CH3 domain. In some embodiments according to (or as applied to) any of the above embodiments, the Fc region of H1 and / or H2 is human IgG1, human IgG2, or human IgG4 Fc. In some embodiments according to (or as applied to) any of the above embodiments, the Fc region of H1 and / or H2 is mouse IgG1, mouse IgG2, or mouse IgG4 Fc. In some embodiments according to (or as applied to) any of the above embodiments, the CH3 domains of H1 and H2 each meet at an interface, and each CH3 domain comprises an amino acid substitution such that the Fc region of H1 preferentially pairs with the Fc region of H2 compared to H1. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitution within the CH3 domain results in higher electrostatic complementarity. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitution within the CH3 domain results in higher steric complementarity. In some embodiments according to (or as applied to) any of the above embodiments, the CH3 domain of H1 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with one or more amino acid residues having a larger side chain volume, thereby creating a protuberance on the surface of the CH3 domain of H1 that interacts with the CH3 domain of H2, and the CH3 domain of H2 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the CH3 domain of H2 that interacts with the CH3 domain of H1.In some embodiments according to (or applied to) any of the above embodiments, the CH3 domain of H2 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with one or more amino acid residues having a larger side chain volume, thereby creating a protuberance on the surface of the CH3 domain of H2 that interacts with the CH3 domain of H1, and the CH3 domain of H1 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the CH3 domain of H1 that interacts with the CH3 domain of H2. In some embodiments according to (or applied to) any of the above embodiments, the protuberance is a knob mutation. In some embodiments according to (or applied to) any of the above embodiments, the modification to create the knob is T366W (EU numbering). In some embodiments according to (or applied to) any of the above embodiments, the cavity is a hole mutation (EU numbering). In some embodiments according to (or applied to) any of the above embodiments, the hole-generating modification is at least one of T366S, L368A, and Y407V.

[0035] In some embodiments according to (or as applied to) any of the above embodiments, the knob comprises T366W (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the hole mutations comprise at least one, at least two, or all three of T366S, L368A, and Y407V.

[0036] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 is further modified so that two or more amino acid residues within the CH1 / CL interface are replaced with an equivalent number of amino acid residues having larger side chain volumes, thereby creating a protuberance on the surface of the CH1 domain of H1 that interacts with the CL domain of L1, and the CL domain of L1 is further modified so that two or more amino acid residues within the CH1 / CL interface are replaced with an equivalent number of amino acid residues having smaller side chain volumes, thereby creating a cavity on the surface of the CL domain of L1 that interacts with the CH1 domain of H1.

[0037] In some embodiments according to (or as applied to) any of the above embodiments, the CL domain is further modified such that two or more amino acid residues within the CH1 / CL interface are replaced with an equivalent number of amino acid residues having larger side chain volumes, thereby creating a protuberance on the surface of the CL domain that interacts with the CH1 domain of H1, and the CH1 domain of H1 is further modified such that two or more amino acid residues within the CH1 / CL interface are replaced with an equivalent number of amino acid residues having smaller side chain volumes, thereby creating a cavity on the surface of the CH1 domain of H1 that interacts with the CL domain.

[0038] Also provided herein is a multispecific antigen-binding protein, or antigen-binding fragment thereof, comprising: a) a first heavy chain / light chain pair comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises an amino acid substitution at F170 (EU numbering) and the CL domain of L1 comprises an amino acid substitution at S176 (EU numbering). In certain embodiments according to (or as applied to) any of the above embodiments, L1 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L2 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L1 and L2 are each a kappa chain.

[0039] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 further comprises one or more amino acid substitutions at positions selected from the group consisting of A141, S181, S183, and V185 (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the CL domain of L1 further comprises one or more amino acid substitutions at positions selected from the group consisting of F116, S131, V133, L135, S162, S174, and T178 (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitutions result in steric complementarity.

[0040] In some embodiments according to (or as applied to) any of the above embodiments, one or more amino acid substitutions at positions selected from the group consisting of A141, F170, S181, S183, and V185 (EU numbering) in CH1 and / or one or more amino acid substitutions at positions selected from the group consisting of F116, S131, V133, L135, S162, S174, S176, and T178 (EU numbering) in CL are not substituted with a charged amino acid residue.

[0041] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises an amino acid substitution selected from the group consisting of F170S and F170A, and the CL domain of L1 comprises an amino acid substitution of S176F. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises an S176F mutation, or the CH1 domain of H1 comprises an F170A mutation and the CL domain of L1 comprises an S176F mutation. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises an S176F mutation.

[0042] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183V, and V185A mutations, and the CL domain of L1 comprises F116A, V133I, L135V, S162M, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, S131D, L135V, S162M, S174A, S176F, and T178V mutations. the CH1 domain of H1 contains A141I, F170S, S181M, S183A, and V185A mutations and the CL domain of L1 contains F116A, L135V, S174A, S176F, and T178V mutations; or the CH1 domain of H1 contains A141I, F170A, S181M, S183V, and V185A mutations and the CL domain of L1 contains F116A, L135V, S162M, S174A, S176F, and T178V mutations. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations.

[0043] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations, the CH1 domain of H2 comprises an amino acid substitution mutation at S183 (EU numbering), and the CL domain of L2 comprises an amino acid substitution mutation V133K (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations, the CH1 domain of H2 comprises an amino acid substitution mutation at S183 (EU numbering), and the CL domain of L2 comprises an amino acid substitution mutation V133 (EU numbering). In particular embodiments according to (or as applied to) any of the above embodiments, the amino acid at position S183 in the CH1 domain of H2 is replaced with a positively charged amino acid, and the amino acid at position V133 in the CL domain of L2 is replaced with a negatively charged amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, the amino acid at position S183 in the CH1 domain of H2 is replaced with a negatively charged amino acid, and the amino acid at position V133 in the CL domain of L2 is replaced with a positively charged amino acid.

[0044] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), the VH domain of H1 comprises a Q39E mutation (Kabat numbering), the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations (EU numbering), the VL domain of L1 comprises a Q38K mutation (Kabat numbering), the VH domain of H2 comprises a Q39K mutation (Kabat numbering), and the VL domain of L2 comprises a Q38E mutation (Kabat numbering).

[0045] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations (EU numbering), the CH1 domain of H2 comprises a S183E mutation (EU numbering), and the CL domain of L2 comprises a V133K mutation (EU numbering).

[0046] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), the VH domain of H1 comprises a Q39E mutation (Kabat numbering), the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations (EU numbering), and the VL domain of L1 comprises a Q38K mutation (Kabat numbering), the CH1 domain of H2 comprises a S183E mutation (EU numbering), the VH domain of H2 comprises a Q39K mutation (Kabat numbering), the CL domain of L2 comprises a V133K mutation (EU numbering), and the VL domain of L2 comprises a Q38E mutation (Kabat numbering).

[0047] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), the VH domain of H1 comprises a Q39K mutation (Kabat numbering), the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations, and the VL domain of L1 comprises a Q38E mutation (Kabat numbering), the VH domain of H2 comprises a Q39E mutation (Kabat numbering), and the VL domain of L2 comprises a Q38K mutation (Kabat numbering).

[0048] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations, the CH1 domain of H2 comprises a S183K mutation (EU numbering), and the CL domain of L2 comprises a V133K mutation (EU numbering).

[0049] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), the VH domain of H1 comprises a Q39K mutation (Kabat numbering), the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations, and the VL domain of L1 comprises a Q38E mutation (Kabat numbering), the CH1 domain of H2 comprises a S183K mutation (EU numbering), the VH domain of H2 comprises a Q39E mutation (Kabat numbering), the CL domain of L2 comprises a V133K mutation (EU numbering), and the VL domain of L2 comprises a Q38K mutation (Kabat numbering).

[0050] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises F170S, S181M, S183A, and V185A mutations, and the CL domain of L1 comprises L135V, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprises A141I, F170S, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprises A141I, F170S, S181M, and the CL domain of L1 comprises a V185A mutation, and the CL domain of L1 comprises F116A, L135V, S176F, and T178V mutations; or the CH1 domain of H1 comprises A141I, F170S, S181M, and S183A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprises a F170S, S183A, and V185A mutation, and the CL domain of L1 comprises F116A, S176F, and T178V mutations; or the CH1 domain of H1 comprises F170S, S181M, and The H1 CH1 domain contains a V185A mutation, and the CL domain of L1 contains F116A, L135V, and S176F mutations; or the H1 CH1 domain contains a F170S, S181M, and S183A mutation, and the L1 CL domain contains F116A, L135V, and S176F mutations; or the H1 CH1 domain contains a A141I, F170S, and V185A mutation, and the L1 CL domain contains a F116A and S176F mutation; or the H1 CH1 domain contains a A141I, F170S, and S183A mutation, and the L1 CL domain contains a F116A and S176F mutation. or the CH1 domain of H1 comprises A141I, F170S, and S181M mutations and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprises F170S and V185A mutations and the CL domain of L1 comprises F116A and S176F mutations; or the CH1 domain of H1 comprises A141I and F170S mutations and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or the CH1 domain of H1 comprisesThe H1 CH1 domain comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S176F, and T178V mutations; or the H1 CH1 domain comprises A141I and F170S mutations, and the L1 CL domain comprises F116A, L135V, S174A, S176F, and T178V mutations; or the H1 CH1 domain comprises A141I and F170S mutations, and the L1 CL domain comprises F116A, L135V, and S176F mutations; or the H1 CH1 domain comprises A141I and F170S mutations, and the L1 CL domain comprises F116A, L135V, and S176F mutations; or the H1 CH1 domain comprises A141I and F170S mutations, and the L1 CL domain comprises F116A, L135V, and S176F mutations; or the H1 CH1 domain comprises F170S mutation, and the L1 CL domain comprises The main domain contains the F116A, L135V, S174A, S176F, and T178V mutations; the CH1 domain of H1 contains the F170S mutation and the CL domain of L1 contains the F116A, L135V, S176F, and T178V mutations; the CH1 domain of H1 contains the F170S mutation and the CL domain of L1 contains the F116A, L135V, S174A, S176F, and T178V mutations; the CH1 domain of H1 contains the F170S mutation and the CL domain of L1 contains the F116A, L135V, and S176F mutations; or the CH1 domain of H1 contains the F170S mutation and the CL domain of L1 contains the F116A, L135V, and S176F mutations.

[0051] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, S181M, and S183A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, and S176F mutations; or the CH1 domain of H1 comprises F170S, S181M, and S183A mutations, and the CL domain of L1 comprises F116A and S176F mutations; or the CH1 domain of H1 comprises A141I and F170S mutations; The CL domain of L1 comprises the F116A, L135V, S174A, and S176F mutations; the CH1 domain of H1 comprises the A141I and F170S mutations and the CL domain of L1 comprises the F116A and S176F mutations; the CH1 domain of H1 comprises the F170S mutation and the CL domain of L1 comprises the F116A, L135V, S174A, and S176F mutations; or the CH1 domain of H1 comprises the F170S mutation and the CL domain of L1 comprises the F116A and S176F mutations.

[0052] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I, F170S, and S181M mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises F170S mutation, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations.

[0053] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S174A, and S176F mutations.

[0054] Provided herein is an antigen-binding protein, or antigen-binding fragment thereof, comprising: a) a first heavy chain / light chain pair comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain sequence (H2) and a second light chain sequence (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises amino acid substitutions at L128 and V185 (EU numbering), and the CL domain of C1 comprises amino acid substitutions at F118 and L135 (EU numbering). In certain embodiments according to (or as applied to) any of the above embodiments, L1 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L2 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L1 and L2 are each a kappa chain.

[0055] Also provided herein is an antigen-binding protein, or antigen-binding fragment thereof, comprising: a) a first heavy chain / light chain pair comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain sequence (H2) and a second light chain sequence (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises an amino acid substitution at L128 (EU numbering), and the CL domain of C1 comprises amino acid substitutions at F118 and L135 (EU numbering). In certain embodiments according to (or as applied to) any of the above embodiments, L1 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L2 is a kappa chain. In certain embodiments according to (or as applied to) any of the above embodiments, L1 and L2 are each a kappa chain. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 further comprises an amino acid substitution at V185 (EU numbering).

[0056] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 further comprises one or more amino acid substitutions at positions selected from the group consisting of A141, F170, S181, and S183 (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the CL domain of L1 further comprises one or more amino acid substitutions at positions selected from the group consisting of S131, V133, S162, T164, S176, and T178 (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitutions result in higher steric complementarity.

[0057] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises an amino acid substitution at a position selected from the group consisting of A141, F170, S181, and S183, and the CL domain comprises an amino acid substitution at a position selected from the group consisting of S131, V133, S162, T164, S176, and T178.

[0058] In some embodiments according to (or as applied to) any of the above embodiments, one or more amino acid substitutions at positions selected from the group consisting of L128 A141, F170, S181, S183, V185 (EU numbering) in CH1 and / or one or more amino acid substitutions at positions selected from the group consisting of F118, S131, V133, and L135 S162, T164, S176, and T178 (EU numbering) in CL are not substituted with a charged amino acid residue.

[0059] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises L128F, A141M, F170M, S181I, and S183A mutations and the CL domain comprises F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and T178L mutations; or the CH1 domain of H1 comprises L128F, A141M, F170Y, S181I, S183A, and V185A mutations and the CL domain comprises F118V, S131T, V133A, L135F, S162A, S176M, and T178L mutations. or the CH1 domain of H1 contains L128F, A141T, F170M, S181T, S183A, and V185L mutations and the CL domain contains F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L mutations; or the CH1 domain of H1 contains L128F, A141M, F170M, S181T, and S183A mutations and the CL domain contains F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L mutations. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H1 comprises L128F, A141M, F170Y, S181I, S183A, and V185A mutations, and the CL domain comprises F118V, S131T, V133A, L135F, S162A, S176A, and T178L mutations.

[0060] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 comprises a S183E mutation and the CL domain of L2 comprises a V133K mutation. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39E mutation, the VL domain of L1 comprises a Q38K mutation, the VH domain of H2 comprises a Q39K mutation, and the VL domain of L2 comprises a Q38E mutation.

[0061] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 comprises a S183K mutation and the CL domain of L2 comprises a V133E mutation. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a Q39K mutation, the VL domain of L1 comprises a Q38E mutation, the VH domain of H2 comprises a Q39E mutation, and the VL domain of L2 comprises a Q38K mutation.

[0062] In some embodiments according to (or applied to) any of the above embodiments, the CH1 domain of H2 comprises an amino acid substitution at EU position S183, and the CL domain of L2 comprises an amino acid substitution at EU position V133. In some embodiments according to (or applied to) any of the above embodiments, the interaction between the amino acid substitution at EU position S183 on the CH1 domain of H2 and the amino acid substitution at EU position V133 on the CL domain of L2 is due to an electrostatic interaction. In some embodiments according to (or applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H2 is replaced with a positively charged residue, and the amino acid at EU position V133 on the CL domain of L2 is replaced with a negatively charged residue. In some embodiments according to (or applied to) any of the above embodiments, the amino acid at EU position S183 on the CH1 domain of H2 is replaced with a negatively charged residue, and the amino acid at EU position V133 on the CL domain of L2 is replaced with a positively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 comprises an amino acid substitution at a position selected from the group consisting of EU positions S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K, and the CL domain of L2 comprises an amino acid substitution at a position selected from the group consisting of EU positions V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

[0063] In some embodiments according to (or as applied to) any of the above embodiments, the CH1 domain of H2 comprises a mutation at EU position S183D and the CL domain of L2 comprises a mutation at EU position V133K; or the CH1 domain of H2 comprises a mutation at EU position S183E and the CL domain of L2 comprises a mutation at EU position V133K; or the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises a mutation at EU position V133E; or the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises , the CH1 domain of H2 contains a mutation at EU position V133S, or the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133L, or the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133W, or the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133K, or the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133R, or the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133R. The main domain contains a mutation at EU position S183A, the CL domain of L2 contains a mutation at EU position V133D, the CH1 domain of H2 contains a mutation at EU position S183T, the CL domain of L2 contains a mutation at EU position V133E, the CH1 domain of H2 contains a mutation at EU position S183T, the CL domain of L2 contains a mutation at EU position V133S, the CH1 domain of H2 contains a mutation at EU position S183T, the CL domain of L2 contains a mutation at EU position V133L, the CH1 domain of H2 contains a mutation at EU position S183T, and the the CL domain contains a mutation at EU position V133W; the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133K; the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133R; the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133D; the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133E;The CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133S; the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133L; the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133W; the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133K; the CH1 domain of H2 contains a mutation at EU position S183V. The CL domain of L2 contains a mutation at EU position V133R; the CH1 domain of H2 contains a mutation at EU position S183V; the CL domain of L2 contains a mutation at EU position V133D; the CH1 domain of H2 contains a mutation at EU position S183Y; the CL domain of L2 contains a mutation at EU position V133E; the CH1 domain of H2 contains a mutation at EU position S183Y; the CL domain of L2 contains a mutation at EU position V133S; or the CH1 domain of H2 contains a mutation at EU position S183Y; the CL domain of L2 contains a mutation at EU position V133 or the CH1 domain of H2 contains a mutation at EU position S183Y and the CL of the L2 domain contains a mutation at EU position V133W; or the CH1 domain of H2 contains a mutation at EU position S183Y and the CL of the L2 domain contains a mutation at EU position V133K; or the CH1 domain of H2 contains a mutation at EU position S183Y and the CL domain of L2 contains a mutation at EU position V133R; or the CH1 domain of H2 contains a mutation at EU position S183Y and the CL domain of L2 contains a mutation at EU position V133D; or the CH1 domain of H2 contains a mutation at EU position V133R. or the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133E; or the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133S; or the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133L; or the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133W; or the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 containsThe CH1 domain of H2 contains a mutation at EU position V133K, or the CH1 domain of H2 contains a mutation at EU position S183F, the CL domain of L2 contains a mutation at EU position V133R, or the CH1 domain of H2 contains a mutation at EU position S183F, the CL of L2 domain contains a mutation at EU position V133D, or the CH1 domain of H2 contains a mutation at EU position S183H, or the CL domain of L2 contains a mutation at EU position V133S, or the CH1 domain of H2 contains a mutation at EU position S183H and the CL domain of L2 comprises a mutation at EU position V133L; or the CH1 domain of H2 comprises a mutation at EU position S183H and the CL domain of L2 comprises a mutation at EU position V133W; or the CH1 domain of H2 comprises a mutation at EU position S183N and the CL domain of L2 comprises a mutation at EU position V133L; or the CH1 domain of H2 comprises a mutation at EU position S183E and the CL domain of L2 comprises a mutation at EU position V133L. In some embodiments according to (or applied to) any of the above embodiments, the CH1 domain of H2 comprises a substitution at EU position S183D and the CL domain of L2 comprises a substitution at EU position V133K. In some embodiments according to (or applied to) any of the above embodiments, the CH1 domain of H2 and the VL domain of L2 do not comprise an amino acid substitution.

[0064] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises an amino acid substitution at Kabat position Q39, and the VL domain of V1 comprises an amino acid substitution at Kabat position Q38. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at Kabat position Q39 on the VH domain of H1 is replaced with a positively charged residue, and the amino acid at Kabat position Q38 on the VL domain of L1 is replaced with a negatively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid at Kabat position Q39 on the VH domain of H1 is replaced with a negatively charged residue, and the amino acid at Kabat position Q38 on the VL domain of L1 is replaced with a positively charged residue. In some embodiments according to (or as applied to) any of the above embodiments, the positively charged residue is selected from the group consisting of R and K. In some embodiments according to (or as applied to) any of the above embodiments, the negatively charged residue is selected from the group consisting of D and E. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39E, the VL domain of L1 comprises a substitution mutation at Kabat position Q38K, the VH domain of H2 comprises a substitution mutation at Kabat position Q39K, and the VL domain of L2 comprises a substitution mutation at Kabat position Q38E.

[0065] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39E, and the VL domain of L1 comprises a substitution at Kabat position Q38K. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39E, the VL domain of L1 comprises a substitution at Kabat position Q38K, and the VH domain of H2 comprises a substitution mutation at Kabat position Q39K. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39E, the VL domain of L1 comprises a substitution mutation at Kabat position Q38K, the VH domain of H2 comprises a substitution mutation at Kabat position Q39K, and the VL domain of L2 comprises a substitution mutation at Kabat position Q38E.

[0066] In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39K, and the VL domain of L1 comprises a substitution at Kabat position Q38E. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39K, the VL domain of L1 comprises a substitution at Kabat position Q38E, and the VH domain of H2 comprises a substitution mutation at Kabat position Q39E. In some embodiments according to (or as applied to) any of the above embodiments, the VH domain of H1 comprises a substitution mutation at Kabat position Q39K, the VL domain of L1 comprises a substitution mutation at Kabat position Q38E, the VH domain of H2 comprises a substitution mutation at Kabat position Q39E, and the VL domain of L2 comprises a substitution mutation at Kabat position Q38K.

[0067] In some embodiments according to (or as applied to) any of the above embodiments, H1 preferentially pairs with L1 relative to L2, and H2 preferentially pairs with L2 relative to L1.

[0068] In some embodiments according to (or as applied to) any of the above embodiments, H1 comprises an Fc region comprising CH2 and CH3 domains. In some embodiments according to (or as applied to) any of the above embodiments, the Fc region of H1 and / or H2 is an IgG1, IgG2, or IgG4 Fc.

[0069] In some embodiments according to (or as applied to) any of the above embodiments, the Fc region of H1 and / or H2 is mouse IgG1, mouse IgG2, or mouse IgG4 Fc.

[0070] In some embodiments according to (or as applied to) any of the above embodiments, the CH3 domains of H1 and H2 each meet at an interface, and each CH3 domain contains amino acid substitutions such that the Fc region of H1 preferentially pairs with the Fc region of H2 compared to H1. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitutions within the CH3 domain result in greater electrostatic complementarity. In some embodiments according to (or as applied to) any of the above embodiments, the amino acid substitutions within the CH3 domain result in greater steric complementarity.

[0071] In some embodiments according to (or as applied to) any of the above embodiments, the CH3 domain of H1 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with one or more amino acid residues having a larger side chain volume, thereby creating a protuberance on the surface of the CH3 domain of H1 that interacts with the CH3 domain of H2, and the CH3 domain of H2 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the CH3 domain of H2 that interacts with the CH3 domain of H1.

[0072] In some embodiments according to (or as applied to) any of the above embodiments, the CH3 domain of H2 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with one or more amino acid residues having a larger side chain volume, thereby creating a protuberance on the surface of the CH3 domain of H2 that interacts with the CH3 domain of H1, and the CH3 domain of H1 is altered such that one or more amino acid residues within the CH3 / CH3 interface are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the CH3 domain of H1 that interacts with the CH3 domain of H2.

[0073] In some embodiments according to (or applied to) any of the above embodiments, the protrusion is a knob. In some embodiments according to (or applied to) any of the above embodiments, the modification to create the knob is T366W. In some embodiments according to (or applied to) any of the above embodiments, the cavity is a hole. In some embodiments according to (or applied to) any of the above embodiments, the modification to create the hole is at least one of T366S, L368A, and Y407V.

[0074] In some embodiments according to (or as applied to) any of the above embodiments, the knob comprises T366W (EU numbering). In some embodiments according to (or as applied to) any of the above embodiments, the hole mutations comprise at least one, at least two, or all three of T366S, L368A, and Y407V.

[0075] In some embodiments according to (or as applied to) any of the above embodiments, H1 preferentially pairs with L1 relative to L2, and H2 preferentially pairs with L2 relative to L1.

[0076] In some embodiments according to (or as applied to) any of the above embodiments, the first antigen and the second antigen are the same. In some embodiments according to (or as applied to) any of the above embodiments, the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to different epitopes on the same antigen. In some embodiments according to (or as applied to) any of the above embodiments, the first antigen and the second antigen are different.

[0077] Also provided herein is a pharmaceutical composition comprising a multispecific antigen-binding protein according to any of the above embodiments and a pharmaceutically acceptable carrier. Further provided herein is a method of treating a disease in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition according to (or adapted to) any of the above embodiments.

[0078] There is provided an isolated nucleic acid(s) encoding at least one polypeptide sequence of a multispecific antigen-binding protein according to any one of the above embodiments. Also provided is a vector comprising a nucleic acid(s) according to (or as applied to) any of the above embodiments. Also provided is an isolated host cell comprising a nucleic acid(s) according to (or as applied to) any of the above embodiments or a vector according to (or as applied to) any of the above embodiments.

[0079] In some embodiments according to (or applied to) any of the above embodiments, the host cell is a prokaryotic host cell, an E. coli cell, a eukaryotic host cell, a yeast cell, a mammalian cell, or a CHO cell.

[0080] Provided herein is a method of producing a multispecific antigen-binding protein according to (or adapted from) any of the above embodiments, the method comprising: (a) obtaining H1, H2, L1, and L2 polypeptides; and (b) preferentially pairing H1 with L1 relative to L2, and preferentially pairing H2 with L2 relative to L1, to form the multispecific antigen-binding protein.

[0081] Also provided is a method for producing a multispecific antigen-binding protein according to (or applied to) any of the above embodiments, the method comprising: (a) introducing a set of polynucleotides encoding H1, L1, H2, and L2 into a host cell; and (b) culturing the host cell to produce the multispecific antigen-binding protein. In some embodiments according to (or applied to) any of the above embodiments, the set of polynucleotides encoding H1, L1, H2, and L2 are introduced into the same host cell. In some embodiments according to (or applied to) any of the above embodiments, the set of polynucleotides encoding H1, L1, H2, and L2 are introduced into a cell line. In some embodiments according to (or applied to) any of the above embodiments, the cell line is a stable cell line that stably co-expresses H1, L1, H2, and L2. In some embodiments according to (or applied to) any of the above embodiments, the cell line is a stable cell line that stably expresses the multispecific antigen-binding protein. In some embodiments according to (or as applied to) any of the above embodiments, the set of polynucleotides encoding H1, L1, H2, and L2 are introduced into the host cell at a predetermined ratio. In some embodiments according to (or as applied to) any of the above embodiments, the method further comprises determining an optimal ratio of polynucleotides for introduction into the host cell. In some embodiments according to (or as applied to) any of the above embodiments, the multispecific antigen-binding protein is produced with a relative yield of 60% or greater.In certain embodiments according to (or as applied to) any of the above embodiments, the multispecific antigen binding protein is produced in a relative yield of at least about 70%, at least about 71%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or greater than about 99%. In certain embodiments according to (or as applied to) any of the above embodiments, the multispecific antigen-binding protein is produced in a relative yield of at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or greater than about 99%. In certain embodiments according to (or as applied to) any of the above embodiments, the multispecific antigen-binding protein is produced in a relative yield of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or greater than about 99%. In certain embodiments according to (or as applied to) any of the above embodiments, the multispecific antigen binding protein is produced in a relative yield of at least about 95%, at least about 96%, at least about 97%, at least about 99%, or greater than about 99%.

[0082] Also provided is a method for producing a multispecific antigen-binding protein, comprising culturing a host cell according to (or as applied to) any of the above embodiments, and producing the multispecific antigen-binding protein. In some embodiments according to (or as applied to) any of the above embodiments, the method further comprises recovering the multispecific antigen-binding protein. Also provided is a multispecific antigen-binding protein produced by a method according to (or as applied to) any of the above embodiments.

[0083] Provided herein is a library comprising a plurality of polynucleotides encoding a plurality of multispecific antigen-binding proteins according to (or adapted to) any of the above embodiments. Also provided is a method of screening for multispecific antigen-binding proteins that bind to a first antigen and a second antigen, the method comprising: (a) obtaining a plurality of multispecific antigen-binding proteins from a library according to (or adapted to) any of the above embodiments; (b) assaying for binding of the plurality of multispecific antigen-binding proteins to the first and second antigens; and (c) identifying a multispecific antigen-binding protein that binds to the first and second antigens.

[0084] Also provided is a computer readable medium for evaluating a multispecific antigen binding protein comprising: 1) a first heavy chain / light chain pair that binds to a first antigen, the first heavy chain sequence comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and 2) a second heavy chain / light chain pair that binds to a second antigen, the first heavy chain sequence comprising a first heavy chain sequence (H2) and a second light chain sequence (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), the computer readable medium comprising: a) a dataset comprising data representing amino acid substitutions within H1, L1, H2, and L2, wherein at least one of the amino acid substitutions within the CH1 domain of H1 is at EU position F170 or L2 and / or a dataset comprising data representing amino acid substitutions within H1, L1, H2, and L2, wherein at least one of the amino acid substitutions in the CH1 domain of H1 comprises an amino acid substitution at EU position S183 and C1 comprises an amino acid substitution at EU position V133; and (b) a computer-readable medium comprising computer-executable code for determining the likelihood that H1 will preferentially pair with L1 compared to L2 and / or the likelihood that H2 will preferentially pair with L2 compared to L1.

[0085] Also provided is a computer readable medium for evaluating a multispecific antigen binding protein comprising: 1) a first heavy chain / light chain pair that binds to a first antigen, the first heavy chain sequence comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and 2) a second heavy chain / light chain pair that binds to a second antigen, the first heavy chain sequence comprising a first heavy chain sequence (H2) and a second light chain sequence (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), the computer readable medium comprising: a) a dataset comprising data representing amino acid substitutions within H1, L1, H2, and L2, wherein at least one of the amino acid substitutions in the CH1 domain of H1 is at EU position F170 or and / or a dataset comprising data representing amino acid substitutions within H1, L1, H2, and L2, wherein at least one of the amino acid substitutions in the CH1 domain of H1 comprises an amino acid substitution at EU position S183 and C1 comprises an amino acid substitution at EU position V133; and (b) a computer-readable medium comprising computer-executable code for determining the likelihood that H1 preferentially pairs with L1 relative to L2 and / or the likelihood that H2 preferentially pairs with L2 relative to L1. In certain embodiments, in a dataset comprising data representing amino acid substitutions in H1, L1, H2, and L2, at least one of the amino acid substitutions in the CH1 domain of H1 comprises an amino acid substitution at EU position F170 or L128 and V185. [Brief explanation of the drawings]

[0086] [Figure 1A] (Figures 1A and 1B) Protein A recovery from 1 ml of 293T culture of an antibody containing a heavy chain with a substitution mutation at positions L128, G143, L145, S183, or V185 and a light chain with a substitution at F118, V133, or L135. [Figure 1B](Figures 1A and 1B) Protein A recovery from 1 ml of 293T culture of an antibody containing a heavy chain with a substitution mutation at positions L128, G143, L145, S183, or V185 and a light chain with a substitution at F118, V133, or L135. [Figure 2] 1 provides the results of mammalian culture expression assays performed to identify amino acid mutations within CH1 that restore antibody expression when co-expressed with CL bearing F118, V133, or L135 substitution mutations. [Figure 3] 1 provides a schematic diagram of the interior of the interface between the light chain constant region (CL) and the heavy chain CH1 domain. [Figure 4] 1 provides the results of a mammalian culture expression assay performed to analyze antibody expression in cultures expressing light / heavy chain pairs with V133X / S183X substitution mutations. [Figure 5] 1 shows the results of a FACS analysis of the binding of various mutant antibodies with amino acid substitutions in either VH Q39 or VL Q38, or both, to the antigen IL-13. [Figure 6] Results of additional FACS analysis performed to identify Q39X-VH / Q38X-VL mutant pairs that favor VH / VL pairing. [Figure 7] 1 shows the results of a mammalian culture expression assay performed to identify Q39X-VH / Q38X-VL mutant pairs that favor VH / VL pairing. [Figure 8] Figure 1 shows exemplary results of the % abundance of bispecific antibody variants with correct heavy-light chain pairing as measured by Orbitrap mass spectrometry from co-expressed antibodies in mammals. The tested variants are indicated on the X-axis. The four letters in each set of mutations refer to amino acid substitutions in the Q39XHC1 / Q38XLC1 knob / Q39XHC2 / Q38XLC2 hole, respectively. [Figure 9A] 1 shows the results of a QTOF assay performed to assess heavy chain / light chain pairing in the 4D5 / UCHT1 bispecific antibody. [Figure 9B]FIG. 1 shows the results of a QTOF assay performed to assess heavy chain / light chain pairing in a 4D5 / UCHT1 bispecific antibody in which the 4D5 arm was modified to include a VL with a Q38K mutation and a VH with a Q39E mutation, and the UCHT1 arm was modified to include a VL with a Q38E mutation and a VH with a Q39K mutation ("EKKE"). [Figure 10A] An enlarged view of Figure 10B is shown. [Figure 10B] 1 shows the results of high-resolution mass spectrometry performed to assess heavy chain / light chain pairing in a bispecific 4D5 / UCHT1 antibody in which the UCHT1 arm has been modified to include the VL-Q38E, CL-V133K, VH-Q39K, and CH-1-S183E mutations, and the 4D5 arm of the 4D5 / UCHT1 antibody has been modified to include the VL-Q38K and VH-Q39E mutations. [Figure 10C] An enlarged view of Figure 10D is shown. [Figure 10D] 1 shows the results of high-resolution mass spectrometry performed to assess heavy chain / light chain pairing in a bispecific 4D5 / UCHT1 antibody in which the UCHT1 arm has been modified to contain the CL-V133K and CH-1-S183E mutations. [Figure 10E] 1 shows the results of high-resolution mass spectrometry performed to assess heavy / light chain pairing in the wild-type (WT) bispecific 4D5 / UCHT1 antibody. [Figure 11A] 1 shows the crystal structure superposition of wild-type 4D5 Fab and 4D5 Fab modified to have the VL-Q38K, CL-V133E, VH-Q39E, and CH1-S183K mutations. [Figure 11B] The bonds (eg, salt bridges and hydrogen bonds) formed by VL-Q38K and VH-Q39E of the modified 4D5 Fab are shown. [Figure 11C] The bonds (eg, salt bridges and hydrogen bonds) formed by CL-V133E and CH1-S183K of the modified 4D5 Fab are shown. [Figure 12A]1 shows the crystal structure superposition of wild-type 4D5 Fab and 4D5 Fab modified to have the VL-Q38E, CL-V133K, VH-Q39K, and CH1-S183E mutations. [Figure 12B] The bond formed by modified 4D5 Fab VL-Q38E and VH-Q39K is shown. [Figure 12C] The bond formed by modified 4D5 Fab CL-V133K and CH1-S183E is shown. [Figure 13A] One design approach (i.e., "Approach A") for generating mutant CH1 / CL pairs is illustrated. [Figure 13B] 1 illustrates a second design approach (i.e., "Approach B") for generating variant CH1 / CL pairs of the 4D5 / UCHT1 bispecific antibody (anti-HER2 / anti-CD3). [Figure 14] FIG. 1 shows a schematic diagram of the sandwich ELISA experiment used to determine bispecific IgG content from single cell co-expression. [Figure 15] Shown are the results of sandwich ELISAs performed on the JS20, JS78, YS18, and YT65 variants. [Figure 16A] Figure 16A provides the amino acid sequence of a portion of the heavy chains of JS20 (SEQ ID NO:49), JS78 (SEQ ID NO:50), JT20 (SEQ ID NO:51), JT25 (SEQ ID NO:52), YS08 (SEQ ID NO:28), YS18 (SEQ ID NO:29), YT65 (SEQ ID NO:29), and the YT34 variant (SEQ ID NO:44). Figure 16A also provides the amino acid sequence of a portion of the 4D5 wild-type heavy chain "4D5wt-Hc" (SEQ ID NO:67). [Figure 16B] Figure 16B provides the amino acid sequence of a portion of the light chain of JS20 (SEQ ID NO: 69), JS78 (SEQ ID NO: 70), JT20 (SEQ ID NO: 71), JT25 (SEQ ID NO: 72), YS08 (SEQ ID NO: 73), YS18 (SEQ ID NO: 74), YT65 (SEQ ID NO: 75), and the YT34 variant (SEQ ID NO: 76). Figure 16B also provides the amino acid sequence of a portion of the 4D5 wild-type light chain "4D5wt-Lc" (SEQ ID NO: 68). [Figure 17A]1 shows the results of mass spectrometry performed on wild-type 4D5 / UCHT1 co-expressed bispecific antibodies. [Figure 17B] 1 shows the results of mass spectrometry performed on a 4D5 / UCHT1 co-expressed antibody containing the YT65 CH1 / CL mutation and the VH-Q39E and VL-Q38K mutations on the 4D5 arm, and the VL-Q38E and VH-Q39K mutations on the UCHT1 arm. [Figure 18A] 1 shows the results of mass spectrometry performed on the wild-type 4D5 / UCHT1 bispecific antibody. [Figure 18B] 1 shows the results of mass spectrometry performed on the 4D5 / UCHT1 antibody containing the YT65 CH1 / CL mutation and the VH-Q39E and VL-Q38K mutations on the 4D5 arm, and the VL-Q38E and VH-Q39K mutations on the UCHT1 arm. [Figure 19A] The amino acid sequences of portions of the heavy chains of YT65 (SEQ ID NO:78), YT65.1 (SEQ ID NO:79), YT65.2 (SEQ ID NO:80), YT65.3 (SEQ ID NO:81), YT65.4 (SEQ ID NO:82), YT65.5 (SEQ ID NO:83), YT65.6 (SEQ ID NO:84), YT65.7 (SEQ ID NO:85), YT65.8 (SEQ ID NO:86), YT65.9 (SEQ ID NO:87), YT65.10 (SEQ ID NO:88), YT65.11 (SEQ ID NO:89), YT65.12 (SEQ ID NO:90), and the YT65.13 variant (SEQ ID NO:91) are provided. [Figure 19B] A portion of the 4D5 wild-type heavy chain sequence "4D5wt-Hc" is disclosed as SEQ ID NO: 77. Figure 19B provides the amino acid sequences of portions of the light chains of YT65 (SEQ ID NO: 93), YT65.1 (SEQ ID NO: 94), YT65.2 (SEQ ID NO: 95), YT65.3 (SEQ ID NO: 96), YT65.4 (SEQ ID NO: 97), YT65.5 (SEQ ID NO: 98), YT65.6 (SEQ ID NO: 99), and the YT65.7 variant (SEQ ID NO: 100). Figure 19B discloses a portion of the 4D5 wild-type light chain sequence "4D5wt-Lc" as SEQ ID NO: 92. [Figure 20A]1 shows the results of pH-neutral native mass spectrometry performed on the 4D5 / UCHT1 antibody containing the YT65 CH1 / CL mutation and the VH-Q39E and VL-Q38K mutations on the 4D5 arm, and the VH-Q39K and VL-Q38E mutations on the UCHT1 arm. [Figure 20B] 20A and 20B show the results of experiments performed to confirm the sensitivity of the assay used to generate the data in FIG. 20A. [Figure 21] Quantitative results of high-resolution mass spectrometry experiments performed to assess the effects of YT65, EKKE, and both YT65 and EKKE on heavy chain / light chain pairing in anti-IL4 / IL13, anti-EGFR / MET, anti-VEGFA / ANG2 anti-VEGFA / VEGFC, and anti-HER2 / CD3 (i.e., 4D5 / UCHT1) bispecific antibodies are shown. [Figure 22A] 1 shows the crystal structure of 4D5 Fab modified to have the VL-Q38K mutation, the VH-Q39E mutation, and the YT65 mutation. [Figure 22B] 1 shows a crystal structure superposition of the CH1 domain of wild-type 4D5 Fab and the CH1 domain of 4D5 Fab modified to contain the YT65 mutation. [Figure 23] 1 shows the biological activity of anti-HER2 / CD3+EKKE+V133K / S183E hole, anti-HER2 / CD3+EKKE+YT65 knob, and unmodified anti-HER2 / CD3 in an in vitro T cell-mediated B cell cytotoxicity assay. [Figure 24] Shown are the mean serum levels of (i) anti-HER2, (ii) anti-gD / CD3, (iii) anti-HER2 / CD3, (iv) anti-HER2 / CD3+EKKE+YT65 knob, and (iv) anti-HER2 / CD3+EKKE+V133E / S183K knob+V133K / S183E hole at various time points after intravenous administration of 5 mg / kg of antibody to CB-17 SCID mice. DETAILED DESCRIPTION OF THE INVENTION

[0087] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D Ed., John Wiley and Sons, New York (1994), and Hale & Margham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. Numerical ranges include the numerical boundaries defining the range. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. For definitions and terminology of the art, practitioners are referred to, inter alia, Sambrook et al., 1989, and Ausubel FM et al., 1993. It is understood that the invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0088] Numeric ranges are inclusive of the numerical limits defining the range.

[0089] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0090] The headings provided herein are not limitations of the various aspects or embodiments that can be had by reference to the specification as a whole, and accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0091] definition The term "multispecific antigen-binding protein" as used herein is used in the broadest sense to refer to a binding protein capable of binding to two or more antigens. In certain aspects, a multispecific binding protein refers to a bispecific antibody, such as a human bispecific antibody, a humanized bispecific antibody, a chimeric bispecific antibody, or a murine bispecific antibody. In certain embodiments, the multispecific antigen-binding proteins provided herein, such as bispecific antibodies, bind to two different antigens. In certain embodiments, the multispecific antigen-binding proteins, such as bispecific antibodies, bind to different epitopes on a single antigen.

[0092] The term "antibody" as used herein is used in the broadest sense to refer to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, as well as any fragment, mutant, variant, or derivative thereof, so long as it exhibits the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments described herein. Antibodies can be murine, chimeric, human, humanized, and / or affinity matured.

[0093] As a frame of reference, antibody, as used herein, refers to the structure of immunoglobulin G (IgG). However, those skilled in the art will understand / appreciate that antibodies of any immunoglobulin class can be utilized in the inventive methods described herein. For clarity, an IgG molecule comprises a pair of heavy chains (HC) and a pair of light chains (LC). Each LC has one variable domain (VL) and one constant domain (CL), and each HC has one variable domain (VH) and three constant domains (CH1, CH2, and CH3). The CH1 and CH2 domains are connected by a hinge region. This structure is well known in the art.

[0094] Briefly, the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two light (L) chains and two heavy (H) chains (IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units together with the J chain). In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains (C H ), and four C for μ and ε isotypes H Each L chain has a variable domain (V L ) followed at the other end by a constant domain (C L ) V L is V H Along with C L is the first constant domain of the heavy chain (C H 1). Specific amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. V H and V L and form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0095] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. H Depending on the amino acid sequence of the immunoglobulins, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, γ, ε, and μ, respectively. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0096] The term "VL" domain includes the amino-terminal variable domain of an immunoglobulin light chain.

[0097] The term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain.

[0098] The term "CL domain" includes, for example, the constant region domain of an immunoglobulin light chain stretching from approximately Kabat positions 107A to 216 (EU positions 108 to 214 (kappa)). An Eu / Kabat conversion table for kappa C domains is provided below. The CL domain is adjacent to the VL domain and comprises the carboxy terminus of the immunoglobulin light chain. An exemplary amino acid sequence of the CL domain of a human kappa light chain, including the N-terminal R residue present in the mature CL domain as a result of gene splicing, is shown below in SEQ ID NO: 30. TIFF2025000659000001.tif248170TIFF2025000659000002.tif255170TIFF2025000659000003.tif255170TIFF2025000659000004.tif223170

[0099] As used herein, the term "CH1 domain" of human IgG includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, e.g., extending from about positions 114 to 223 of the Kabat numbering system (EU positions 118-215). The CH1 domain is adjacent to the VH domain, amino-terminal to the hinge region of the immunoglobulin heavy chain molecule, does not form part of the Fc region of the immunoglobulin heavy chain, and can dimerize with an immunoglobulin light chain constant domain (i.e., "CL").

[0100] An EU / Kabat conversion table for the IgG1 heavy chain (SEQ ID NO: 65) is provided below: An exemplary CH1 sequence is set forth in SEQ ID NO: 53, the hinge sequence is set forth in SEQ ID NO: 129, the CH2 sequence is set forth in SEQ ID NO: 130, and the CH3 sequence is set forth in SEQ ID NO: 131. TIFF2025000659000005.tif255170TIFF2025000659000006.tif248170TIFF2025000659000007.tif248170TIFF20250006590 00008.tif243170TIFF2025000659000009.tif255170TIFF2025000659000010.tif255170TIFF2025000659000011.tif161170

[0101] As used herein, the term "complementarity" refers to the combination of interactions that affect heavy chain / light chain pairing, for example, at the interface between the heavy chain CH1 and light chain CL of a multispecific antigen-binding protein described herein. "Steric complementarity" or "conformational complementarity" refers to the compatibility of the three-dimensional structures, for example, at the interaction surface between the heavy chain CH1 domain and the light chain CL domain. "Electrostatic complementarity" refers to the compatibility of the arrangement of negatively and / or positively charged atoms, for example, at the interaction surface between the heavy chain CH1 domain and the light chain CL domain, and / or the heavy chain VH domain and the light chain VL domain.

[0102] The term "CH2 domain" of the human IgG Fc region typically includes residues approximately 231 to approximately 340 of IgG according to the EU numbering system. The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in intact, native IgG molecules. It has been speculated that this carbohydrate may provide an alternative for domain-domain pairing and help stabilize the CH2 domains. Burton, Mol. Immunol. 22:161-206 (1985).

[0103] The term "CH3 domain" includes residues C-terminal to the CH2 domain within the Fc region (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0104] As used herein, the term "Fc region" generally refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence can be obtained by papain digestion of an intact antibody. The Fc region can comprise a native or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence comprises from about Cys226, or from about Pro230, to the carboxyl terminus of the Fc sequence. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises two constant domains, one CH2 domain, one CH3 domain, and optionally a CH4 domain. As used herein, "Fc polypeptide" refers to one of the polypeptides that make up the Fc region, e.g., a monomeric Fc. The Fc polypeptide can be derived from any suitable immunoglobulin, such as human IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. The Fc polypeptide may also be derived from a mouse, e.g., a mouse IgG2a. The Fc region comprises the carboxy-terminal portions of both heavy chains linked together by disulfides. The effector functions of an antibody are determined by sequences within the Fc region, and this region is also the portion recognized by Fc receptors (FcRs) found on certain types of cells. In some embodiments, the Fc polypeptide comprises part or all of a wild-type hinge sequence (generally at its N-terminus). In some embodiments, the Fc polypeptide does not comprise a functional or wild-type hinge sequence.

[0105] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require the association of an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays disclosed in the definitions herein.

[0106] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc regions also include native sequence murine IgG2a.

[0107] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, within the native-sequence Fc region or within the Fc region of the parent polypeptide. A variant Fc region herein preferably retains at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology therewith.

[0108] As used herein, "Fc component" refers to the hinge region, CH2 domain, or CH3 domain of the Fc region.

[0109] In certain embodiments, the Fc region comprises an IgG Fc region, preferably derived from a wild-type human IgG Fc region. In certain embodiments, the Fc region is derived from a "wild-type" mouse IgG, such as mouse IgG2a. "Wild-type" human IgG Fc or "wild-type" mouse IgG Fc refers to a sequence of amino acids that naturally occur in the human or mouse population, respectively. Of course, just as Fc sequences can vary slightly between individuals, one or more alterations to the wild-type sequence can still remain within the scope of the present invention. For example, the Fc region may include alterations such as mutations of glycosylation sites or the inclusion of unnatural amino acids.

[0110] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 61st ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0111] As used herein, the expressions "antigen-binding arm," "target molecule-binding arm," "target-binding arm," and variations thereof refer to a component of a multispecific antigen-binding protein provided herein that has the ability to specifically bind to a target of interest. Typically and preferably, an antigen-binding arm is a complex of immunoglobulin polypeptide sequences, e.g., immunoglobulin light and heavy chain CDR and / or variable domain sequences.

[0112] "Target" or "target molecule" refers to the moiety recognized by the binding arms of a multispecific antigen-binding protein. For example, if the multispecific antigen-binding protein is an antibody, the target may be an epitope on a single molecule or on different molecules, or a pathogen or tumor cell, depending on the context. Similarly, if the multispecific antigen-binding protein is a receptor-Fc fusion protein, the target is the cognate binding partner for that receptor. Those skilled in the art will understand that the target is determined by the binding specificity of the target-binding arm, and that different target-binding arms may recognize different targets. Preferably, the target binds to the multispecific antigen-binding proteins provided herein with an affinity greater than 1 μM Kd (by Scatchard analysis). Examples of target molecules include, but are not limited to, serum-soluble proteins and / or their receptors, such as cytokines and / or cytokine receptors, adhesins, growth factors and / or their receptors, hormones, viral particles (e.g., RSV F protein, CMV, Staph A, influenza, Hepatitis C virus), microorganisms (e.g., bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.

[0113] As used herein, the term "interface" refers to the association surface resulting from the interaction of one or more amino acids in a first antibody domain with one or more amino acids in a second antibody domain. Exemplary interfaces include, for example, CH1 / CL, VH / VL, and CH3 / CH3. In some embodiments, the interface comprises, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids that form the interface.

[0114] An example of an "intact" or "full length" antibody is one which comprises an antigen-binding arm, as well as a CL and at least heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0115] As used herein, the term "coupling" refers to the steps necessary to link the first and second heavy chain polypeptides (i.e., H1 and H2) to one another, e.g., the formation of a covalent bond. Such steps include reduction, annealing, and / or oxidation of cysteine ​​residues in the first and second heavy chain polypeptides (i.e., H1 and H2) to form interchain disulfide bonds. Coupling can be achieved by chemical crosslinking or the use of redox systems. See, e.g., Humphreys et al., J. Immunol. Methods (1998) 217:1-10, and Zhu et al., Cancer Lett., (1994) 86:127-134.

[0116] A "monospecific" antigen-binding protein refers to the ability of an antigen-binding protein, such as an antibody, to bind to only one epitope. A "bispecific" antigen-binding protein refers to the ability of an antigen-binding protein to bind to two different epitopes. A "multispecific" antigen-binding protein refers to the ability of an antigen-binding protein to bind to more than one epitope. In certain embodiments, multispecific antigen-binding proteins, such as multispecific antibodies, encompass bispecific antigen-binding proteins or bispecific antibodies. For bispecific and multispecific antigen-binding proteins provided herein, the epitopes may be present on the same antigen, or each epitope may be present on a different antigen. Thus, in certain embodiments, multispecific antigen-binding proteins provided herein, such as bispecific antibodies, bind to two different antigens. In certain embodiments, multispecific antigen-binding proteins, such as bispecific antibodies, bind to different epitopes on a single antigen. In certain embodiments, the multispecific antigen-binding proteins provided herein have an antibody titer of about ≦1 μM, about ≦100 nM, about ≦10 nM, about ≦1 nM, about ≦0.1 nM, about ≦0.01 nM, or about ≦0.001 nM (e.g., about 10 -8 M or less, e.g., about 10 -8 M ~ about 10 -13 M, for example, about 10 -9 M ~ about 10 -13 It binds to each epitope with a dissociation constant (Kd) of 1 M.

[0117] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies (Db); tandem diabodies (taDb); linear antibodies (e.g., U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, and (scFV)4-Fc, but are not limited to these).

[0118] The antibodies provided herein can be "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0119] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Most frequently, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0120] As used herein, "complex" or "complexed" refers to an association of two or more molecules that interact with each other through bonds and / or forces other than peptide bonds (e.g., van der Waals, hydrophobic, hydrophilic forces). In one embodiment, the complex is a heteromultimer. It should be understood that the term "protein complex" or "polypeptide complex" as used herein includes complexes in which a non-protein element (e.g., including, but not limited to, a chemical molecule such as a toxin or a detection agent) is conjugated to a protein in the protein complex.

[0121] As provided herein, a multispecific antigen-binding protein that "binds to an antigen of interest" is one that binds to an antigen, e.g., a protein, with sufficient affinity so that the multispecific antigen-binding protein is useful as a diagnostic and / or therapeutic agent in targeting the protein or cells or tissues expressing that protein, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antigen-binding protein to a "non-target" protein will be less than about 10% of the binding of an antibody to its particular target protein, as determined by fluorescence-activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), or ELISA. With respect to the binding of a multispecific antigen-binding protein to a target molecule, the terms "specific binding" of, or "specifically binding to," or "specific for," a particular polypeptide or epitope on a particular polypeptide target refers to binding that is measurably different from non-specific interactions (e.g., non-specific interactions can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to target, for example, excess unlabeled target.In this case, specific binding is shown when the binding of labeled target to probe is competitively inhibited by excess unlabeled target.As used herein, the term " specific binding " of a specific polypeptide or epitope on a specific polypeptide target, or " specifically binds " to it, or " specific " to it, can be shown by the molecule having Kd for target with higher affinity, for example, at least about 200nM, alternatively at least about 150nM, alternatively at least about 100nM, alternatively at least about 60nM, alternatively at least about 50nM, alternatively at least about 40nM, alternatively at least about 30nM, alternatively at least about 20nM, alternatively at least about 10nM, alternatively at least about 8nM, alternatively at least about 6nM, alternatively at least about 4nM, alternatively at least about 2nM, alternatively at least about 1nM, or alternatively at least about 1nM.In one embodiment, the term "specific binding" refers to the multispecific antigen-binding protein binding to a particular polypeptide or epitope on a particular polypeptide and not substantially binding to any other polypeptide or epitope of a polypeptide.

[0122] "Binding affinity" generally refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody or multispecific antigen-binding protein) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). For example, Kd can be about 200 nM or less, about 150 nM or less, about 100 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, or about 1 nM or less. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used in the present invention.

[0123] In one embodiment, "Kd" or "Kd value" is measured at -10 response units (RU) using a surface plasmon resonance assay with an immobilized target (e.g., antigen) CM5 chip at 25°C using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextrin biosensor chip (CM5, BIAcore Inc.) is run with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of the coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on-rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1If greater than , this on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured with a spectrometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0124] "Biologically active" and "biological activity" and "biological characteristics" with respect to multispecific antigen-binding proteins provided herein, such as antibodies (e.g., bispecific antibodies), fragments, or derivatives thereof, mean, unless otherwise specified, having the ability to bind to a biological molecule.

[0125] "Isolated," when used to describe various heteromultimeric polypeptides, means that the heteromultimer has been separated and / or recovered from the cell or cell culture in which it was expressed. Contaminating components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the heteromultimer, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the heteromultimer is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the protein as determined by the Lowry method; (2) to an extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, isolated polypeptides are prepared by at least one purification step.

[0126] The multispecific antigen-binding proteins provided herein are generally purified to substantial homogeneity. The terms "substantially homogeneous," "substantially homogeneous form," and "substantial homogeneity" are used to indicate a product that is substantially devoid of by-products resulting from undesired polypeptide combinations (e.g., homomultimers).

[0127] Substantial homogeneity, expressed in terms of purity, means that the amount of by-products does not exceed or is less than 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2%, or 1% by weight, hi one embodiment, the amount of by-products is less than 5%.

[0128] "Biomolecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, a biomolecule is naturally occurring.

[0129] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (a native-sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0130] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxic agents. Antibodies "arm" the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998).

[0131] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR. Further, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, for review, M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. This term also includes FcRn, the neonatal receptor involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0132] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood.

[0133] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) can be performed.

[0134] The term "therapeutically effective amount" refers to an amount of an antibody (including a multispecific antibody), its antigen-binding antibody fragment, or derivative thereof to treat a disease or disorder in a subject. In the case of tumors (e.g., cancerous tumors), a therapeutically effective amount of an antibody or antibody fragment (e.g., a multispecific antibody or antibody fragment) can reduce the number of cancer cells, reduce primary tumor size, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion to peripheral organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent the antibody or antibody fragment or derivative thereof can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. With respect to cancer therapy, in vivo efficacy can be measured, for example, by assessing survival time, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0135] Here, "reduce or inhibit" refers to the ability to cause an overall decrease, preferably of 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95% or more. Reduction or inhibition may refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in an angiogenic disorder.

[0136] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. This definition includes both benign and malignant cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal cell carcinoma), liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various types of head and neck cancer. As used herein, "early cancer" refers to a cancer that is not invasive or metastatic, or that is classified as a stage 0, stage I, or stage II cancer. The term "precancerous" refers to a condition or growth that typically precedes or develops into a cancer. As used herein, "non-metastatic" refers to a cancer that is benign or that remains at the primary site and has not penetrated the lymphatic or vascular system or tissues outside the primary site. Generally, a non-metastatic cancer is any cancer that is a stage 0, stage I, or stage II cancer, and sometimes a stage III cancer.

[0137] As used herein, an "autoimmune disease" refers to a disease or disorder arising from and directed against an individual's own tissues, or a co-segregation or manifestation thereof, or a condition resulting therefrom. Examples of autoimmune diseases or disorders include, but are not limited to, rheumatoid arthritis, autoimmune hemolytic anemia (e.g., immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (e.g., Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus type 1 or insulin-dependent diabetes mellitus, demyelinating diseases of the central and peripheral nervous system (e.g., multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain-Barré syndrome), chronic inflammatory demyelinating polyneuropathy, bullous dermatosis, erythema multiforme, contact dermatitis, and autoimmune chronic active hepatitis.

[0138] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , Ra 223 , P 32 , and radioactive isotopes of Lu), chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes such as nucleases and fragments thereof, antibiotics, and toxins (including fragments and / or variants thereof) such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, as well as the various anti-tumor, anti-cancer, and chemotherapeutic agents disclosed herein. Other cytotoxic agents are described herein. Tumoricidal agents cause the destruction of tumor cells.

[0139] As used herein, "pharmaceutically acceptable" or "pharmaceutically compatible" means a material that is not biologically or otherwise undesirable; for example, the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Preferably, a pharmaceutically acceptable carrier or excipient will have met required standards of toxicological and manufacturing testing and / or will be included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0140] As used herein, "anti-cancer therapy" refers to a treatment that reduces or inhibits cancer in a subject. Examples of anti-cancer therapy include cytotoxic radiation therapy, and administering to a subject a therapeutically effective amount of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, a corticosteroid, an immunosuppressant, an antiemetic, an antibody or antibody fragment, or an analgesic.

[0141] The term "prodrug," as used herein, refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986), and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use herein include, but are not limited to, the chemotherapeutic agents described above.

[0142] A "subject" is a vertebrate, such as a mammal, e.g., a human. Mammals include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats.

[0143] Unless the context dictates otherwise, the terms "first" polypeptide (e.g., heavy chain (H1) or light chain (L1)), and "second" polypeptide (e.g., heavy chain (H2) or light chain (L2)), and variations thereof, are merely generic identifiers and are not to be construed as identifying particular or specific polypeptides or components of the multispecific antigen binding proteins provided herein.

[0144] Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise noted. The source of cells identified by ATCC accession numbers in the following examples and throughout the specification is the American Type Culture Collection, Manassas, VA. Unless otherwise noted, the present invention employs standard procedures of recombinant DNA technology, such as those described herein and in the following textbooks: Sambrook et al. (supra), Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989), Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc., NY, 1990), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988), Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984), Freshney, Animal Cell Culture, 1987, and Coligan et al., Current Protocols in Immunology, 1991.

[0145] Reference herein to "about" a value or parameter refers to the normal error range of the respective value, which is readily apparent to those skilled in the art. Reference herein to "about" a value or parameter includes (and represents) aspects related to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0146] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0147] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0148] Multispecific antigen-binding proteins comprising mutant or modified heavy / light chain pairs The present application is based on the identification of novel mutations at the CH1 / CL interface and / or VH / VL interface that improve heavy / light chain pairing selectivity.

[0149] The multispecific antigen-binding proteins provided herein comprise amino acid modifications at specific residues within the variable and / or constant domains of the heavy and light chain polypeptides. As will be understood by those skilled in the art, various numbering conventions can be used to designate specific amino acid residues within an IgG variable region sequence. Commonly used numbering conventions include the Kabat and EU index numbering (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Other conventions involving correct or alternative numbering systems for variable domains include Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196:901-917; Chothia, et al. (1989), Nature 342:877-883), IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27:55-77), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309:657-670). These references provide immunoglobulin variable region amino acid sequence numbering schemes that define the positions of variable region amino acid residues in the antibody sequence.

[0150] Unless otherwise expressly stated herein, all references to immunoglobulin heavy chain variable region (i.e., VH) amino acid residues (i.e., numbers) in the examples and claims are based on the Kabat numbering system, as are all references to VL residues. All references to immunoglobulin heavy chain constant region CH1 residues (i.e., numbers) in the examples and claims are based on the EU system, as are all references to CL residues. With knowledge of the residue numbers according to Kabat or EU index numbering, one of skill in the art can identify amino acid sequence modifications described herein according to any commonly used numbering convention.

[0151] Although the Examples and claims herein identify particular amino acid residues using the Kabat or EU index, it is understood that the SEQ ID NOs set forth in the Sequence Listing, which is incorporated herein in its entirety, provide consecutive numbering of amino acids within a given polypeptide and therefore do not match the corresponding amino acid numbering provided by the Kabat or EU index. For example, serine residue number 66 in SEQ ID NO: 53 corresponds to S183 of CH1 in the EU numbering system.

[0152] Although items, components, or elements provided herein (e.g., "multispecific antigen binding protein") may be described or claimed in the singular, the plural is also contemplated within its scope unless limitation to the singular is expressly stated.

[0153] As described in more detail below, provided herein are modified multispecific antigen-binding proteins that can be produced in a single cell, with improved and correct heavy chain / light chain pairing and / or improved yield when compared to a multispecific antigen-binding protein having a sequence without the modifications. In certain embodiments, the multispecific antigen-binding protein comprises modification(s) in the VH / VL and / or CH1 / CL regions that promote correct heavy chain / light chain pairing (i.e., the pairing of a first heavy chain H1 (or a fragment thereof) with a first light chain L1 to form a first heavy chain / light chain pair (i.e., H1 / L1) that can bind to a first epitope, and the pairing of a second heavy chain H2 (or a fragment thereof) with a second light chain L2 to form a second heavy chain / light chain pair (i.e., H2 / L2) that can bind to a second (e.g., different) epitope). It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be reversed. That is, any of the mutations described above as being in the CH1 domain of H1 and the CL domain of L1 can alternatively be in the CH1 domain of H2 and the CL domain of L2.

[0154] In certain other embodiments, the multispecific antigen binding protein further comprises modification(s) in the Fc region that promote heterodimerization of the two arms of the multispecific antigen binding protein.

[0155] Strategy Number 1 In one aspect, it was surprisingly found that a single amino acid modification at position S183 (EU numbering) in the CH1 domain of the heavy chain and a single amino acid modification at position V133 (EU numbering) in the CL domain of the light chain exhibit preferential pairing between the modified heavy chain and the modified light chain, and reduced pairing between, for example, the modified heavy chain and the unmodified light chain, or between, for example, the unmodified heavy chain and the modified light chain. These residues were selected from expression data and confirmed by knowledge and study of the structure and function of the heavy chain CH1 domain and the light chain CL domain. See Figures 1-3, 11C, and 12C.

[0156] Thus, in certain embodiments, there is provided a multispecific antigen binding protein comprising: (a) a first heavy chain / light chain pair capable of binding to a first antigen, the first heavy chain polypeptide (H1) and the first light chain polypeptide (L1); and (b) a second heavy chain / light chain pair capable of binding to a second antigen, the second heavy chain polypeptide (H2) and the second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises an amino acid substitution at position S183 (EU numbering), and the CL domain comprises an amino acid substitution mutation at position V133 (EU numbering). In certain embodiments, the CH1 domain of H1 consists (e.g., consists essentially of) an amino acid substitution at position S183 (EU numbering), and the CL domain consists (e.g., consists essentially of) an amino acid substitution mutation at position V133 (EU numbering). In certain embodiments, the first antigen and the second antigen are the same. In certain embodiments, the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to different epitopes on the same antigen. In certain embodiments, the first antigen and the second antigen are different. In certain embodiments, the multispecific antigen-binding protein is a bispecific antibody that binds to two different antigens. In certain embodiments, the bispecific antibody is an antagonist or agonist antibody. In certain embodiments, the bispecific antibody is an antagonist for one or both antigens, while in other embodiments, the bispecific antibody is an agonist for one or both antigens. Antigen-binding fragments of the multispecific antigen-binding proteins (such as bispecific antibodies) provided herein are also contemplated.

[0157] In certain embodiments, the amino acid at position S183 (EU numbering) on ​​the CH1 domain of H1 of the multispecific antigen-binding protein is replaced with a positively charged amino acid residue and the amino acid at position V133 (EU numbering) on ​​the CL domain of the multispecific antigen-binding protein is replaced with a negatively charged residue. In certain embodiments, the amino acid at position S183 (EU numbering) on ​​the CH1 domain of H1 is replaced with a negatively charged amino acid residue and the amino acid at position V133 (EU numbering) on ​​the CL domain is replaced with a positively charged residue. In certain embodiments, the positively charged residue is selected from the group consisting of R or K. In certain embodiments, the negatively charged residue is selected from the group consisting of D and E.

[0158] In certain embodiments, the CH1 domain of the multispecific antigen binding protein comprises an amino acid substitution selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K (EU numbering), and the CL domain comprises an amino acid substitution selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D (EU numbering).

[0159] In certain embodiments, the CH1 domain of the multispecific antigen binding protein consists of (e.g., consists essentially of) an amino acid substitution selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K (EU numbering) and the CL domain consists of (e.g., consists essentially of) an amino acid substitution selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D (EU numbering).

[0160] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be reversed. That is, any of the mutations described above as being in the CH1 domain of H1 and the CL domain of L1 can alternatively be in the CH1 domain of H2 and the CL domain of L2.

[0161] Thus, all possible pairwise combinations of a substitution mutation at position S183 (EU numbering) on ​​the CH1 domain of H1 and a substitution mutation at position V133 (EU numbering) on ​​the CL domain of L1 are contemplated. In certain embodiments, specific combinations of a substitution mutation at position S183 (EU numbering) on ​​the CH1 domain of H1 and a substitution mutation at position V133 (EU numbering) on ​​the CL domain of L1 are contemplated. Such combinations include, but are not limited to, those provided in Table 1 below. TIFF2025000659000012.tif131168

[0162] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises an S183D or S183E mutation (EU numbering) and a CL domain of L1 that comprises a V133K mutation (EU numbering). In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 and a CL domain of L1, wherein the CH1 domain comprises an S183D or S183E mutation (EU numbering) and the CL domain of L1 comprises a V133K mutation (EU numbering).

[0163] Provided below are two exemplary IgG1 CH1 domain amino acid sequences (SEQ ID NO:53 and SEQ ID NO:109), an exemplary IgG2 CH1 domain amino acid sequence (SEQ ID NO:109), an exemplary IgG2 CH1 domain amino acid sequence (SEQ ID NO:110), an exemplary IgG3 CH1 domain amino acid sequence (SEQ ID NO:111), an exemplary IgG4 CH1 domain amino acid sequence (SEQ ID NO:112), an exemplary lambda CL domain amino acid sequence (SEQ ID NO:113), and an exemplary kappa CL domain amino acid sequence (SEQ ID NO:54). TIFF2025000659000013.tif99170

[0164] The amino acid sequences of the full-length 4D5 IgG1 heavy chain (SEQ ID NO: 55) and light chain (SEQ ID NO: 56) are provided below. 4D5 HC (human IgG1): TIFF2025000659000014.tif411704D5 LC(Human Kappa): TIFF2025000659000015.tif24170

[0165] In certain embodiments, the multispecific antigen-binding protein comprises a kappa light chain. In certain embodiments, the multispecific antigen-binding protein comprises the amino acid sequence set forth in SEQ ID NO:1, or the CL domain of L1 comprising amino acids 11-116 of the sequence set forth in SEQ ID NO:1. In certain embodiments, the multispecific antigen-binding protein comprises the amino acid sequence set forth in SEQ ID NO:2, or the CL domain of L1 comprising amino acids 11-116 of the sequence set forth in SEQ ID NO:2. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO:3, or the CL domain of L1 comprising amino acids 11-116 of the sequence set forth in SEQ ID NO:3. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO:4, or the CL domain of L1 comprising amino acids 11-116 of the sequence set forth in SEQ ID NO:4. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO:5, or the CL domain of L1 comprising amino acids 11-116 of the sequence set forth in SEQ ID NO:5. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 6, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 6. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 7, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 7.

[0166] The amino acid sequences of SEQ ID NOs: 1-7 are provided below in Table 2A. In certain embodiments, the multispecific antigen-binding protein comprises a first light chain polypeptide, L1, comprising a CL domain set forth in any of these sequences. The CL domain set forth in SEQ ID NO: 54 corresponds to amino acids 11-116 of SEQ ID NOs: 1-7. Thus, L1 may comprise a CL domain comprising amino acids 11-116 set forth in any of SEQ ID NOs: 1-7. TIFF2025000659000016.tif133170

[0167] Amino acid position 133 (EU numbering) within the CL domain of the kappa light chain corresponds to amino acid position 35 of SEQ ID NOs: 1-7.

[0168] In certain embodiments, the multispecific antigen-binding protein comprises a lambda light chain. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 114. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 115. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 116. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 117. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 118. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 119. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 120.

[0169] The amino acid sequences of SEQ ID NOs: 114-120 are provided below in Table 2B. TIFF2025000659000017.tif104170

[0170] Amino acid position 133 (EU numbering) in the CL domain of the kappa light chain corresponds to amino acid position 133 (Kabat numbering) in the CL domain of the lambda light chain, which corresponds to amino acid position 27 of SEQ ID NOs: 114-120.

[0171] In certain embodiments, the multispecific antigen-binding protein comprises an IgG1 heavy chain, an IgG2 heavy chain, an IgG3 heavy chain, or an IgG4 heavy chain. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 101. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 102. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 121. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 122. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 123. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 124.In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 125. In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 126.

[0172] The amino acid sequences of SEQ ID NOs: 8-16, 101-102, and 121-126 are provided in Table 3 below. TIFF2025000659000018.tif255170TIFF2025000659000019.tif19170

[0173] Amino acid position 183 in the CH1 domain of IgG1, IgG2, IgG3, and IgG4 corresponds to amino acid position 64 in SEQ ID NOs: 8-16, 101-102, and 121-126.

[0174] All possible pairwise combinations of amino acids 11 to 116 of SEQ ID NOs: 1 to 7 or SEQ ID NOs: 1 to 7 and 114 to 120 with SEQ ID NOs: 8 to 15, 101 to 102, and 121 to 126 are contemplated. In certain embodiments, specific combinations are contemplated of amino acids 11 to 116 of SEQ ID NOs: 1 to 7 or SEQ ID NOs: 1 to 7 and 114 to 120 with SEQ ID NOs: 8 to 15 and 101 and 102. Such combinations include, but are not limited to, those provided in Table 4 below (unless otherwise specified, HC is IgG1 HC). TIFF2025000659000020.tif251170TIFF2025000659000021.tif251170

[0175] The first SEQ ID NO in each pair in Table 4 refers to the CH1 domain sequence, and the second SEQ ID NO in each pair in Table 4 refers to the CL domain sequence.

[0176] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be reversed. That is, any of the mutations described above as being in the CH1 domain of H1 and the CL domain of L1 can alternatively be in the CH1 domain of H2 and the CL domain of L2.

[0177] In certain embodiments, the multispecific antigen binding protein comprises CH1 / CL mutations and exhibits correct protein folding and / or expression levels that are comparable to or superior to the multispecific antigen binding protein without the mutations or to each parent monospecific antigen binding protein without the mutations.

[0178] In certain embodiments, the CH1 domain of H2 and the CL domain of L2 of the multispecific antigen binding protein do not comprise an amino acid substitution, hi certain embodiments, the CH1 of H2 of the multispecific antigen binding protein does not comprise a substitution at S183 (EU numbering) and the CL of L2 of the multispecific antigen binding protein does not comprise a substitution at V133 (EU numbering).

[0179] In certain embodiments, the parental H1 from which the H1 of a multispecific antigen-binding protein provided herein is derived does not show a significant preference over the parental L1 from which the L1 of a multispecific antigen-binding protein provided herein is derived. In certain embodiments, the parental H1 from which the H1 of a multispecific antigen-binding protein provided herein is derived shows a preference over the parental L1 from which the L1 of a multispecific antigen-binding protein provided herein is derived.

[0180] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be reversed. That is, any of the mutations described above as being in the CH1 domain of H1 and the CL domain of L1 can alternatively be in the CH1 domain of H2 and the CL domain of L2.

[0181] Strategy Number 2 Using a second strategy aided by computer-guided design and human-guided design, the applicants redesigned the interaction surfaces of the heavy chain CH1 domain and the light chain CL domain to generate CH1 / CL mutant pairs that are sterically compatible (e.g., conformationally compatible) with each other. The modified CH1 domain in each CH1 / CL mutant pair is less sterically compatible (e.g., conformationally compatible) with wild-type CL and exhibits reduced pairing with wild-type CL. Correspondingly, the modified CL domain in each CH1 / CL mutant pair is less sterically compatible (e.g., conformationally compatible) with wild-type CH1 and exhibits reduced pairing with wild-type CH1. Surprisingly, it was discovered that the Tm of Fabs containing modifications at the interface between the CH1 and CL domains was the same or substantially the same as the Tm of Fabs lacking the corresponding mutations in the CH1 / CL domains.

[0182] Mutations identified using the design strategies described below may be used independently of, or in addition to, the S183 / V133 mutations detailed above (i.e., strategy no. 1).

[0183] In the first approach (i.e., "Approach A"), one or more amino acid substitution mutations were introduced into the CL domain of the light chain to create a "knob" (or protuberance) on the surface of the CL domain that interacts with the CH1 domain. Correspondingly, one or more amino acid substitution mutations were introduced into the CH1 domain of the heavy chain to create a "hole" (or cavity) on the surface of the CH1 domain that interacts with the CL domain. See, for example, Figure 11A. In certain embodiments, multispecific antigen binding proteins comprising modified CH1 / CL sequences result in preferential pairing of heavy and light chains that have amino acid substitution mutations in the CH1 and CL domains. In certain embodiments, multispecific antigen binding proteins comprising modified CH1 / CL sequences exhibit increased protein expression levels compared to multispecific antigen binding proteins that do not have the corresponding CH1 / CL mutations. In certain embodiments, the substituted amino acid(s) are not replaced with charged residue(s).

[0184] In the second approach (i.e., "Approach B"), two or more amino acid substitution mutations were introduced into the CL domain of the light chain to create a "knob" (or protuberance) and a "hole" (or cavity) on the surface of the CL domain that interacts with the CH1 domain. Correspondingly, two or more mutations were introduced into the CH1 domain of the heavy chain to create a "hole" and a "knob" on the surface of the CH1 domain that interacts with the CL domain. See, for example, Figure 11B. In certain embodiments, multispecific antigen-binding proteins comprising modified CH1 / CL sequences exhibit preferential pairing of heavy and light chains having amino acid substitution mutations in the CH1 and CL domains. In certain embodiments, multispecific antigen-binding proteins comprising modified CH1 / CL sequences exhibit increased protein expression compared to multispecific antigen-binding proteins without the corresponding CH1 / CL mutations. In certain embodiments, the substituted amino acid(s) are not replaced by charged residue(s).

[0185] Multispecific antigen-binding proteins comprising a modified CH1 domain of H1 and a modified CL domain of L1 exhibit improved stability and improved specificity with respect to heavy chain / light chain pairing. Provided herein are multispecific antigen-binding proteins comprising: a) a first heavy chain / light chain pair capable of binding to a first antigen, the first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and b) a second heavy chain / light chain pair capable of binding to a second antigen, the second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of H1 comprises an amino acid substitution at position F170 (EU numbering) and the CL domain comprises an amino acid substitution at position S176 (EU numbering). In certain embodiments, the first antigen and the second antigen are the same. In certain embodiments, the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to different epitopes on the same antigen. In certain embodiments, the first antigen and the second antigen are different. In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of A141, S181, S183, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of F116, S131, V133, L135, S162, S174, and T178 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of F116, V133, L135, S162, S174, and T178 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of F116, S131, L135, S162, S174, and T178 (all EU numbering).In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S174, and T178 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S162, S174, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity. In certain embodiments, the substituted amino acid(s) are not replaced with charged residue(s).

[0186] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141 and F170 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0187] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of F170, S181, and S183 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116 and S176 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0188] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) the amino acid substitution F170 (EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S174, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0189] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of F170, S183, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0190] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of F170, S181, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, and S176 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0191] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141 and F170 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S174, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0192] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141, F170, and S181 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S174, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0193] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of F170, S181, S183, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of L135, S174, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0194] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141, F170, S183, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, S174, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0195] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141, F170, S181, and V185 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0196] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141, F170, S181, and S183 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S174, and S176 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0197] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) the amino acid substitution F170 (EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116, L135, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity. In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0198] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141 F170, and V185 (EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116 and S176 mutations (all EU numbering). In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0199] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of A141, F170, and S183 (EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116 and S176 mutations (all EU numbering). In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0200] In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein comprises (e.g., consists of, or consists essentially of) one or more amino acid substitutions selected from the group consisting of F170 and V185 (EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein comprises one or more amino acid substitutions selected from the group consisting of F116 and S176 mutations (all EU numbering). In certain embodiments, the amino acid substitutions result in conformational complementarity.

[0201] In certain embodiments, the CH1 domain of H1 and the CL domain of L1 of a multispecific antigen-binding protein are altered such that, within the CH1 / CL interface, one or more amino acid residues in the CH1 domain are replaced with an equivalent number of amino acid residues, some or all of which have a larger side chain volume, thereby creating a protuberance on the surface of the CH1 domain, and one or more amino acid residues in the CL domain are replaced with an equivalent number of amino acid residues, some or all of which have a smaller side chain volume, thereby creating a cavity on the surface of the CL domain. In certain embodiments, the modifications on the CH1 domain and the CL domain provide steric complementarity at the CH1 / CL interface. In certain embodiments, the modifications on the CH1 domain and the CL domain provide steric complementarity at the CH1 / CL interface.

[0202] In certain embodiments, the CH1 domain of H1 and the CL domain of L1 of a multispecific antigen-binding protein are altered such that, within the CH1 / Cl interface, one or more amino acid residues in the CL domain are replaced, some or all, with an equivalent number of amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the CL domain, and one or more amino acid residues in the CH1 domain are replaced, some or all, with an equivalent number of amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the CH1 domain. In certain embodiments, the one or more substituted amino acid residues in the CL domain comprise S176. In certain embodiments, the one or more substituted amino acid residues in the CH1 domain comprise F170. In certain embodiments, the modifications on the CH1 domain and the CL domain provide steric complementarity at the interface. In certain embodiments, the modifications on the CH1 domain and the CL domain provide steric complementarity at the interface.

[0203] In certain embodiments, the CH1 domain of H1 comprises an amino acid substitution selected from the group consisting of F170S and F170A (EU numbering), and the CL domain of L1 comprises an amino acid substitution selected from the group consisting of S176F (EU numbering).

[0204] In certain embodiments, the CH1 domain of H1 comprises a F170S mutation (EU numbering) and the CL domain of L1 comprises a S176F mutation (EU numbering). In certain embodiments, the CH1 domain of H1 comprises a F170A mutation (EU numbering) and the CL domain of L1 comprises a S176F mutation (EU numbering).

[0205] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S181M, S183V, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, V133I, L135V, S162M, S174A, S176F, and T178V mutations (EU numbering).

[0206] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, S131D, L135V, S162A, S174A, S176F, and T178I mutations (EU numbering).

[0207] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering). In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 consisting of (e.g. consisting essentially of) A141I, F170S, S181M, S183A, and V185A mutations (EU numbering), and a CL domain of L1 consisting of (e.g. consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering).

[0208] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170A, S181M, S183V, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, L135V, S162M, S174A, S176F, and T178V mutations (EU numbering).

[0209] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) F170S, S181M, S183A, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) L135V, S174A, S176F, and T178V mutations (EU numbering).

[0210] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S183A, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, S174A, S176F, and T178V mutations (EU numbering).

[0211] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S181M, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, L135V, S176F, and T178V mutations (EU numbering).

[0212] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, S181M, and S183A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, L135V, S174A, and S176F mutations (EU numbering).

[0213] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) F170S, S183A, and V185A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, S176F, and T178V mutations (EU numbering).

[0214] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) F170S, S181M, and V185A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A, L135V, and S176F mutations (EU numbering).

[0215] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) F170S, S181M, and S183A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A and S176F mutations (EU numbering).

[0216] In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising (including consisting of, including consisting essentially of) A141I, F170S, and V185A mutations (EU numbering) and an L1 CL domain comprising (including consisting of, including consisting essentially of) F116A and S176F mutations.

[0217] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, and S183A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F116A and S176F mutations.

[0218] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I, F170S, and S181M mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In a specific embodiment, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0219] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) F170S and V185A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A and S176F mutations.

[0220] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In a specific embodiment, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0221] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, S176F, and T178V mutations (EU numbering).

[0222] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, S174A, and S176F mutations (EU numbering).

[0223] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, S176F, and T178V mutations (EU numbering).

[0224] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, and S176F mutations (EU numbering).

[0225] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) A141I and F170S mutations (EU numbering) and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A and S176F mutations (EU numbering).

[0226] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) an F170S mutation (EU numbering), and a CL domain of L1 comprising (including consisting of, or consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In a specific embodiment, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0227] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) the F170S mutation (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) the F116A, L135V, S176F, and T178V mutations (EU numbering).

[0228] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) the F170S mutation (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) the F116A, L135V, S174A, and S176F mutations (EU numbering).

[0229] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) the F170S mutation (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) the F116A, S176F, and T178V mutations (EU numbering).

[0230] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) the F170S mutation (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) the F116A, L135V, and S176F mutations (EU numbering).

[0231] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) the F170S mutation (EU numbering) and a CL domain of L1 comprising (including consisting of, and consisting essentially of) the F116A and S176F mutations (EU numbering).

[0232] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of A141I, F170S, S181M, S183V, and V185A mutations (EU numbering).

[0233] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of A141I, F170S, S181M, S183A, and V185A mutations (EU numbering).

[0234] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 consisting of no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of A141I, F170S, S181M, S183A, and V185A mutations (EU numbering).

[0235] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of A141I, F170A, S181M, S183V, and V185A mutations (EU numbering).

[0236] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, or no more than 7 amino acid mutations selected from the group consisting of F116A, V133I, L135V, S162M, S174A, S176F, and T178V mutations (EU numbering).

[0237] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, or no more than 7 amino acid mutations selected from the group consisting of F116A, S131D, L135V, S162A, S174A, S176F, and T178I mutations (EU numbering).

[0238] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of F116A, L135V, S174A, S176F, and T178V mutations (EU numbering).

[0239] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 consisting of no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of F116A, L135V, S174A, S176F, and T178V mutations (EU numbering).

[0240] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 amino acid mutations selected from the group consisting of F116A, L135V, S162M, S174A, S176F, and T178V mutations (EU numbering).

[0241] All possible combinations of pairs of CH1 and L1 domains mentioned above are contemplated.

[0242] In certain embodiments, the multispecific antigen-binding protein comprises a kappa light chain. In certain embodiments, the multispecific antigen-binding protein comprises a first light chain polypeptide, L1, comprising a CL domain set forth in any of SEQ ID NOs: 17 to 27. The CL domain set forth in SEQ ID NO: 54 corresponds to amino acids 11 to 116 of SEQ ID NOs: 17 to 27. Thus, L1 may comprise a CL domain comprising amino acids 11 to 116 of any of SEQ ID NOs: 17 to 27. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 17, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 17. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 18, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 18. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 19, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 19. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 20. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 21, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 21. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 22, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 22. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 23, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 23. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 24, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 24.In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 25, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 25. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 26, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 26. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 27, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 27.

[0243] In certain embodiments, the multispecific antigen binding protein comprises a lambda light chain. In certain embodiments, the multispecific antigen binding protein comprises the CL domain of L1, which comprises the amino acid sequence set forth in SEQ ID NO:127.

[0244] The amino acid sequences of SEQ ID NOs: 17-27 and 127 are provided in Table 5 below (LC is kappa LC unless otherwise stated). TIFF2025000659000022.tif251170TIFF2025000659000023.tif27169

[0245] Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (EU numbering) in CL correspond to amino acids 18, 20, 33, 35, 37, 64, 66, 76, 78, and 80, respectively, in SEQ ID NOs: 17 to 27. Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (EU numbering) in the CL domain of the kappa chain correspond to amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (Kabat numbering) in the CL domain of the lambda light chain. Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (Kabat numbering) within the CL domain of the lambda light chain correspond to amino acids 10, 12, 25, 27, 29, 56, 59, 67, 69, and 71, respectively, in SEQ ID NO: 127.

[0246] In certain embodiments, the multispecific antigen-binding protein comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the multispecific antigen binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 43.In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 104. In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the amino acid sequence set forth in SEQ ID NO: 105. The amino acid sequences of SEQ ID NOs: 28-44 are provided in Table 6 below (unless otherwise stated, HC is of the IgG1 isotype). TIFF2025000659000024.tif255170TIFF2025000659000025.tif219170

[0247] Amino acid positions 141, 170, 181, 183, and 185 (EU numbering) within CH1 correspond to amino acid positions 22, 51, 62, 64, and 66 of SEQ ID NOs: 28-44 and 103-105, respectively.

[0248] All possible pairwise combinations of amino acids 11 to 116 of SEQ ID NOs: 17 to 27 or SEQ ID NOs: 17 to 27 and 127 with the above-mentioned SEQ ID NOs: 28 to 44 and 103 to 105 are contemplated. In certain embodiments, specific combinations of amino acids 11 to 116 of SEQ ID NOs: 17 to 27 or SEQ ID NOs: 17 to 27 and 127 with SEQ ID NOs: 28 to 44 and 103 to 105 are contemplated. Such combinations include, but are not limited to, those provided in Table 7 below. TIFF2025000659000026.tif202168

[0249] The first SEQ ID NO in each pair in Table 7 refers to the CH1 domain sequence, and the second SEQ ID NO in each pair in Table 7 refers to the CL domain sequence.

[0250] In a related aspect, there is provided a multispecific antigen binding protein comprising: a) a first heavy chain / light chain pair capable of binding to a first antigen, the first heavy chain sequence (H1) and the first light chain sequence (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain sequence (H2) and a second light chain sequence (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein one or more amino acid residues of the CH1 domain are located within the CH1 / CL interface. The CH1 domain of H1 and the CL domain of L1 of the multispecific antigen-binding protein are altered so that groups are replaced with an equivalent number of amino acid residues, some with larger side chain volumes and some with smaller side chain volumes, thereby creating protuberances and cavities on the surface of the CH1 domain, and one or more amino acid residues in the CL domain are replaced with an equivalent number of amino acid residues, some with smaller side chain volumes and some with larger side chain volumes, thereby creating cavities and protuberances on the surface of the CL domain. In certain embodiments, the modifications on the CH1 and CL domains provide steric complementarity (e.g., conformational complementarity) at the interface.

[0251] In certain embodiments, the CH1 domain of H1 comprises an amino acid substitution at position L128, and the CL domain of L1 comprises amino acid substitutions at positions F118 and L135 (EU numbering). In certain embodiments, the first antigen and the second antigen are the same. In certain embodiments, the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to different epitopes on the same antigen. In certain embodiments, the first antigen and the second antigen are different. In certain embodiments, the CH1 domain of H1 comprises amino acid substitutions at positions L128 and V185 (EU numbering), and the CL domain of L1 comprises amino acid substitutions at positions F118 and L135 (EU numbering). In certain embodiments, the first antigen and the second antigen are the same. In certain embodiments, the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to different epitopes on the same antigen. In certain embodiments, the first antigen and the second antigen are different. In certain embodiments, the CH1 domain of H1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of A141, F170, S181, and S183 (all EU numbering). In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of S131, V133, S162, S176, and T178 (all EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity (e.g., conformational complementarity) that is comparable to or greater than that of the wild-type sequence. In certain embodiments, the CL domain of L1 of the multispecific antigen-binding protein further comprises one or more amino acid substitutions selected from the group consisting of S131, V133, S162, T164, S176, and T178 (all EU numbering).

[0252] In certain embodiments, the multispecific antigen-binding protein comprises a CH1 domain of H1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from F128, A141, F170, S181, and S183 (EU numbering). In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from V118, S131, V133, S135, S162, T164, S176, and T178 (EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity (e.g., conformational complementarity) comparable to or greater than that of the wild-type sequence.

[0253] In certain embodiments, the multispecific antigen-binding protein comprises a CH1 domain of H1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from F128, A141, F170, S181, S183, and V185 (EU numbering). In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from V118, S131, V133, S135, S162, T164, S176, and T178 (EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity (e.g., conformational complementarity) comparable to or greater than that of the wild-type sequence.

[0254] In certain embodiments, the multispecific antigen-binding protein comprises a CH1 domain of H1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from F128, A141, F170, S181, S183, and V185 (EU numbering). In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising (including consisting of, including consisting essentially of) one or more amino acid substitutions selected from V118, S131, V133, S135, S162, S176, and T178 (EU numbering). In certain embodiments, the amino acid substitutions result in steric complementarity (e.g., conformational complementarity) that is comparable to or greater than that of the wild-type sequence.

[0255] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) L128F, A141M, F170M, S181I, and S183A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and T178L mutations (EU numbering).

[0256] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) L128F, A141M, F170Y, S181I, S183A, and V185A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F118V, S131T, V133A, L135F, S162A, S176A, and T178L mutations (EU numbering).

[0257] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) L128F, A141T, F170M, S181T, S183A, and V185L mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L mutations (EU numbering).

[0258] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (including consisting of, and consisting essentially of) L128F, A141M, F170M, S181T, and S183A mutations (EU numbering), and a CL domain of L1 comprising (including consisting of, and consisting essentially of) F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L mutations (EU numbering).

[0259] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of L128F, A141M, F170M, S181I, and S183A mutations (EU numbering).

[0260] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 amino acid mutations selected from the group consisting of L128F, A141M, F170Y, S181I, S183A, and V185A mutations (EU numbering).

[0261] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 amino acid mutations selected from the group consisting of L128F, A141T, F170M, S181T, S183A, and V185L mutations (EU numbering).

[0262] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acid mutations selected from the group consisting of L128F, A141M, F170M, S181T, and S183A mutations (EU numbering).

[0263] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, or no more than 8 amino acid mutations selected from the group consisting of F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and T178L mutations (EU numbering).

[0264] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, or no more than 7 amino acid mutations selected from the group consisting of F118V, S131T, V133A, L135F, S162A, S176A, and T178L mutations (EU numbering).

[0265] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, or no more than 8 amino acid mutations selected from the group consisting of F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L mutations (EU numbering).

[0266] In certain embodiments, the multispecific antigen binding protein comprises a CL domain of L1 that comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, or no more than 8 amino acid mutations selected from the group consisting of F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L mutations (EU numbering).

[0267] All possible combinations of pairs of CH1 and L1 domains mentioned above are contemplated.

[0268] In certain embodiments, the multispecific antigen-binding protein comprises a kappa light chain. In certain embodiments, the multispecific antigen-binding protein comprises a first light chain polypeptide, L1, comprising a CL domain set forth in any of SEQ ID NOs: 45 to 48. The CL domain set forth in SEQ ID NO: 54 corresponds to amino acids 11 to 116 of SEQ ID NOs: 45 to 48. Thus, L1 may comprise a CL domain comprising amino acids 11 to 116 of any of SEQ ID NOs: 45 to 48. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 45, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 45. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 46, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 46. In certain embodiments, the multispecific antigen-binding protein comprises a CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO: 47, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO: 47. In certain embodiments, the multispecific antigen-binding protein comprises the CL domain of L1 comprising the amino acid sequence set forth in SEQ ID NO:48, or amino acids 11 to 116 of the sequence set forth in SEQ ID NO:48.

[0269] In certain embodiments, the multispecific antigen binding protein comprises a lambda light chain. In certain embodiments, the multispecific antigen binding protein comprises the CL domain of L1, which comprises the amino acid sequence set forth in SEQ ID NO: 128.

[0270] In certain embodiments, the multispecific antigen-binding protein comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 51. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 106. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 107. In certain embodiments, the multispecific antigen-binding protein comprises an H1 CH1 domain comprising the amino acid sequence set forth in SEQ ID NO: 108. The amino acid sequences of SEQ ID NOs: 45-52 and 106-108 are provided in Table 8 below (LC is a kappa LC unless otherwise stated, and HC is of the IgG1 isotype unless otherwise stated). TIFF2025000659000027.tif196170

[0271] Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (EU numbering) in CL correspond to amino acids 18, 20, 33, 35, 37, 64, 66, 76, 78, and 80, respectively, in SEQ ID NOs: 45 to 48. Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (EU numbering) in the CL domain of the kappa chain correspond to amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (Kabat numbering) in the CL domain of the lambda light chain. Amino acids 116, 118, 131, 133, 135, 162, 164, 174, 176, and 178 (Kabat numbering) within the CL domain of the lambda light chain correspond to amino acids 10, 12, 25, 27, 29, 56, 59, 67, 69, and 71, respectively, in SEQ ID NO: 128.

[0272] Amino acids 128, 141, 170, 181, 183, and 185 (EU numbering) in CH1 correspond to amino acids 9, 22, 51, 62, 64, and 66 in SEQ ID NOs: 49 to 52 and 106 to 108, respectively.

[0273] All possible pairwise combinations of SEQ ID NOs: 45-48 or amino acids 11-116 of SEQ ID NOs: 45-48 with SEQ ID NOs: 49-52 are contemplated. In certain embodiments, the multispecific antigen-binding protein comprises a first light chain polypeptide L1 comprising a CL domain set forth in any of SEQ ID NOs: 45-48. The CL domain set forth in SEQ ID NO: 54 corresponds to amino acids 11-116 of SEQ ID NOs: 45-48. Thus, L1 may comprise a CL domain comprising amino acids 11-116 of any of SEQ ID NOs: 45-48. In certain embodiments, specific combinations of amino acids 11-116 of SEQ ID NOs: 45-48 or SEQ ID NOs: 45-48 and 128 with SEQ ID NOs: 49-52 and 106-108 are contemplated. Such combinations include, but are not limited to, SEQ ID NO:49 / SEQ ID NO:45 or amino acids 11 to 116 of SEQ ID NO:45, SEQ ID NO:50 / SEQ ID NO:46 or amino acids 11 to 116 of SEQ ID NO:46, SEQ ID NO:106 / SEQ ID NO:46 or amino acids 11 to 116 of SEQ ID NO:46, SEQ ID NO:107 / SEQ ID NO:46 or amino acids 11 to 116 of SEQ ID NO:46, SEQ ID NO:108 / SEQ ID NO:46 or amino acids 11 to 116 of SEQ ID NO:51 / SEQ ID NO:47 or amino acids 11 to 116 of SEQ ID NO:47, SEQ ID NO:50 / SEQ ID NO:128, SEQ ID NO:106 / SEQ ID NO:128, SEQ ID NO:107 / SEQ ID NO:128, SEQ ID NO:108 / SEQ ID NO:128, and SEQ ID NO:52 / SEQ ID NO:48 or amino acids 11 to 116 of SEQ ID NO:48.

[0274] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be reversed. That is, any of the mutations described above as being in the CH1 domain of H1 and the CL domain of L1 can alternatively be in the CH1 domain of H2 and the CL domain of L2.

[0275] As noted above, the mutations in the CH1 / CL interface specified by strategy number 2 may be used independently of or in addition to the mutations in the CH1 / CL interface specified by strategy number 1.

[0276] Thus, for example, there is provided herein a multispecific antigen binding protein comprising a CH1 domain of H1 that comprises A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 that comprises F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 that comprises a S183E substitution mutation (EU numbering); and a CL domain of L2 that comprises a V133K substitution mutation (EU numbering). Additionally, in certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 consisting of a S183E substitution mutation (EU numbering); and a CL domain of L2 consisting of a V133K substitution mutation (EU numbering). Additionally, in certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering).In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 consisting of A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering), a CL domain of L1 consisting of F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering), a CH1 domain of H2 consisting of an S183E substitution mutation (EU numbering), and a CL domain of L2 consisting of a V133K substitution mutation (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). Additionally, in certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In a specific embodiment, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:29; a CL domain of L1 comprising (or consisting of, or consisting essentially of) SEQ ID NO:19 or amino acids 11 to 116 of the sequence set forth in SEQ ID NO:19; a CH1 domain of H2 comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:15; and a CL domain of L2 comprising (or consisting of, or consisting essentially of) SEQ ID NO:5 or amino acids 11 to 116 of the sequence set forth in SEQ ID NO:5.

[0277] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising a S183K substitution mutation (EU numbering); and a CL domain of L2 comprising a V133E substitution mutation (EU numbering). In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 consisting of a S183K substitution mutation (EU numbering); and a CL domain of L2 consisting of a V133E substitution mutation (EU numbering). In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 consisting of A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering), a CL domain of L1 consisting of F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering), a CH1 domain of H2 consisting of a S183K substitution mutation (EU numbering), and a CL domain of L2 consisting of a V133E substitution mutation (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering). In a specific embodiment, the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering).In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0278] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141M, F170M, S181I, and S183A substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and S178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0279] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0280] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141T, F170M, S181T, S183A, and V185L substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0281] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141M, F170M, S181T, and S183A substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0282] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170S, S181M, S183V, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, V133I, L135V, S162M, S174A, S176F, and T178V substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0283] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, S131D, L135V, S162A, S174A, S176F, and T178I substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0284] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170A, S181M, S183V, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, L135V, S162M, S174A, S176F, and T178V substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0285] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the L128F, A141M, F170Y, S181I, S183A, and V185A mutations (EU numbering); a CL domain of L1 comprising the F118V, S131T, V133A, L135F, S162A, S176A, and T178L mutations (EU numbering); a CH1 domain of H2 comprising an S183E substitution mutation (EU numbering); and a CL domain of L2 comprising a V133K substitution mutation (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0286] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising the L128F, A141M, F170Y, S181I, S183A, and V185A mutations (EU numbering); a CL domain of L1 comprising the F118V, S131T, V133A, L135F, S162A, S176A, and T178L mutations (EU numbering); a CH1 domain of H2 comprising a S183K substitution mutation (EU numbering); and a CL domain of L2 comprising a V133E substitution mutation (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0287] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141M, F170M, S181I, and S183A substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and S178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0288] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, L135V, S174A, S176F, and T178V substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0289] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141T, F170M, S181T, S183A, and V185L substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0290] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising L128F, A141M, F170M, S181T, and S183A substitution mutations (EU numbering); and a CL domain of L2 comprising F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0291] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170S, S181M, S183V, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, V133I, L135V, S162M, S174A, S176F, and T178V substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0292] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be inverted. That is, any of the mutations described above as being within the CH1 and / or VH domains of H1 and the CL and / or VL domains of L1 can alternatively be present in the CH1 and / or VH domains of H2 and the CL and / or VL domains of L2.

[0293] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170S, S181M, S183A, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, S131D, L135V, S162A, S174A, S176F, and T178I substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0294] In certain embodiments, the multispecific antigen binding protein comprises a CH1 domain of H1 comprising L128F, A141M, F170Y, S181I, S183A, and V185A substitution mutations (EU numbering); a CL domain of L1 comprising F118V, S131T, V133A, L135F, S162A, S176A, and T178L substitution mutations (EU numbering); a CH1 domain of H2 comprising A141I, F170A, S181M, S183V, and V185A substitution mutations (EU numbering); and a CL domain of L2 comprising F116A, L135V, S162M, S174A, S176F, and T178V substitution mutations (EU numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering). In certain embodiments, the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering).

[0295] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be inverted. That is, any of the mutations described above as being within the CH1 and / or VH domains of H1 and the CL and / or VL domains of L1 can alternatively be present in the CH1 and / or VH domains of H2 and the CL and / or VL domains of L2.

[0296] In certain embodiments, the multispecific antigen binding proteins provided herein comprise a CH1 domain of H1 that comprises no more than 1 amino acid substitution mutation, no more than 2 amino acid substitution mutations, no more than 3 amino acid substitution mutations, no more than 5 amino acid substitution mutations, or no more than 6 amino acid substitutions, no more than 7 amino acid substitutions, no more than 8 amino acid substitutions, no more than 9 amino acid substitutions, no more than 10 amino acid substitutions, no more than 11 amino acid substitutions, or no more than 12 amino acid substitutions.

[0297] In certain embodiments, the multispecific antigen binding proteins provided herein comprise a CL domain of L1 that comprises no more than 1 amino acid substitution mutation, no more than 2 amino acid substitution mutations, no more than 3 amino acid substitution mutations, no more than 5 amino acid substitution mutations, or no more than 6 amino acid substitutions, no more than 7 amino acid substitutions, no more than 8 amino acid substitutions, no more than 9 amino acid substitutions, no more than 10 amino acid substitutions, no more than 11 amino acid substitutions, or no more than 12 amino acid substitutions. In certain embodiments, the CH1 domain of H1 is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline or allotype heavy chain CH1 domain. See Jefferis et al. (2009) Mabs 1:332-338. Provided below are the amino acid sequence of the human IgG1 CH1 domain (ie, SEQ ID NO: 57) and the amino acid sequence of the human IgG4 S228P CH1 domain (ie, SEQ ID NO: 58). TIFF2025000659000028.tif65170

[0298] In some embodiments, LC1 is a kappa light chain. In some embodiments, the CL domain of LC1 is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the CL domain of a human germline or allotype kappa chain. The amino acid sequence of a human kappa light chain constant domain (i.e., SEQ ID NO: 59) is provided below.

[0299] TIFF2025000659000029.tif13170 In some embodiments, LC1 is a lambda light chain. In some embodiments, the CL domain of LC1 is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline or allotypic lambda CL domain. Provided below is the amino acid sequence of the human lambda light chain constant domain (ie, SEQ ID NO: 113). TIFF2025000659000030.tif13170

[0300] In certain embodiments, the parental H1 from which the H1 of a multispecific antigen-binding protein provided herein is derived does not show a significant preference over the parental L1 from which the L1 of a multispecific antigen-binding protein provided herein is derived. In certain embodiments, the parental H1 from which the H1 of a multispecific antigen-binding protein provided herein is derived shows a preference over the parental L1 from which the L1 of a multispecific antigen-binding protein provided herein is derived.

[0301] It will be apparent to one of skill in the art that the terms "H1" and "H2" are arbitrary designations, and that "H1" and "H2" in any of the above embodiments can be inverted. That is, any of the mutations described above as being within the CH1 and / or VH domains of H1 and the CL and / or VL domains of L1 can alternatively be present in the CH1 and / or VH domains of H2 and the CL and / or VL domains of L2.

[0302] Modifications to the VH and VL domains In certain embodiments, the multispecific antigen binding protein, or antigen-binding fragment thereof, comprises amino acid modifications in the VH and VL domain(s), i.e., independent of the modifications in the CH1 and CL domains described herein. In certain embodiments, the multispecific antigen binding proteins provided herein comprise amino acid modifications in the VH and VL domain(s) in addition, i.e., in combination with mutations in the CH1 / CL interface identified by strategy no. 1 and / or mutations in the CH1 / CL interface identified by strategy no. 1.

[0303] In certain embodiments, the VH domain of H1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q39 (Kabat numbering), and the VL domain of L1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q38 (Kabat numbering). In certain embodiments, the amino acid at position Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, and the amino acid at position Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue. In certain embodiments, the amino acid at position Q39 (Kabat numbering) in the VH domain of H1 is replaced by a negatively charged residue, and the amino acid at position Q38 (Kabat numbering) in the VL domain of L1 is replaced by a positively charged residue. In certain embodiments, the positively charged residue is selected from the group consisting of R, H, and K. In certain embodiments, the negatively charged residue is selected from the group consisting of D and E.

[0304] Additionally or alternatively, in certain embodiments, the VH domain of H2 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q39 (Kabat numbering), and the VL domain of L2 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q38 (Kabat numbering). In certain embodiments, the amino acid at position Q39 (Kabat numbering) in the VH domain of H2 is replaced by a positively charged residue, and the amino acid at position Q38 (Kabat numbering) in the VL domain of L2 is replaced by a negatively charged residue. In certain embodiments, the amino acid at position Q39 (Kabat numbering) in the VH domain of H2 is replaced by a negatively charged residue, and the amino acid at position Q38 (Kabat numbering) in the VL domain of L2 is replaced by a positively charged residue. In certain embodiments, the positively charged residue is selected from the group consisting of R, H, and K. In certain embodiments, the negatively charged residue is selected from the group consisting of D and E.

[0305] In certain embodiments, specific combinations are envisaged of a substitution mutation at position Q39 (Kabat numbering) on ​​the VH domain of H1 with a substitution mutation at position Q38 (Kabat numbering) on ​​the VL domain of L1, and / or a substitution mutation at position Q39 (Kabat numbering) on ​​the VH domain of H2 with a substitution mutation at position Q38 (Kabat numbering) on ​​the VL domain of L2, including, but not limited to, those shown in Tables 9A and 9B below. TIFF2025000659000031.tif46170

[0306] The first mutation in each pair in Table 9A refers to a modification in the VH domain sequence, and the second mutation in each pair in Table 9A refers to a modification in the VL domain sequence. TIFF2025000659000032.tif58170

[0307] The four letter mutation in Table 9B is Q39X VH1 / Q38X VL1 / Q39X VH2 / Q38X VL2 "VH1" refers to the amino acid substitutions in the VH domain of H1, "VL" refers to the VL domain of L1, "VH2" refers to the VH domain of H2, and "VL2" refers to the VL domain of L2. It will be apparent to those skilled in the art that the terms "H1" / "L1" and "H2" / "L2" are arbitrary designations, and that "H1" and "L1" in any of the above embodiments can be inverted to "H2" and "L2," respectively. That is, any of the mutations described above as being within the VH domain of H1 and the VL domain of L1 can alternatively be present in the VH domain of H2 and the VL domain of L2.

[0308] In certain embodiments, the VH domain of H1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q39E (Kabat numbering) and the VL domain of L1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q38K (Kabat numbering). In certain embodiments, the VH domain of H2 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q39K (Kabat numbering) and the VL domain of L2 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q38E (Kabat numbering). In certain embodiments, the VH domain of H1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q39K (Kabat numbering) and the VL domain of L1 of the multispecific antigen-binding protein comprises an amino acid substitution at position Q38E (Kabat numbering). In certain embodiments, the VH domain of H2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q39E (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q38K (Kabat numbering). In certain embodiments, the VH domain of H1 of the multispecific antigen binding protein comprises an amino acid substitution at position Q39E (Kabat numbering) and the VL domain of L1 of the multispecific antigen binding protein comprises an amino acid substitution at position Q38K (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q39K (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q38E (Kabat numbering). In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises an amino acid substitution at position Q39K (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises an amino acid substitution at position Q38E (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q39E (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises an amino acid substitution at position Q38K (Kabat numbering).

[0309] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0310] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0311] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0312] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0313] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0314] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0315] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises the substitution mutation Q39D (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0316] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0317] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0318] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0319] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0320] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0321] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0322] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0323] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0324] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0325] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0326] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0327] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0328] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0329] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0330] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0331] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0332] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0333] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0334] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0335] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0336] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0337] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0338] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39R substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38R substitution mutation (Kabat numbering).

[0339] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39H substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38H substitution mutation (Kabat numbering).

[0340] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39H substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38H substitution mutation (Kabat numbering).

[0341] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39D substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38H substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39H substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38D substitution mutation (Kabat numbering).

[0342] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38H substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39H substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0343] It will be apparent to those skilled in the art that the terms "H1" / "L1" and "H2" / "L2" are arbitrary designations, and that "H1" and "L1" in any of the above embodiments can be inverted to "H2" and "L2," respectively. That is, any of the mutations described above as being in the VH domain of H1 and the VL domain of L1 can alternatively be in the VH domain of H2 and the VL domain of L2.

[0344] As noted elsewhere herein, the multispecific antigen binding proteins provided herein in some embodiments comprise amino acid modifications in the VH and VL domain(s), such as those described above, in combination with mutations in the CH1 / CL interface specified by strategy no. 1 and / or mutations in the CH1 / CL interface specified by strategy no. 2.

[0345] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0346] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0347] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0348] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0349] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0350] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183K substitution mutation (EU numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133E substitution mutation (EU numbering).

[0351] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0352] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0353] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183F substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0354] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183F substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0355] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183T substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0356] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183T substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0357] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183Y substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0358] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183Y substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0359] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133K substitution mutation (EU numbering).

[0360] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183K substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133E substitution mutation (EU numbering).

[0361] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183K substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133E substitution mutation (EU numbering).

[0362] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a V133K substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0363] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a V133K substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0364] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering).

[0365] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a S183K substitution mutation (EU numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of or consists essentially of) a V133E substitution mutation (EU numbering).

[0366] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises (e.g. consists of, or consists essentially of) a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering) and the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering).

[0367] In certain embodiments, the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering). In certain embodiments, the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0368] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0369] In a specific embodiment, the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0370] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0371] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0372] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0373] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering).

[0374] In a specific embodiment, the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering).

[0375] In a specific embodiment, the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering) and the CL domain of L2 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering).

[0376] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering), the CL domain of L1 of the multispecific antigen binding protein comprises a V133K substitution mutation (EU numbering), the VH domain of H2 of the multispecific antigen binding protein comprises a Q39K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen binding protein comprises a S183K substitution mutation (EU numbering), the VL domain of L2 of the multispecific antigen binding protein comprises a Q38E substitution mutation (Kabat numbering), and the CL domain of L2 of the multispecific antigen binding protein comprises a V133E substitution mutation (EU numbering).

[0377] In a specific embodiment, the VH domain of H1 of the multispecific antigen binding protein comprises a Q39E substitution mutation (Kabat numbering), the CH1 domain of H1 of the multispecific antigen binding protein comprises a S183E substitution mutation (EU numbering), the VL domain of L1 of the multispecific antigen binding protein comprises a Q38K substitution mutation (Kabat numbering) and the CL domain of L1 of the multispecific antigen binding protein comprises a V133K subs...

Claims

1. 1. A multispecific antigen binding protein comprising: a) a first heavy chain / light chain pair which binds to a first antigen comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and b) a second heavy chain / light chain pair which binds to a second antigen comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of said H1 comprises an amino acid substitution at S183 (EU numbering) and the CL domain of said L1 comprises an amino acid substitution at V133 (EU numbering).

2. 2. The multispecific antigen binding protein of claim 1, wherein the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K, and the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

3. 3. The multispecific antigen-binding protein of claim 1 or 2, wherein the amino acid at EU position S183 on the CH1 domain of H1 is replaced with a positively charged residue and the amino acid at V133 on the CL domain of L1 is replaced with a negatively charged residue.

4. 3. The multispecific antigen-binding protein of claim 1 or 2, wherein the amino acid at EU position S183 on the CH1 domain of H1 is replaced with a negatively charged residue and the amino acid at V133 on the CL domain of L1 is replaced with a positively charged residue.

5. 5. The multispecific antigen-binding protein of claim 3 or 4, wherein the positively charged residues are selected from the group consisting of R and K.

6. The multispecific antigen-binding protein of any one of claims 3 to 5, wherein the negatively charged residue is selected from the group consisting of D and E.

7. 7. The multispecific antigen-binding protein of any one of claims 1 to 6, wherein the CH1 domain consists of an amino acid substitution at S183 (EU numbering) and the L1 CL domain consists of an amino acid substitution at V133 (EU numbering).

8. (a) the CH1 domain of H1 comprises a S183D mutation and the CL domain of L1 comprises a V133K mutation; or (b) the CH1 domain of H1 comprises an S183E mutation and the CL domain of L1 comprises a V133K mutation; or (c) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133E mutation; or (d) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133S mutation; or (e) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133L mutation; or (f) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133W mutation; or (g) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133K mutation; or (h) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133R mutation; or (i) the CH1 domain of H1 comprises a S183A mutation and the CL domain of L1 comprises a V133D mutation; or (j) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133E mutation; or (k) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133S mutation; or (l) the CH1 domain of H1 comprises an S183T mutation and the CL domain of L1 comprises a V133L mutation; or (m) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133W mutation; or (n) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133K mutation; or (o) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133R mutation; or (p) the CH1 domain of H1 comprises a S183T mutation and the CL domain of L1 comprises a V133D mutation; or (q) the CH1 domain of H1 comprises an S183V mutation and the CL domain of L1 comprises a V133E mutation; or (r) the CH1 domain of H1 comprises a S183V mutation and the CL domain of L1 comprises a V133S mutation; or (s) the CH1 domain of H1 comprises a S183V mutation and the CL domain of L1 comprises a V133L mutation; or (t) the CH1 domain of H1 comprises an S183V mutation and the CL domain of L1 comprises a V133W mutation; or (u) the CH1 domain of H1 comprises an S183V mutation and the CL domain of L1 comprises a V133K mutation; or (v) the CH1 domain of H1 comprises a S183V mutation and the CL domain of L1 comprises a V133R mutation; or (w) the CH1 domain of H1 comprises an S183V mutation and the CL domain of L1 comprises a V133D mutation; (x) the CH1 domain of H1 comprises a S183Y mutation and the CL domain of L1 comprises a V133E mutation; or (y) the CH1 domain of H1 comprises a S183Y mutation and the CL domain of L1 comprises a V133S mutation; or (z) the CH1 domain of H1 comprises a S183Y mutation and the CL domain of L1 comprises a V133L mutation; or (aa) the CH1 domain of the H1 comprises a S183Y mutation and the CL of the L1 domain comprises a V133W mutation; or (bb) the CH1 domain of the H1 comprises a S183Y mutation and the CL of the L1 domain comprises a V133K mutation; or (cc) the CH1 domain of H1 comprises a S183Y mutation and the CL domain of L1 comprises a V133R mutation; or (dd) the CH1 domain of H1 comprises a S183Y mutation and the CL domain of L1 comprises a V133D mutation; or (ee) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133E mutation; or (ff) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133S mutation; or (gg) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133L mutation; (hh) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133W mutation; or (ii) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133K mutation; or (jj) the CH1 domain of H1 comprises a S183F mutation and the CL domain of L1 comprises a V133R mutation; or (kk) the CH1 domain of the H1 comprises a S183F mutation and the CL of the L1 domain comprises a V133D mutation; (ll) the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133S mutation; or (mm) the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133L mutation; or (nn) the CH1 domain of H1 comprises a S183H mutation and the CL domain of L1 comprises a V133W mutation; (oo) the CH1 domain of H1 comprises a S183N mutation and the CL domain of L1 comprises a V133L mutation; or (pp) the H1 CH1 domain comprises a S183E mutation and the L1 CL domain comprises a V133L mutation, or the H1 CH1 domain comprises a S183K mutation and the L1 CL domain comprises a V133E mutation.

9. (a) the CH1 domain of H1 comprises or consists of a S183D mutation and the CL domain of L1 comprises or consists of a V133K mutation; (b) the CH1 domain of H1 comprises or consists of a S183E mutation and the CL domain of L1 comprises or consists of a V133K mutation; (c) the CH1 domain of H1 comprises or consists of a S183T mutation and the CL domain of L1 comprises or consists of a V133K mutation; or (d) the CH1 domain of H1 comprises or consists of a S183V mutation and the CL domain of L1 comprises or consists of a V133E mutation; (e) the CH1 domain of H1 comprises or consists of a S183K mutation and the CL domain of L1 comprises or consists of a V133E mutation.

10. 10. The multispecific antigen-binding protein of any one of claims 1 to 9, wherein the CH1 domain of H2 and / or the CL domain of L2 does not comprise an amino acid substitution.

11. 10. The multispecific antigen-binding protein of any one of claims 1 to 9, wherein the CH1 domain of H2 does not comprise a substitution at S183 and the CL domain of L2 does not comprise a substitution at V133.

12. 12. The multispecific antigen-binding protein of any one of claims 1 to 11, wherein the H1 and / or H2 VH domain comprises an amino acid substitution at position Q39 (Kabat numbering) and the L1 and / or L2 VL domain comprises an amino acid substitution at position Q38 (Kabat numbering).

13. 13. The multispecific antigen binding protein of claim 12, wherein the amino acid at Q39 in the VH domain is replaced by a positively charged residue and the amino acid at Q38 in the VL domain is replaced by a negatively charged residue (Kabat numbering).

14. 5. The multispecific antigen-binding protein of claim 4, wherein the amino acid at Q39 in the VH domain is replaced by a positively charged residue and the amino acid at Q38 in the VL domain is replaced by a negatively charged residue (Kabat numbering).

15. 13. The multispecific antigen binding protein of claim 12, wherein the amino acid at Q39 in the VH domain is replaced by a negatively charged residue and the amino acid at Q38 in the VL domain is replaced by a positively charged residue (Kabat numbering).

16. 4. The multispecific antigen binding protein of claim 3, wherein the amino acid at Q39 in the VH domain is replaced by a negatively charged residue and the amino acid at Q38 in the VL domain is replaced by a positively charged residue (Kabat numbering).

17. 17. The multispecific antigen-binding protein of any one of claims 13 to 16, wherein the positively charged residues are selected from the group consisting of R and K.

18. 18. The multispecific antigen-binding protein of any one of claims 13 to 17, wherein the negatively charged residue is selected from the group consisting of D and E.

19. 13. The multispecific antigen-binding protein of any one of claims 1 to 12, wherein the H1 VH domain comprises a Q39E substitution mutation (Kabat numbering) and the L1 VL domain comprises a Q38K substitution mutation (Kabat numbering).

20. 13. The multispecific antigen binding protein of any one of claims 1 to 12, wherein the H1 VH domain comprises a Q39E substitution mutation, the L1 VL domain comprises a Q38K substitution mutation and the H2 VH domain comprises a Q39K substitution mutation (Kabat numbering).

21. 13. The multispecific antigen binding protein of any one of claims 1 to 12, wherein the H1 VH domain comprises a Q39E substitution mutation, the L1 VL domain comprises a Q38K substitution mutation, the H2 VH domain comprises a Q39K substitution mutation and the L2 VL domain comprises a Q38E substitution mutation (Kabat numbering).

22. 13. The multispecific antigen-binding protein of any one of claims 1 to 12, wherein the H1 VH domain comprises a Q39K substitution mutation (Kabat numbering) and the L1 VL domain comprises a Q38E substitution mutation (Kabat numbering).

23. 21. The multispecific antigen binding protein of any one of claims 1 to 12 and 20, wherein the H1 VH domain comprises a Q39K substitution mutation, the L1 VL domain comprises a Q38E substitution mutation and the H2 VH domain comprises a Q39E substitution mutation (all Kabat numbering).

24. 13. The multispecific antigen binding protein of any one of claims 1 to 12, wherein the H1 VH domain comprises a Q39K substitution mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation (all Kabat numbering).

25. 22. The multispecific antigen binding protein of any one of claims 1 to 12 and 20 to 21, wherein the H1 VH domain comprises a Q39K substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation (all Kabat numbering).

26. 26. The multispecific antigen-binding protein of any one of claims 1 to 25, wherein the interaction between the two substituted amino acids is through a hydrogen bond.

27. 27. The multispecific antigen-binding protein of any one of claims 1 to 26, wherein the interaction between the two substituted amino acids is through electrostatic interaction.

28. 27. The multispecific antigen binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the H1 VH domain comprises a Q39K substitution mutation, the H1 CH1 domain comprises a S183E substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the L1 CL domain comprises a V133K mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation.

29. 27. The multispecific antigen binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the H1 VH domain comprises a Q39K substitution mutation, the H1 CH1 domain comprises a S183T substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the L1 CL domain comprises a V133K mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation.

30. 27. The multispecific antigen binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the H1 VH domain comprises a Q39K substitution mutation, the H1 CH1 domain comprises a S183Y substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the L1 CL domain comprises a V133K mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation.

31. 27. The multispecific antigen binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the H1 VH domain comprises a Q39K substitution mutation, the H1 CH1 domain comprises a S183F substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the L1 CL domain comprises a V133K mutation, the H2 VH domain comprises a Q39E substitution mutation and the L2 VL domain comprises a Q38K substitution mutation.

32. 27. The multispecific antigen binding protein of any one of claims 1 to 9, 12 to 13, 15, 17 to 21 and 25 to 26, wherein the H1 VH domain comprises a Q39E substitution mutation, the H1 CH1 domain comprises a S183E substitution mutation, the L1 VL domain comprises a Q38K substitution mutation, the L1 CL domain comprises a V133K mutation, the H2 VH domain comprises a Q39K substitution mutation and the L2 VL domain comprises a Q38E substitution mutation.

33. 27. The multispecific antigen binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the H1 VH domain comprises a Q39K substitution mutation, the L1 VL domain comprises a Q38E substitution mutation, the H2 VH domain comprises a Q39E substitution mutation, the H2 CH1 domain comprises a S183E substitution mutation, the L2 VL domain comprises a Q38K substitution mutation and the L1 CL domain comprises a V133K mutation.

34. 27. The multispecific antigen binding protein of any one of claims 1 to 9, 12 to 13, 15, 17 to 21 and 25 to 26, wherein the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183K substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, the CL domain of L1 comprises a V133E substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38E substitution mutation and the CL domain of L2 comprises a V133K mutation.

35. 27. The multispecific antigen-binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the VH domain of H1 comprises a Q39E substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38K substitution mutation, the CL domain of L1 comprises a V133K substitution mutation, the VH domain of H2 comprises a Q39K substitution mutation, the CH1 domain of H2 comprises a S183K substitution mutation, the VL domain of L2 comprises a Q38E substitution mutation, and the CL domain of L2 comprises a V133E mutation.

36. 27. The multispecific antigen-binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183K substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133E substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183E substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L2 comprises a V133K mutation.

37. 27. The multispecific antigen-binding protein of any one of claims 1-9, 12-13, 15, 17-18, 22-23 and 25-26, wherein the VH domain of H1 comprises a Q39K substitution mutation, the CH1 domain of H1 comprises a S183E substitution mutation, the VL domain of L1 comprises a Q38E substitution mutation, the CL domain of L1 comprises a V133K substitution mutation, the VH domain of H2 comprises a Q39E substitution mutation, the CH1 domain of H2 comprises a S183K substitution mutation, the VL domain of L2 comprises a Q38K substitution mutation, and the CL domain of L2 comprises a V133E mutation.

38. 38. The multispecific antigen-binding protein of any one of claims 1 to 37, wherein each of H1 and / or H2 comprises an Fc region comprising a CH2 and a CH3 domain.

39. 39. The multispecific antigen-binding protein of claim 38, wherein the H1 and / or H2 Fc region is human IgG1, human IgG2, or human IgG4 Fc.

40. 39. The multispecific antigen-binding protein of claim 38, wherein the H1 and / or H2 Fc region is mouse IgG1, mouse IgG2, or mouse IgG4 Fc.

41. 41. The multispecific antigen-binding protein of any one of claims 1 to 40, wherein the H1 and H2 CH3 domains each meet at an interface, and each of the CH3 domains comprises amino acid substitutions such that the H1 Fc region preferentially pairs with the H2 Fc region compared to H1.

42. 42. The multispecific antigen-binding protein of claim 41 , wherein said amino acid substitutions in said CH3 domain result in greater electrostatic complementarity.

43. 42. The multispecific antigen-binding protein of claim 41 , wherein the amino acid substitutions in the CH3 domain result in greater steric complementarity.

44. the H1 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the H1 CH3 domain that interacts with the H2 CH3 domain; 42. The multispecific antigen binding protein of claim 41 , wherein the H2 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the H2 CH3 domain which interacts with the H1 CH3 domain.

45. the H2 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the H2 CH3 domain that interacts with the H1 CH3 domain; and, 42. The multispecific antigen binding protein of claim 41 , wherein the H1 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the H1 CH3 domain which interacts with the H2 CH3 domain.

46. 46. ​​The multispecific antigen-binding protein of claim 44 or 45, wherein the protuberance is a knob mutation.

47. 47. The multispecific binding protein of claim 46, wherein the knob mutation comprises T366W (EU numbering).

48. 46. ​​The multispecific antigen-binding protein of claim 44 or 45, wherein the cavity is a hole mutation.

49. 49. The multispecific antigen-binding protein of claim 48, wherein said hole mutations include at least one, at least two, or all three of T366S, L368A, and Y407V (EU numbering).

50. 1. A multispecific antigen binding protein comprising: a) a first heavy chain / light chain pair comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of said H1 comprises an amino acid substitution at F170 (EU numbering) and wherein the CL domain of said L1 comprises an amino acid substitution at S176 (EU numbering).

51. 51. The multispecific antigen-binding protein of claim 50, wherein the H1 CH1 domain further comprises one or more amino acid substitutions at positions selected from the group consisting of A141, S181, S183, and V185 (EU numbering).

52. 52. A multispecific antigen-binding protein according to claim 50 or 51, wherein the L1 CL domain further comprises one or more amino acid substitutions at positions selected from the group consisting of F116, S131, V133, L135, S162, S174 and T178 (EU numbering).

53. 53. The multispecific antigen-binding protein of any one of claims 50 to 52, wherein said one or more amino acid substitutions at positions selected from the group consisting of A141, F170, S181, S183 and V185 (EU numbering) in CH1 and / or said one or more amino acid substitutions at positions selected from the group consisting of F116, S131, V133, L135, S162, S174, S176 and T178 (EU numbering) in CL1 are not substituted with a charged amino acid residue.

54. 54. The multispecific antigen-binding protein of any one of claims 50 to 53, wherein the amino acid substitutions result in steric complementarity.

55. 55. The multispecific antigen-binding protein of any one of claims 50 to 54, wherein the CH1 domain of H1 comprises an amino acid substitution selected from the group consisting of F170S and F170A, and the CL domain of L1 comprises an amino acid substitution of S176F.

56. (a) the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises an S176F mutation; or (b) the CH1 domain of H1 comprises a F170A mutation and the CL domain of L1 comprises a S176F mutation.

57. 57. The multispecific antigen-binding protein of claim 56, wherein the CH1 domain of H1 comprises a F170S mutation and the CL domain of L1 comprises a S176F mutation.

58. (a) the H1 CH1 domain comprises A141I, F170S, S181M, S183V, and V185A mutations, and the L1 CL domain comprises F116A, V133I, L135V, S162M, S174A, S176F, and T178V mutations; or (b) the H1 CH1 domain comprises A141I, F170S, S181M, S183A, and V185A mutations, and the L1 CL domain comprises F116A, S131D, L135V, S162A, S174A, S176F, and T178I mutations; or (c) the CH1 domain of H1 comprises A141I, F170S, S181M, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or (d) the H1 CH1 domain comprises the A141I, F170A, S181M, S183V and V185A mutations and the L1 CL domain comprises the F116A, L135V, S162M, S174A, S176F and T178V mutations.

59. 58. The multispecific antigen-binding protein of any one of claims 50 to 57, wherein the CH1 domain of H1 comprises the A141I, F170S, S181M, S183A and V185A mutations and the CL domain of L1 comprises the F116A, L135V, S174A, S176F and T178V mutations.

60. 60. The multispecific antigen binding protein of claim 59, wherein the VH domain of H1 comprises a Q39E mutation (Kabat numbering), the VL domain of L1 comprises a Q38K mutation (Kabat numbering), the VH domain of H2 comprises a Q39K mutation (Kabat numbering), and the VL domain of L2 comprises a Q38E mutation (Kabat numbering).

61. 61. The multispecific antigen-binding protein of claim 59 or 60, wherein the CH1 domain of H2 comprises a S183E mutation (EU numbering) and the CL domain of L2 comprises a V133K mutation (EU numbering).

62. 60. The multispecific antigen binding protein of claim 59, wherein the VH domain of H1 comprises a Q39K mutation (Kabat numbering), the VL domain of L1 comprises a Q38E mutation (Kabat numbering), the VH domain of H2 comprises a Q39E mutation (Kabat numbering), and the VL domain of L2 comprises a Q38K mutation (Kabat numbering).

63. 63. The multispecific antigen-binding protein of claim 59 or 62, wherein the CH1 domain of H2 comprises a S183K mutation (EU numbering) and the CL domain of L2 comprises a V133K mutation (EU numbering).

64. (a) the H1 CH1 domain comprises F170S, S181M, S183A, and V185A mutations, and the L1 CL domain comprises L135V, S174A, S176F, and T178V mutations; or (b) the CH1 domain of H1 comprises A141I, F170S, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, S174A, S176F, and T178V mutations; or (c) the H1 CH1 domain comprises A141I, F170S, S181M, and V185A mutations, and the L1 CL domain comprises F116A, L135V, S176F, and T178V mutations; or (d) the CH1 domain of H1 comprises A141I, F170S, S181M, and S183A mutations, and the CL domain of L1 comprises F116A, L135V, S174A, and S176F mutations; or (e) the CH1 domain of H1 comprises F170S, S183A, and V185A mutations, and the CL domain of L1 comprises F116A, S176F, and T178V mutations; or (f) the CH1 domain of H1 comprises F170S, S181M, and V185A mutations, and the CL domain of L1 comprises F116A, L135V, and S176F mutations; or (g) the CH1 domain of H1 comprises F170S, S181M, and S183A mutations, and the CL domain of L1 comprises F116A and S176F mutations; or (h) the CH1 domain of H1 comprises A141I, F170S, and V185A mutations, and the CL domain of L1 comprises F116A and S176F mutations; or (i) the CH1 domain of H1 comprises A141I, F170S, and S183A mutations, and the CL domain of L1 comprises F116A and S176F mutations; or (j) the CH1 domain of H1 comprises A141I, F170S, and S181M mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or (k) the CH1 domain of H1 comprises F170S and V185A mutations, and the CL domain of L1 comprises F116A and S176F mutations; or (l) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or (m) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S176F, and T178V mutations; or (n) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, S174A, and S176F mutations; or (o) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, S176F, and T178V mutations; or (p) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A, L135V, and S176F mutations; or (q) the CH1 domain of H1 comprises A141I and F170S mutations, and the CL domain of L1 comprises F116A and S176F mutations; or (r) the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations; or (s) the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises F116A, L135V, S176F, and T178V mutations; or t) the CH1 domain of H1 comprises a F170S mutation and the CL domain of L1 comprises F116A, L135V, S174A, and S176F mutations; (u) the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises an F116A, S176F, and T178V mutation; or (v) the CH1 domain of H1 comprises an F170S mutation and the CL domain of L1 comprises an F116A, an L135V, and an S176F mutation; or 56. The multispecific antigen-binding protein of any one of claims 50 to 55, wherein (w) the CH1 domain of H1 comprises a F170S mutation and the CL domain of L1 comprises a F116A and a S176F mutation.

65. 65. The multispecific antigen-binding protein of claim 64, wherein the CH1 domain of H1 comprises the A141I, F170S, and S181M mutations and the CL domain of L1 comprises the F116A, L135V, S174A, S176F, and T178V mutations.

66. 65. The multispecific antigen-binding protein of claim 64, wherein the CH1 domain of H1 comprises A141I and F170S mutations and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations.

67. 65. The multispecific antigen-binding protein of claim 64, wherein the CH1 domain of H1 comprises a F170S mutation and the CL domain of L1 comprises F116A, L135V, S174A, S176F, and T178V mutations.

68. 1. A multispecific antigen binding protein comprising: a) a first heavy chain / light chain pair comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and b) a second heavy chain / light chain pair comprising a second heavy chain sequence (H2) and a second light chain sequence (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the CH1 domain of said H1 comprises an amino acid substitution at L128 (EU numbering) and the CL domain of said C1 comprises amino acid substitutions at F118 and L135 (EU numbering).

69. 69. The multispecific antigen-binding protein of claim 68, wherein the CH1 domain of H1 further comprises an amino acid substitution at V185 (EU numbering).

70. 70. A multispecific antigen-binding protein according to claim 68 or 69, wherein the H1 CH1 domain further comprises one or more amino acid substitutions at positions selected from the group consisting of A141, F170, S181 and S183 (EU numbering).

71. 71. The multispecific antigen-binding protein of any one of claims 68 to 70, wherein the L1 CL domain further comprises one or more amino acid substitutions at positions selected from the group consisting of S131, V133, S162, T164, S176, and T178 (EU numbering).

72. 72. The multispecific antigen-binding protein of any one of claims 68 to 71, wherein said one or more amino acid substitutions at positions selected from the group consisting of L128 A141, F170, S181, S183, V185 of CH1 (EU numbering) and / or said one or more amino acid substitutions at positions selected from the group consisting of F118 S131, V133, and L135 S162, T164, S176, and T178 of CL1 (EU numbering) are not substituted with a charged amino acid residue.

73. 73. The multispecific antigen-binding protein of any one of claims 68 to 72, wherein the amino acid substitutions result in higher steric complementarity.

74. 74. The multispecific antigen-binding protein of any one of claims 68 to 73, wherein the H1 CH1 domain comprises an amino acid substitution at a position selected from the group consisting of A141, F170, S181, and S183, and the CL domain comprises an amino acid substitution at a position selected from the group consisting of S131, V133, S162, T164, S176, and T178.

75. (a) the H1 CH1 domain comprises the following mutations: L128F, A141M, F170M, S181I, and S183A, and the CL domain comprises the following mutations: F118V, S131T, V133A, L135Y, S162A, T164S, S176M, and T178L; or (b) the H1 CH1 domain comprises the following mutations: L128F, A141M, F170Y, S181I, S183A, and V185A, and the CL domain comprises the following mutations: F118V, S131T, V133A, L135F, S162A, S176A, and T178L; or (c) the H1 CH1 domain comprises the following mutations: L128F, A141T, F170M, S181T, S183A, and V185L, and the CL domain comprises the following mutations: F118V, S131T, V133A, L135F, S162A, T164S, S176T, and T178L; or (d) the H1 CH1 domain comprises the following mutations: L128F, A141M, F170M, S181T, and S183A, and the CL domain comprises the following mutations: F118V, S131T, V133A, L135F, S162M, T164S, S176M, and T178L.

76. 76. The multispecific antigen binding protein of claim 75, wherein the H1 CH1 domain comprises the following mutations: L128F, A141M, F170Y, S181I, S183A, and V185A, and the CL domain comprises the following mutations: F118V, S131T, V133A, L135F, S162A, S176A, and T178L.

77. the CH1 domain of H2 is altered such that within the CH1 / CL interface, two or more amino acid residues are replaced with an equivalent number of amino acid residues having larger side chain volume, thereby generating a protuberance on the surface of the CH1 domain of H2 that interacts with the CL domain of L2; and, 50. The multispecific antigen-binding protein of any one of claims 1 to 49, wherein the CL domain of L2 is altered such that within the CH1 / CL interface, two or more amino acid residues are replaced with an equivalent number of amino acid residues having smaller side chain volumes, thereby generating a cavity on the surface of the CL domain of L2 which interacts with the CH1 domain of H2.

78. the CL domain of L2 is altered such that within the CH1 / CL interface, two or more amino acid residues are replaced with amino acid residues having larger side chain volume, thereby generating a protuberance on the surface of the CL domain that interacts with the CH1 domain of H2; and, 50. The multispecific antigen binding protein of any one of claims 1 to 49, wherein the H2 CH1 domain is altered such that within the CH1 / CL interface, two or more amino acid residues are replaced with amino acid residues having smaller side chain volumes, thereby generating a cavity on the surface of the H2 CH1 domain which interacts with the CL domain.

79. 60. The multispecific antigen-binding protein of claim 59, wherein the CH1 domain of H2 comprises an amino acid substitution mutation at S183 (EU numbering) and the CL domain of L2 comprises an amino acid substitution mutation V133K (EU numbering).

80. 80. The multispecific antigen-binding protein of claim 79, wherein the amino acid at position S183 in the CH1 domain of H2 is replaced with a positively charged amino acid and the amino acid at position V133 in the CL domain of L2 is replaced with a negatively charged amino acid.

81. 80. The multispecific antigen-binding protein of claim 79, wherein the amino acid at position S183 in the CH1 domain of H2 is replaced with a negatively charged amino acid and the amino acid at position V133 in the CL domain of L2 is replaced with a positively charged amino acid.

82. 80. The multispecific antigen binding protein of claim 79, wherein the CH1 domain of H2 comprises a S183E mutation and the CL domain of L2 comprises a V133K mutation.

83. 83. The multispecific antigen binding protein of claim 82, further comprising a Q39E mutation in the VH domain of H1, a Q38K mutation in the VL domain of L1, a Q39K mutation in the VH domain of H2, and a Q38E mutation in the VL domain of L2.

84. 80. The multispecific antigen binding protein of claim 79, wherein the CH1 domain of H2 comprises a S183K mutation and the CL domain of L2 comprises a V133E mutation.

85. 85. The multispecific antigen binding protein of claim 84, further comprising a Q39K mutation in the VH domain of H1, a Q38E mutation in the VL domain of L1, a Q39E mutation in the VH domain of H2, and a Q38K mutation in the VL domain of L2.

86. 80. The multispecific antigen-binding protein of any one of claims 50 to 59 and 64 to 78, wherein the CH1 domain of H2 comprises an amino acid substitution at EU position S183 and the CL domain of L2 comprises an amino acid substitution at EU position V133.

87. 86. The multispecific antigen-binding protein of claim 85, wherein the interaction between the amino acid substitution at EU position S183 on the CH1 domain of H2 and the amino acid substitution at EU position V133 on the CL domain of L2 is by electrostatic interaction.

88. 88. The multispecific antigen-binding protein of claim 87, wherein the amino acid at EU position S183 on the CH1 domain of H2 is replaced by a positively charged residue and the amino acid at EU position V133 on the CL domain of L2 is replaced by a negatively charged residue.

89. 88. The multispecific antigen-binding protein of claim 87, wherein the amino acid at EU position S183 on the CH1 domain of H2 is replaced with a negatively charged residue and the amino acid at EU position V133 on the CL domain of L2 is replaced with a positively charged residue.

90. 90. The multispecific antigen-binding protein of any one of claims 86 to 89, wherein the CH1 domain of H2 comprises an amino acid substitution at a position selected from the group consisting of EU positions S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K, and the CL domain of L2 comprises an amino acid substitution at a position selected from the group consisting of EU positions V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

91. (a) the CH1 domain of H2 comprises a mutation at EU position S183D and the CL domain of L2 comprises a mutation at EU position V133K; or (b) the CH1 domain of H2 comprises a mutation at EU position S183E and the CL domain of L1 comprises a mutation at EU position V133K; or (c) the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises a mutation at EU position V133E; or (d) the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133S; or (e) the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises a mutation at EU position V133L; or (f) the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133W; or (g) the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises a mutation at EU position V133K; or (h) the CH1 domain of H2 comprises a mutation at EU position S183A and the CL domain of L2 comprises a mutation at EU position V133R; or (i) the CH1 domain of H2 contains a mutation at EU position S183A and the CL domain of L2 contains a mutation at EU position V133D; or (j) the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133E; or (k) the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133S; or (l) the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133L; or (m) the CH1 domain of H2 comprises a mutation at EU position S183T and the CL domain of L2 comprises a mutation at EU position V133W; or (n) the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133K; or (o) the CH1 domain of H2 comprises a mutation at EU position S183T and the CL domain of L2 comprises a mutation at EU position V133R; or (p) the CH1 domain of H2 contains a mutation at EU position S183T and the CL domain of L2 contains a mutation at EU position V133D; or (q) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133E; or (r) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133S; or (s) the CH1 domain of H2 comprises a mutation at EU position S183V and the CL domain of L2 comprises a mutation at EU position V133L; or (t) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133W; or (u) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133K; or (v) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133R; or (w) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133D; (x) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL domain of L2 comprises a mutation at EU position V133E; or (y) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL domain of L2 comprises a mutation at EU position V133S; or (z) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL domain of L2 comprises a mutation at EU position V133L; or (aa) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL of the L2 domain comprises a mutation at EU position V133W; or (bb) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL of the L2 domain comprises a mutation at EU position V133K; or (cc) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL domain of L2 comprises a mutation at EU position V133R; or (dd) the CH1 domain of H2 comprises a mutation at EU position S183Y and the CL domain of L2 comprises a mutation at EU position V133D; or (ee) the CH1 domain of H2 comprises a mutation at EU position S183F and the CL domain of L2 comprises a mutation at EU position V133E; or (ff) the CH1 domain of H2 comprises a mutation at EU position S183F and the CL domain of L2 comprises a mutation at EU position V133S; or (gg) the CH1 domain of H2 comprises a mutation at EU position S183F and the CL domain of L2 comprises a mutation at EU position V133L; (hh) the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133W; or (ii) the CH1 domain of H2 comprises a mutation at EU position S183F and the CL domain of L2 comprises a mutation at EU position V133K; or (jj) the CH1 domain of H2 contains a mutation at EU position S183F and the CL domain of L2 contains a mutation at EU position V133R; or (kk) the CH1 domain of H2 comprises a mutation at EU position S183F and the CL of the L2 domain comprises a mutation at EU position V133D; or (ll) the CH1 domain of H2 comprises a mutation at EU position S183H and the CL domain of L2 comprises a mutation at EU position V133S; or (mm) the CH1 domain of H2 contains a mutation at EU position S183H and the CL domain of L2 contains a mutation at EU position V133L; (nn) the CH1 domain of H2 comprises a mutation at EU position S183H and the CL domain of L2 comprises a mutation at EU position V133W; (oo) the CH1 domain of H2 comprises a mutation at EU position S183N and the CL domain of L2 comprises a mutation at EU position V133L; or (pp) the CH1 domain of H2 comprises a mutation at EU position S183E and the CL domain of L2 comprises a mutation at EU position V133L; or (qq) The multispecific antigen binding protein of claim 90, wherein the CH1 domain of H2 comprises a S183K mutation and the CL domain of L2 comprises a V133E mutation.

92. (a) the CH1 domain of H2 comprises a mutation at EU position S183D and the CL domain of L2 comprises a mutation at EU position V133K; or (b) the CH1 domain of H2 comprises a mutation at EU position S183E and the CL domain of L2 comprises a mutation at EU position V133K; or (c) the CH1 domain of H2 comprises a mutation at EU position S183T and the CL domain of L2 comprises a mutation at EU position V133K; or (d) the CH1 domain of H2 contains a mutation at EU position S183V and the CL domain of L2 contains a mutation at EU position V133E; or (e) the CH1 domain of H2 comprises a mutation at EU position S183K and the CL domain of L2 comprises a mutation at EU position V133E.

93. 80. The multispecific antigen-binding protein of any one of claims 50 to 59 and 64 to 78, wherein the CH1 domain of H2 and the CL domain of L2 do not comprise an amino acid substitution.

94. 94. The multispecific antigen-binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises an amino acid substitution at Kabat position Q39 and the V1 VL domain comprises an amino acid substitution at Kabat position Q38.

95. 95. The multispecific antigen binding protein of claim 94, wherein the amino acid at Kabat position Q39 on the VH domain of H1 is replaced with a positively charged residue and the amino acid at Kabat position Q38 on the VL domain of L1 is replaced with a negatively charged residue.

96. 95. The multispecific antigen binding protein of claim 94, wherein the amino acid at Kabat position Q39 on the VH domain of H1 is replaced with a negatively charged residue and the amino acid at Kabat position Q38 on the VL domain of L1 is replaced with a positively charged residue.

97. 97. The multispecific antigen-binding protein of any one of claims 94 to 96, wherein said positively charged residues are selected from the group consisting of R and K.

98. 98. The multispecific antigen-binding protein of any one of claims 94 to 97, wherein said negatively charged residues are selected from the group consisting of D and E.

99. 94. The multispecific antigen binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39E and the L1 VL domain comprises a substitution at Kabat position Q38K.

100. 94. The multispecific antigen binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39E, the L1 VL domain comprises a substitution mutation at Kabat position Q38K, and the H2 VH domain comprises a substitution mutation at Kabat position Q39K.

101. 94. The multispecific antigen binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39E, the L1 VL domain comprises a substitution mutation at Kabat position Q38K, the H2 VH domain comprises a substitution mutation at Kabat position Q39K, and the L2 VL domain comprises a substitution mutation at Kabat position Q38E.

102. 94. The multispecific antigen-binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39K and the L1 VL domain comprises a substitution at Kabat position Q38E.

103. 94. The multispecific antigen binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39K, the L1 VL domain comprises a substitution mutation at Kabat position Q38E, and the H2 VH domain comprises a substitution mutation at Kabat position Q39E.

104. 94. The multispecific antigen binding protein of any one of claims 50 to 59 and 64 to 93, wherein the H1 VH domain comprises a substitution mutation at Kabat position Q39K, the L1 VL domain comprises a substitution mutation at Kabat position Q38E, the H2 VH domain comprises a substitution mutation at Kabat position Q39E, and the L2 VL domain comprises a substitution mutation at Kabat position Q38K.

105. 104. The multispecific antigen-binding protein of any one of claims 50 to 103, wherein each of the H1 comprises an Fc region comprising a CH2 and a CH3 domain.

106. 106. The multispecific antigen-binding protein of any one of claims 50 to 105, wherein the H1 and / or H2 Fc region is human IgG1, human IgG2, or human IgG4 Fc.

107. 106. The multispecific antigen-binding protein of any one of claims 50 to 105, wherein the H1 and / or H2 Fc region is murine IgG1, murine IgG2, or murine IgG4 Fc.

108. 108. A multispecific antigen-binding protein as described in claim 106 or 107, wherein the H1 and H2 CH3 domains each meet at an interface, and each of the CH3 domains comprises amino acid substitutions such that the Fc region of H1 preferentially pairs with the Fc region of H2 compared to H1.

109. 109. The multispecific antigen-binding protein of claim 108, wherein said amino acid substitutions in said CH3 domain result in greater electrostatic complementarity.

110. 109. The multispecific antigen-binding protein of claim 108, wherein the amino acid substitutions in the CH3 domain result in greater steric complementarity.

111. the H1 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the H1 CH3 domain that interacts with the H2 CH3 domain; 109. The multispecific antigen binding protein of claim 108, wherein the H2 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the H2 CH3 domain which interacts with the H1 CH3 domain.

112. the H2 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the H2 CH3 domain that interacts with the H1 CH3 domain; and, 109. The multispecific antigen binding protein of claim 108, wherein the H1 CH3 domain is altered such that within the CH3 / CH3 interface, one or more amino acid residues are replaced amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the H1 CH3 domain which interacts with the H2 CH3 domain.

113. 113. The multispecific antigen-binding protein of claim 111 or 112, wherein the protuberance is a knob mutation.

114. 114. The multispecific binding protein of claim 113, wherein the knob mutation comprises T366W (EU numbering).

115. 113. A multispecific antigen-binding protein according to claim 111 or 112, wherein the cavity is a hole mutation.

116. 116. The multispecific antigen-binding protein of claim 115, wherein the hole mutations include at least one, at least two, or all three of T366S, L368A, and Y407V (EU numbering).

117. 117. The multispecific antigen-binding protein of any one of claims 1 to 116, wherein H1 preferentially pairs with L1 compared to L2 and H2 preferentially pairs with L2 compared to L1.

118. 118. The multispecific antigen-binding protein of any one of claims 1 to 117, wherein the first antigen and the second antigen are the same.

119. 119. The multispecific antigen-binding protein of any one of claims 1 to 118, wherein the first heavy chain / light chain pair and the second heavy chain / light chain pair each bind to a different epitope on the same antigen.

120. 118. The multispecific antigen-binding protein of any one of claims 1 to 117, wherein the first antigen and the second antigen are different.

121. 121. A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1 to 120 and a pharma- ceutically acceptable carrier.

122. 122. A method for treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 121.

123. 123. An isolated nucleic acid(s) encoding at least one polypeptide sequence of a multispecific antigen binding protein according to any one of claims 1 to 122.

124. A vector comprising the nucleic acid(s) of claim 123.

125. 125. An isolated host cell comprising the nucleic acid(s) of claim 123 or the vector of claim 124.

126. 126. The host cell of claim 125, wherein the host cell is a prokaryotic host cell, an E. coli cell, a eukaryotic host cell, a yeast cell, a mammalian cell, or a CHO cell.

127. A method for producing a multispecific antigen-binding protein according to any one of claims 1 to 120, comprising the steps of: (a) obtaining the H1, H2, L1, and L2 polypeptides; (b) allowing H1 to preferentially pair with L1 relative to L2, and allowing H2 to preferentially pair with L2 relative to L1 to form the multispecific antigen-binding protein.

128. A method for producing a multispecific antigen-binding protein according to any one of claims 1 to 120, comprising the steps of: (a) introducing into a host cell a set of polynucleotides encoding H1, L1, H2, and L2; (b) culturing said host cell to produce said multispecific antigen binding protein.

129. 129. The method of claim 128, wherein the set of polynucleotides encoding H1, L1, H2, and L2 are introduced into the host cell in a predetermined ratio.

130. 130. The method of claim 129, further comprising determining an optimal ratio of said polynucleotides for introduction into said host cell.

131. 131. The method of any one of claims 127 to 130, wherein the multispecific antigen binding protein is produced in a relative yield of 60% or greater.

132. 132. The method of any one of claims 127-131, wherein the multispecific antigen binding protein is produced in a relative yield of at least about 70%, at least about 71%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or greater than about 99%.

133. 127. A method of producing a multispecific antigen binding protein comprising culturing a host cell according to claim 125 or 126 and producing said multispecific antigen binding protein.

134. 134. The method of any one of claims 127-133, further comprising recovering the multispecific antigen binding protein.

135. 135. A multispecific antigen-binding protein produced by the method of any one of claims 127 to 134.

136. A library comprising a plurality of polynucleotides encoding a plurality of multispecific antigen-binding proteins according to any one of claims 1 to 120.

137. 1. A method for screening for a multispecific antigen-binding protein that binds to a first antigen and a second antigen, comprising the steps of: (a) obtaining a plurality of multispecific antigen binding proteins from the library of claim 123; (b) assaying for binding of said plurality of multispecific antigen binding proteins to said first and second antigens; and (c) identifying said multispecific antigen binding protein that binds to said first and second antigens.

138. 1. A computer readable medium for evaluating a multispecific antigen binding protein comprising: 1) a first heavy chain / light chain pair that binds to a first antigen comprising a first heavy chain sequence (H1) and a first light chain sequence (L1); and 2) a second heavy chain / light chain pair that binds to a second antigen comprising a second heavy chain sequence (H2) and a second light chain sequence (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), a) a dataset comprising data representing amino acid substitutions in H1, L1, H2, and L2, wherein at least one of the amino acid substitutions in the CH1 domain of H1 comprises an amino acid substitution at EU position F170 or L128, and the CL domain comprises amino acid substitutions at EU position S176 or F118 and L135; and / or b) a dataset comprising data representing amino acid substitutions in H1, L1, H2, and L2, wherein at least one of the amino acid substitutions in the CH1 domain of H1 comprises an amino acid substitution at EU position S183, and the CL domain of L1 comprises an amino acid substitution at EU position V133; and (b) computer-executable code for determining the likelihood that H1 preferentially pairs with L1 compared to L2 and / or the likelihood that H2 preferentially pairs with L2 compared to L1.

139. 139. The computer readable medium of claim 138, wherein said at least one of said amino acid substitutions in the CH1 domain of said H1 comprises an amino acid substitution at EU position F170 or L128 and V185.

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