Ch1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising the same

Abstract

To provide an engineered bispecific antibody having appropriate heavy chain-light chain pairing.SOLUTION: The present invention provides CH1 domain variants engineered for preferential binding to either a kappa CL domain or a lambda CL domain, as well as polypeptides, e.g., antibody heavy chains or antibodies, comprising such engineered CH1 domain variants, and pharmaceutical compositions comprising such CH1 domain variants and / or such polypeptides, and methods for making and using such CH1 domain variants. The CH1 domain variants minimize heavy chain-light chain mispairing and promote cognate heavy chain-light chain pairing, thereby improving the generation of multispecific, e.g., bispecific, antibodies. Also provided are methods of making CH1 domain variant libraries and methods of identifying one or more CH1 domain variants.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 908,367, filed September 30, 2019, and entitled "CH1 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL LIGHT CHAIN ​​PAIRING AND MULTISPECIFIC ANTIBODIES COMPRISING THE SAME," the contents of which are incorporated by reference in their entirety.

[0002] The present invention relates to CH1 domain variants containing at least one amino acid substitution that promotes proper heavy-light chain pairing, and antibody heavy chains and antibodies, particularly multispecific antibodies, comprising same. The present invention further relates to compositions comprising such antibodies and uses thereof, e.g., therapeutic or diagnostic methods. The present invention further relates to methods for generating CH1 domain variant libraries and for identifying one or more CH1 domain variants. [Background technology]

[0003] There is a continuing effort to develop antibody therapeutics with multiple antigen-binding specificities, e.g., bispecific antibodies. Bispecific antibodies can be used to interfere with multiple surface receptors associated with cancer, inflammatory processes, or other disease states. Bispecific antibodies can also be used to place targets in close proximity, regulate protein complex formation, or drive cell-cell contact. The production of bispecific antibodies was first reported in the early 1960s (Nisonoff et al., Arch Biochem Biophys 1961 93(2):460-462), and the first monoclonal bispecific antibodies were generated using hybridoma technology in the 1980s (Milstein et al., Nature 1983 305(5934):537-540). Interest in bispecific antibodies has grown significantly in the past decade due to their therapeutic potential, and bispecific antibodies are now being used in the clinic. For example, blinatumomab and emicizumab have been approved for the treatment of certain cancers (for recent reviews of bispecific antibody production methods and characteristics of bispecific antibodies approved for pharmaceutical use, see Sedykh et al., Drug Des Devel Ther 12:195-208 (2018) and Labrijn et al. Nature Reviews Drug Discovery 18:585-608 (2019)).

[0004] While bispecific antibodies have shown significant advantages over monospecific antibodies, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, their lack of stability, and their short half-life in humans. A wide variety of methods have been developed over the past decades to improve the production of bispecific antibodies. These include recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knob-in-hole" engineering (e.g., U.S. Pat. No. 5,731,168), immunoglobulin crossover technology (also known as Fab domain swapping or CrossMab format) (see, e.g., WO 2009 / 080253; Schaefer et al., EMBO J. 10:3655 (1991)), and the use of Fab domains in the cross-over process (see, e.g., WO 2009 / 080253; Schaefer et al., EMBO J. 10:3655 (1991)). al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), engineering electrostatic steering effects for the creation of antibody Fc heterodimeric molecules (WO 2009 / 089004A1), cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)), "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2020, 2021, 2021, 2022, 2023, 2023, 2024, 2025, 2026, 2027, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 203 al., J. Immunol, 152:5368 (1994)), as well as trispecific antibodies, as described, for example, in Tutt et al. J. Immunol 147:60 (1991).

[0005] Despite these improvements, the generation of bispecific antibodies with correct heavy-light chain pairing remains a challenge. Bispecific antibodies can be formed by co-expression of two different heavy chains and two different light chains. Because heavy chains have evolved to bind to light chains in a relatively promiscuous manner, it remains a challenge to properly form bispecific antibodies in the desired format. As a result, co-expression of two heavy chains and two light chains can result in a scrambled heavy-light chain pairing - a complex mixture of 16 possible combinations, representing 10 different antibodies, of which only one corresponds to the desired bispecific antibody (maximum yield of 12.5% ​​in the mixture if there is complete randomness). This mispairing (also called the chain pairing problem) remains a major challenge for the generation of bispecific antibodies, since homogeneous pairing is essential for manufacturability and efficacy.

[0006] One strategy used to mitigate mispairing is to generate bispecific antibodies with a common light chain (see, e.g., Merchant et al., Nat. Biotech. 16:677-681 (1998)). Alternatively, a single common heavy chain and two different light chains (one kappa and one lambda) may be used (see, e.g., Fischer et al., Nature Commun. 6:6113 (2015)). However, this strategy requires identifying antibodies with a common chain, which is difficult and tends to compromise the specificity of each binding arm, substantially reducing diversity (see, e.g., Wang et al., MABS 10(8):1226-1235 (2018)).

[0007] Other approaches to improve correct heavy-light chain pairing include the CrossMab technology (Roche), in which the light chain of one antigen-binding fragment (Fab) arm or one of the subdomains therein is exchanged for the corresponding region of the heavy chain Fd region, and the DuetMab technology (MedImmune), in which the native disulfide bond of one Fab arm is replaced with an engineered disulfide bond. However, these approaches require significant modifications to the native IgG format, which may result in compounds that do not adequately resemble natural antibodies.

[0008] Another strategy is to utilize amino acid substitutions in the constant and / or variable regions of the heavy and light chains of the IgG format to reduce or eliminate mispairing of the heavy and light chains. To the best of the inventors' knowledge, modifying only the CH1 domain has not been demonstrated to solve the chain pairing or mispairing problems often observed during the expression of multispecific antibodies. Rather, multispecific antibodies engineered to include CH1 domain variants have also required modifications outside the CH1 domain to address chain pairing issues, such as the CL domain and in certain instances the VH, CH2, CH3, and / or VL domains. Examples include Lewis et al., Nature Biotech. 32(2):191-198 (2014), who used mutated CH1 and CL domains, CRD1 (heavy chain substitutions at D148K, F170T, V185F, and light chain substitutions K129D, L135F; in an attempt to drive preferential pairing of engineered heavy and light chains and prevent pairing of heavy and light chain domains with wild-type constant domains;They generated CRD1 (with heavy chain substitutions H168A and F170T, and light chain substitutions L135Y, S176W) and CRD2 (with heavy chain substitutions H168A and F170T, and light chain substitutions L135Y, S176W). However, Lewis et al. reported that any pairing specificity obtained by the mutated CH1 and CL domains in the absence of variable domains did not translate to a full-length IgG format without further engineering in the VH-VL interface, i.e., substitutions in the VH-VL interface were required along with the CL and CH1 domain substitutions to achieve preferential heavy-light chain pairing. Engineering the CH1 and CL domains to contain charged amino acid residues has also been shown to promote preferential heavy-light chain pairing (see, e.g., U.S. Pat. No. 10,047,163). Bispecific antibodies with at least two Fab fragments with different CH1 and CL domains are also known, where one Fab fragment has substitutions in the CH1 and Cκ domains to drive preferential pairing (see US Patent Application Publication No. 20180022829 and US Patent No. 9,631,031, which disclose CH1:T187E and Cκ:N137K+S114A, CH1:L145Q+S183V and Cκ:V133T+S176V, CH1:L128A+L145E and Cκ:V133W, CH1:V185A and Cκ:L135W+N137A). Further examples of specific CH1 domain substitutions that are claimed to promote preferential heavy-light chain pairing when the light chain, or optionally CH2, CH3, and / or VH, is appropriately substituted to promote preferential heavy-light chain pairing include: A141C / L, K147D, G166D, G166K, or substitutions with cysteines at positions 128, 129, 162, or 171 (WO2019183406 (Invenra Inc.)); substitutions of cysteine ​​at positions 126 or 220 with valine or alanine, or substitutions of non-cysteine ​​at positions 128, 141, or 168 with cysteine, L145F, K147A, F170V, S183F, or V185W / F (U.S. Pat. No. 9,527,927 (MedImmune));172A and 174G (WO 2020060924 (Dualogics)); A172R and 174G, or substitution of residue 190 with M or I (U.S. Pat. No. 10,047,167 (University of North Carolina at Chapel Hill and Eli Lilly)); L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and V185A / L (U.S. Pat. App. Pub. No. 20180177873 (Genentech)); 131C / S, 133R / K, 137E / G, 138S / G 178S / Y, 192N / S, and / or 193F / L (U.S. Pat. No. 10,487,156 (Argenx) 145D / E / R / H / K (IMGT position 26) (WO 2018141894 (Merck)); 124K / E / R / D (U.S. Pat. No. 10,392,438 (Pfizer)); 133V, 150A, 150D, 152D, 173D, or 188W (U.S. Pat. App. Pub. No. 201900 No. 23810 (MIT); 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L, and / or 190S / A / G / Y (U.S. Patent Application Publication No. 20180179296 and U.S. Patent No. 9,914,785 (Zymeworks); 143A / E / R / K / D and 145T / L (U.S. Patent No. 10,077,298 (Zymeworks)); 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, 139W / G / C, 179E, or 186R (U.S. Patent Application Publication No. 20170204199 (Zymeworks)); substitutions with cysteine ​​at positions 126, 127, 128, 134, 141, 171, or 173 (Zenyaku Kogyosha); L145Q, H168A, F170G, S183V, and T187E (WO 2020127354 (Alligator Bioscience));143D / E, 145T, 190E / D and 124R (WO 2017 / 059551 (Zymeworks)). Also, U.S. Patent No. 9,150,639, Kyowa Hakko Kirin Co., Ltd. reports that they have generated heavy chains containing A140C, K147C, or S183C for the purpose of introducing cysteines that allow for chemical tuning. Kyowa Hakko Kirin Co., Ltd. suggests that antibody variants containing these heavy chain mutations may contain wild-type light chains, although there is no indication that this would promote preferential heavy-light chain pairing.

[0009] Yet another strategy used to minimize heavy-light chain mismatching is to utilize different light chains, such as light chains with different constant domains.For example, Loew et al. have generated multispecific antibodies with kappa and lambda light chains and observed minimal mismatching, because certain naturally occurring kappa light chains have high fidelity and do not pair with heavy chains from lambda antibodies, and vice versa (WO2018057955).Unfortunately, the applicability of this methodology is limited to those light chains with high fidelity. Others have generated multispecific antibodies using kappa and lambda light chains that utilize amino acid substitutions in both the heavy and light chains to electrostatically or conformationally drive preferential pairing (see, e.g., WO2017059551 (Zymeworks), US20140154254 (Amgen), and US10,047,163 (AbbVie Stemcentrx)). However, introducing multiple amino acid substitutions in both the heavy and light chains represents an additional technical hurdle and may have deleterious effects on antibody function and / or immunogenicity. Summary of the Invention

[0010] The object of the present invention is to provide an engineered bispecific antibody with proper heavy-light chain pairing. In one aspect, provided herein is a CH1 domain variant polypeptide (also referred to herein as a CH1 domain variant) that promotes preferential pairing of a heavy chain with a specific light chain, and a polypeptide, such as an antibody, comprising the same. The CH1 domain variant contains at least one amino acid substitution (e.g., relative to a parent sequence, such as a wild type).

[0011] In some embodiments, the CH1 domain variant contains at least one amino acid substitution at a CH1 domain position that forms an interface with the CL domain of the light chain, including but not limited to, positions 140 and / or 141 or 147 and / or 183 (EU numbering), which promotes preferential pairing of the CH1 domain variant-containing heavy chain with a particular light chain, e.g., CH1 domain variant 141 preferentially pairs with lambda CL domains as opposed to kappa CL domains, whereas CH1 domain variants 147F and / or 183R, 183K or 183Y preferentially pairs with kappa CL domains as opposed to lambda CL domains.

[0012] In some embodiments, the CH1 domain variant contains at least one amino acid substitution in a CH1 domain position that forms the interface between the CH1 domain and VH, such as at position 151 of CH1 (EU numbering).

[0013] This preferential pairing of constant domains is expected to drive pairing of full-length light chains, e.g., variable domains, with heavy chains, thereby resulting in a solution to the chain pairing problem for bispecificity. In particular, the CH1 domain variant polypeptides contain amino acid substitutions at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering. Optionally, such CH1 domain variant polypeptides preferentially pair (i) with a kappa light chain constant region ("CL") domain compared to a lambda CL domain and / or with a kappa light chain polypeptide compared to a lambda light chain polypeptide, or (ii) with a lambda CL domain compared to a kappa CL domain and / or with a lambda light chain polypeptide compared to a kappa light chain polypeptide.

[0014] Optionally, in some embodiments, certain CH1 domain variants may be excluded, and CH1 domain variants according to the present invention may satisfy the following:

[0015] (a) the CL domain that is preferentially paired by the CH1 domain variant does not contain an amino acid substitution if residue 141 on CH1 is substituted with C or L, residue 166 is substituted with D or K, residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or residue 147 is substituted with D;

[0016] (b) the CL domain that is preferentially paired with the CH1 domain variant does not contain an amino acid substitution if position 126 or 220 on CH1 is substituted with valine or alanine, if the non-cysteines at positions 128, 141, or 168 are substituted with cysteine, or if the CH1 substitution is L145F, K147A, F170V, S183F, or V185W / F;

[0017] (c) if residue 172 on CH1 is substituted with 172R, if residue 174 is mutated to 174G, or if residue 190 is substituted with 190M or 190I, these are not the only substitutions that said CH1 contains;

[0018] (d) when the CH1 substitution consists of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V, and / or V185A / L, the CL domain with which it is preferentially paired by the CH1 domain variant is unaltered;

[0019] (e) if the CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S, and / or 193F / L, these are not the only CH1 substitutions and / or, in the bispecific antibody, the CH1 domains are of the same human immunoglobulin subtype or allotype;

[0020] (f) if the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), the corresponding LC substitution, 129D / E / R / H / K (IMGT position 18), is not present;

[0021] (g) if the CH1 substitution consists of 124K / E / R / D, there is no corresponding substitution at position 176 of LC with which the CH1 domain variant preferentially pairs;

[0022] (h) if the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D, and / or 188W, there is no corresponding substitution in the LC that is preferentially paired by the CH1 domain variant;

[0023] (i) if the CH1 substitution consists of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L, and / or 190S / A / G / Y, there is no corresponding substitution in the LC that is preferentially paired by the CH1 domain variant;

[0024] (j) if the CH1 substitutions consist of 143A / E / R / K / D and 145T / L, there are no corresponding substitutions in the LC that are preferentially paired by the CH1 domain variant;

[0025] (k) if the CH1 substitutions consist of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, and 139W / G / C, 179E, and / or 186R, there are no corresponding substitutions in the LC that are preferentially paired by the CH1 domain variant;

[0026] (l) if the CH1 substitution consists of a substitution with a cysteine ​​at position 126, 127, 128, 134, 141, 171, or 173, the corresponding LC position is not modified to form a disulfide bond;

[0027] (m) when the CH1 substitution consists of L145Q, H168A, F170G, S183V and / or T187E, there is no corresponding substitution in the kappa LC or lambda LC with which the CH1 domain variant is preferentially paired;

[0028] (n) when the CH1 substitution consists of 143D / E, 145T, 190E / D, and / or 124R, there is no corresponding substitution in the LC with which the CH1 domain variant preferentially pairs; or

[0029] (o) If the CH1 substitution consists of A140C, K147C and / or S183C, the substitution is in the LC that is preferentially paired with the CH1 domain variant.

[0030] In some embodiments, the CH1 domain variant polypeptide comprises an amino acid substitution at one or more of positions 118, 124, 126-129, 131, 132, 134, 136, 139, 143, 145, 147-151, 153, 154, 170, 172, 175, 176, 181, 183, 185, 190, 191, 197, 201, 203-206, 210, 212-214, and 218 according to EU numbering, optionally whereby the CH1 domain variant polypeptide preferentially pairs (i) with a kappa CL domain (or a kappa CL containing polypeptide) compared to a lambda CL domain (or a lambda CL containing polypeptide); and / or (ii) with a kappa light chain polypeptide compared to a lambda light chain polypeptide.

[0031] In certain embodiments, such CH1 domain variants comprise amino acid substitutions at positions 147, 183, or 147 and 183.

[0032] In certain embodiments, such CH1 domain variants contain one or more of the following amino acid substitutions: position 118 is substituted with G, position 124 is substituted with H, R, E, L, or V, position 126 is substituted with A, T, or L, position 127 is substituted with V or L, position 128 is substituted with H, position 129 is substituted with P, position 131 is substituted with A, position 132 is substituted with P, position 134 is substituted with H, position 135 is substituted with H, position 136 is substituted with H, position 137 is substituted with H, position 138 is substituted with H, position 139 is substituted with H, position 140 is substituted with H, position 141 is substituted with H, position 142 is substituted with H, position 143 is substituted with H, position 144 is substituted with H, position 145 is substituted with H, position 146 is substituted with H, position 147 is substituted with H, position 148 is substituted with H, position 149 is substituted with H, position 150 is substituted with H, position 151 is substituted with H, position 152 is substituted with H, position 153 is substituted with H, position 154 is substituted with H, position 155 is substituted with H, position 156 is substituted with H, position 157 is substituted with H, position 158 is substituted with H, position 159 is substituted with H, position 160 is substituted with H, position 161 is substituted with H, position 162 is substituted with H, position 163 is substituted with H, position 164 is substituted with H, position 165 is substituted with H, position 166 is substituted with H, position 167 is substituted with H, position 168 is substituted with H, is substituted with G, position 136 is substituted with E, position 139 is substituted with I, position 143 is substituted with V or S, position 145 is substituted with F, I, N or T, position 147 is substituted with F, I, L, R, T, S, M, V, N, E, H, Y, Q, A or G, position 148 is substituted with I, Q, Y or G, position 149 is substituted with C, S or H, position 150 is substituted with L or S, 15 Position 1 is substituted with A or L, position 153 is substituted with S, position 154 is substituted with M or G, position 170 is substituted with G or L, position 172 is substituted with V, position 175 is substituted with G, L, E, A, position 176 is substituted with P, position 181 is substituted with Y, Q, or G, position 183 is substituted with I, W, F, E, Y, L, K, Q, N, R, or H, position 185 is substituted with W, Position 90 is substituted with P, position 191 is substituted with I, position 197 is substituted with A, position 201 is substituted with S, position 203 is substituted with S, position 204 is substituted with Y, position 205 is substituted with Q, position 206 is substituted with S, position 210 is substituted with R, position 212 is substituted with G, position 213 is substituted with E or R, position 214 is substituted with R, and position 218 is substituted with Q. In an embodiment, a kappa-preferring CH1 domain variant polypeptide may comprise: (i) amino acid residues F, I, L, R, T, S, M, V, N, E, H, Y, or Q at position 147; and / or (ii) amino acid residues I, W, F, E, Y, L, K, Q, N, or R at position 183.

[0033] In some preferred embodiments of the kappa-preferring CH1 domain variant, the CH1 domain variant polypeptide may comprise (i) amino acid residue R, K, or Y at position 183 and / or (ii) amino acid residue F at position 147.

[0034] In further embodiments, the CH1 domain variant polypeptide comprises (i) amino acid residue F at position 147 and amino acid residue R at position 183, (ii) amino acid residue F at position 147 and amino acid residue K at position 183, (iii) amino acid residue F at position 147 and amino acid residue Y at position 183, (iv) amino acid residue R at position 183, (v) amino acid residue K at position 183, or (vi) amino acid residue Y at position 183. Optionally, such a CH1 domain variant may comprise the amino acid sequence of (i) SEQ ID NO:137, (ii) SEQ ID NO:138, (iii) SEQ ID NO:139, (iv) SEQ ID NO:60, (v) SEQ ID NO:41, or (vi) SEQ ID NO:136.

[0035] In some embodiments, the CH1 domain variant polypeptide comprises an amino acid substitution at a CH1 amino acid position in the interface between CH1 and VH. Optionally, the CH1 amino acid position in such interface is position 151. Further optionally, such a CH1 domain variant can comprise amino acid residue A or L at position 151.

[0036] In some embodiments, the CH1 domain variant polypeptide further comprises one or more amino acid substitutions that increase pairing of the CH1 domain with (i) a kappa CL domain compared to a lambda CL domain, and / or (ii) a kappa light chain polypeptide compared to a lambda light chain polypeptide.

[0037] In some embodiments, the CH1 domain variant polypeptide of any one of claims 2-10 provides at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, or a 100% increase in pairing with a kappa CL domain compared to a lambda CL domain, and / or with a kappa light chain polypeptide compared to a lambda light chain polypeptide. The increase in kappa pairing may optionally be measured by liquid chromatography-mass spectrometry (LCMS).

[0038] In some embodiments a CH1 domain variant polypeptide of any one of claims 2-10, which provides at least a 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold increase in (i) association with a kappa CL domain compared to a lambda CL domain, and / or (ii) association with a kappa light chain polypeptide compared to a lambda light chain polypeptide. The increase in kappa synapsis may optionally be quantified by flow cytometry, for example, by comparing the ratio of mean fluorescent intensity (MFI) of kappa CL staining to lambda CL staining.

[0039] In some embodiments, a CH1 domain variant polypeptide according to the invention comprises an amino acid substitution at one or more of positions 119, 124, 126, 127, 130, 131, 133, 134, 138-142, 152, 163, 168, 170, 171, 175, 176, 181, 183-185, 187, 197, 203, 208, 210-214, 216, and 218 according to EU numbering. Optionally, the CH1 domain variant preferentially pairs (i) with a lambda CL domain compared to a kappa CL domain, and / or (ii) with a lambda light chain polypeptide compared to a kappa light chain polypeptide.

[0040] In certain embodiments, the lambda-preferring CH1 domain variant polypeptide comprises an amino acid substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218.

[0041] In certain embodiments, the lambda-preferring CH1 domain variant polypeptide comprises one or more of the following amino acid substitutions: position 119 is substituted with R, position 124 is substituted with V, position 126 is substituted with V, position 127 is substituted with G, position 130 is substituted with H or S, position 131 is substituted with Q, T, N, R, V, or D, position 133 is substituted with D, T, L, E, S, or P. position 134 is substituted with A, H, I, P, V, N, or L, position 138 is substituted with R, position 139 is substituted with A, position 140 is substituted with I, V, D, Y, K, S, W, R, L, or P, position 141 is substituted with D, K, E, T, R, Q, V, or M, position 142 is substituted with M, position 152 is substituted with G, position 163 is substituted with M, position 168 is substituted with F, I, or V. Position 170 is substituted with N, G, E, S, or T; position 171 is substituted with N, E, G, S, A, or D; position 175 is substituted with D or M; position 176 is substituted with R or M; position 181 is substituted with V, L, A, K, or T; position 183 is substituted with L or V; position 184 is substituted with R; position 185 is substituted with M, L, S, R, or T; position 187 is substituted with R, D, E, Y, or S, position 197 is substituted with S, position 203 is substituted with D, position 208 is substituted with I, position 210 is substituted with T, position 211 is substituted with A, position 212 is substituted with N, position 213 is substituted with E, position 214 is substituted with R, position 216 is substituted with G, and position 218 is substituted with L, E, D, P, A, H, S, Q, N, T, I, M, G, C, K, or W.

[0042] In further embodiments, the lambda-preferring CH1 domain variant polypeptide comprises any one or more of the following: (i) amino acid residue V at position 126; (ii) amino acid residue G at position 127; (iii) amino acid residue V at position 131; (iv) amino acid residue S at position 133; (v) amino acid residue R at position 138; (vi) amino acid residue I or V at position 140; (vii) amino acid residue D, K, E, or T at position 141; (viii) amino acid residue M at position 142; (ix) amino acid residue N at position 168; (x) amino acid residue I, (x) amino acid residue E, G, or S at position 170, (xi) amino acid residue E, D, G, S, or A at position 171, (xii) amino acid residue M at position 175, (xiii) amino acid residue R at position 176, (xiv) amino acid residue K, V, A, or L at position 181, (xv) amino acid residue R at position 184, (xvi) amino acid residue R at position 185, (xvii) amino acid residue R at position 187, and (xviii) amino acid residue L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W at position 218.

[0043] In a preferred embodiment, a lambda-preferring CH1 domain variant polypeptide according to the invention comprises or consists of one or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

[0044] In a preferred embodiment, a lambda-preferring CH1 domain variant polypeptide according to the invention comprises or consists of two or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

[0045] In a preferred embodiment, a lambda-preferring CH1 domain variant polypeptide according to the invention comprises or consists of three or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

[0046] In a preferred embodiment, a lambda-preferring CH1 domain variant polypeptide according to the invention comprises or consists of the following substitutions: (i) 141E and 185R; (ii) 141E and 187R; (iii) 141E, 170E or 171E, and 185R; (iv) 141E, 170E or 171E, and 187R; (v) 141D and 185R; (vi) 141D and 187R; (vii) 141D, 170E or 171E, and 185R; (viii) 141D, 170E or 171E, and 187R; (ix) 141E, 185R, and 187R; or (x) 141D, 185R, and 187R.

[0047] Further in some embodiments, lambda-preferring CH1 domain variant polypeptides according to the invention comprise one or more substitutions at position 141 to D, K, or E, optionally paired with a substitution at position 181 to K, and further optionally with a substitution at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W.

[0048] Further in some embodiments, lambda-preferring CH1 domain variant polypeptides according to the invention comprise a D, K, or E substitution at position 141, paired with a K substitution at position 181 and / or a L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W substitution at position 218.

[0049] In a further embodiment, the lambda-preferring CH1 domain variant polypeptide according to the invention comprises any one or more of the following (i) to (xvii): (i) amino acid residue D, E or K at position 141; (ii) amino acid residue E at position 170, (iii) amino acid residue E at position 171, (iv) amino acid residue M at position 175, (v) amino acid residue K at position 181, (vi) amino acid residue R at position 184, (vii) amino acid residue R at position 185, (viii) amino acid residue R at position 187, and (ix) amino acid residue P, A or E at position 218.

[0050] In a further embodiment, the lambda-preferring CH1 domain variant polypeptide according to the invention comprises: (i) amino acid residue D at position 141; (ii) amino acid residue D at position 141 and amino acid residue K at position 181; (iii) amino acid residue D at position 141, amino acid residue K at position 181 and amino acid residue A at position 218; (iv) amino acid residue D at position 141, amino acid residue K at position 181 and amino acid residue P at position 218; (v) amino acid residue E at position 141; (vi) amino acid residue E at position 141 and amino acid residue K at position 181; (vii) amino acid residue E at position 141 and amino acid residue K at position 181; (viii) amino acid residue K at position 141 and amino acid residue K at position 181; (ix) amino acid residue K at position 141, amino acid residue K at position 181 and amino acid residue E at position 218; (x) amino acid residue K at position 141, amino acid residue K at position 181 and amino acid residue P at position 218; (xi) amino acid residue E at position 141, amino acid residue E at position 170, amino acid residue V at position 181 and amino acid residue R at position 187; (xii) amino acid residue E at position 141, amino acid residue D at position 171 and amino acid residue R at position 185; (xiii) amino acid residue E at position 141, amino acid residue D at position 171 and amino acid residue R at position 185; (xiv) amino acid residue E at position 141, amino acid residue G at position 171, amino acid residue R at position 185, and amino acid residue R at position 187; (xv) amino acid residue E at position 141, amino acid residue R at position 185, and amino acid residue R at position 187; (xvi) amino acid residue E at position 141, amino acid residue S at position 171, and amino acid residue K at position 181; (xvii) amino acid residue E at position 141, amino acid residue G at position 170, amino acid residue M at position 175, and amino acid residue V at position 181. , an amino acid residue R at position 184 and an amino acid residue R at position 187; (xviii) an amino acid residue E at position 141 and an amino acid residue R at position 185; (xix) an amino acid residue E at position 141 and an amino acid residue R at position 187; (xx) an amino acid residue E at position 141, an amino acid residue E at position 170 and an amino acid residue R at position 185; (xxi) an amino acid residue E at position 141, an amino acid residue E at position 170 and an amino acid residue R at position 187; (xxii) an amino acid residue D at position 141 and an amino acid residue R at position 185; (xxiii) an amino acid residue D at position 141;and amino acid residue R at position 187, (xxiv) amino acid residue D at position 141, amino acid residue R at position 185, and amino acid residue R at position 187, (xxv) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 185, (xxvi) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187, (xxvii) amino acid residue E at position 141, amino acid residue E at position 171, and amino acid residue R at position 187, (xxiii) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185, or (xxix) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 187.

[0051] Optionally, such CH1 domain variants include (i) SEQ ID NO: 140, (ii) SEQ ID NO: 141, (iii) SEQ ID NO: 142, (iv) SEQ ID NO: 143, (v) SEQ ID NO: 144, (vi) SEQ ID NO: 145, (vii) SEQ ID NO: 146, (viii) SEQ ID NO: 147, (ix) SEQ ID NO: 148, (x) SEQ ID NO: 149, (xi) SEQ ID NO: 155, (xii) SEQ ID NO: 157, (xiii) SEQ ID NO: 159, (xiv) SEQ ID NO: 162, (xv) SEQ ID NO: 163, (xvi) SEQ ID NO: 164, (xvi) SEQ ID NO: 165, (xvi) SEQ ID NO: 166, (xvi) SEQ ID NO: 167, (xvi) SEQ ID NO: 169, (xvi) SEQ ID NO: 170, (xvi) SEQ ID NO: 171, (xvi) SEQ ID NO: 172, (xvi) SEQ ID NO: 173, (xvi) SEQ ID NO: 174, (xvi) SEQ ID NO: 175, (xvi) SEQ ID NO: 176, (xvi) SEQ ID NO: 177, (xvi) SEQ ID NO: 178, (xvi) SEQ ID NO: 179, (xvi) SEQ ID NO: 180, (xvi) SEQ ID NO: 181, (xvi) SEQ ID NO: 182, (xvi) SEQ ID NO: 183, (xvi) SEQ ID NO: 184, (xvi) SEQ ID NO: 185, (xvi) S xvi) SEQ ID NO: 164, (xvii) SEQ ID NO: 165, (xviii) SEQ ID NO: 178, (xix) SEQ ID NO: 179, (xx) SEQ ID NO: 180, (xxi) SEQ ID NO: 181, (xxii) SEQ ID NO: 182, (xxiii) SEQ ID NO: 183, (xxiv) SEQ ID NO: 184, (xxv) SEQ ID NO: 185, (xxvi) SEQ ID NO: 186, (xxvii) SEQ ID NO: 187, (xxviii) SEQ ID NO: 188, or (xxix) SEQ ID NO: 189.

[0052] In some preferred embodiments, the lambda-preferring CH1 domain variant comprises (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0053] In a further preferred embodiment, the lambda-preferring CH1 domain variant comprises the amino acid substitutions consisting of: (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0054] In certain preferred embodiments, the lambda-preferring CH1 domain variant comprises an amino acid substitution consisting of: (i) SEQ ID NO:188; or (ii) SEQ ID NO:186.

[0055] In some embodiments, a lambda-preferring CH1 domain variant polypeptide may further comprise one or more amino acid substitutions that increase pairing of the CH1 domain with (i) a lambda CL domain compared to a kappa CL domain, and / or (ii) a lambda light chain polypeptide compared to a kappa light chain polypeptide.

[0056] In some embodiments, the CH1 domain variant polypeptide may provide at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, or at least a 100% increase in pairing with a lambda CL domain compared to a kappa CL domain, and / or with a lambda light chain polypeptide compared to a kappa light chain polypeptide. The increase in lambda pairing may optionally be measured by liquid chromatography-mass spectrometry (LCMS).

[0057] In some embodiments, a CH1 domain variant polypeptide may provide at least a 1.2-fold, at least a 1.5-fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5-fold, at least a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a 5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, at least a 7.5-fold, at least a 8-fold, at least a 8.5-fold, at least a 9-fold, at least a 9.5-fold, at least a 10-fold, at least a 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold, at least a 15-fold, at least a 16-fold, at least a 17-fold, at least a 18-fold, at least a 19-fold, at least a 20-fold, at least a 21-fold, at least a 22-fold, at least a 23-fold, at least a 24-fold, or at least a 25-fold increase in pairing with a lambda CL domain compared to a kappa CL domain, and / or with a lambda light chain polypeptide compared to a kappa light chain polypeptide. Increased lambda synapsis may be measured, optionally by flow cytometry, and optionally by comparing the ratio of MFI values ​​of lambda CL staining to kappa CL staining.

[0058] In another aspect, further provided herein is an antibody heavy chain polypeptide comprising a variable region and a constant region, wherein the constant region comprises a CH1 domain variant according to any of those described above.

[0059] In some embodiments, the CH1 domain variant of such an antibody heavy chain polypeptide comprises: (I) (i) amino acid residue F at position 147 and amino acid residue R at position 183, (ii) amino acid residue F at position 147 and amino acid residue K at position 183, (iii) amino acid residue F at position 147 and amino acid residue Y at position 183, (iv) amino acid residue R at position 183, (v) amino acid residue K at position 183, or (vi) amino acid residue Y at position 183, or (II) by containing amino acid substitutions consisting of: (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0060] In another aspect, further provided herein is an antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, wherein (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair, and (b) the first heavy chain polypeptide comprises a first CH1 domain variant comprising an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, according to EU numbering, such that the first CH1 domain variant preferentially binds to the first light chain. Optionally, the first light chain polypeptide comprises a first CL domain that is a wild-type CL domain. Further optionally, certain CH1 domain variants may be excluded as described above, and CH1 domain variants according to the invention may satisfy one or more of items (a) to (o) above. Also included is such an antibody or antibody fragment, further comprising a second heavy chain polypeptide and a second light chain polypeptide, wherein (a) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair, and (b) the second heavy chain polypeptide is selected from the group consisting of 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 173-174, 175-176, 177-178, 179-200, 180-182, 183-184, 185-186, 187-188, 189-201, 188-202, 189-203, 189-204, 190-205, 191-206, 192-207, 193-208, 194-209, 195-208, 196-209, 197-204, 198-205, 199-300, 200-301, 201-302, 202-303, 203-304, 204-305, 205-306, 206-307, 207-308, 208-309, 210-309, 211-304, 212-305, 213 Provided herein is an antibody or antibody fragment as described above, comprising a second CH1 domain variant comprising an amino acid substitution at one or more of positions 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, such that the second CH1 domain variant preferentially binds to a second light chain polypeptide comprising a second CL domain. Again optionally, certain CH1 domain variants may be excluded as described above, and a CH1 domain variant according to the invention may satisfy one or more of items (a)-(o) above.Further optionally, such an antibody or antibody fragment comprises one or more of the following features (i) to (vii): (i) the first CL domain is a wild-type CL domain, (ii) the second CL domain is a wild-type CL domain, (iii) the first CL domain is a kappa CL domain, (iv) the first CL domain is a lambda CL domain, (v) the second CL domain is a kappa CL domain, (vi) the second CL domain is a lambda CL domain, (vii) the first CH1 domain variant is a CH1 domain variant according to any one of claims 1 to 20, (viii) the second CH1 domain variant is a CH1 domain variant according to any one of claims 1 to 20, and / or (ix) the amino acid substitutions in the first CH1 domain variant are different from the amino acid substitutions in the second CH1 domain variant.

[0061] Further provided herein is an antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, wherein (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair, (b) the first heavy chain polypeptide comprises a first CH1 domain variant according to any one of the kappa-preferential CH1 domain variants described above, and (c) the first light chain polypeptide comprises a kappa CL domain, and optionally is a kappa light chain polypeptide. Optionally, (i) the kappa CL domain is a wild-type CL domain, and / or (ii) the first light chain polypeptide is a wild-type light chain polypeptide. In certain embodiments, the first heavy chain polypeptide optionally comprises one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with (i) the kappa CL domain compared to the lambda CL domain, and / or (ii) the kappa light chain polypeptide compared to the lambda light chain polypeptide. The one or more amino acid substitutions outside the CH1 domain may be, for example, within the VH.

[0062] Also provided herein is an antibody or antibody fragment comprising a second heavy chain polypeptide and a second light chain polypeptide, wherein (a) the second heavy chain polypeptide and the second light chain polypeptide form a first cognate pair, (b) the second heavy chain polypeptide comprises a second CH1 domain variant according to any one of the lambda-preferential CH1 domain variants described above, and (c) the second light chain polypeptide comprises a lambda CL domain, and optionally is a lambda light chain polypeptide. Optionally, (i) the lambda CL domain is a wild-type CL domain, and / or (ii) the second light chain polypeptide is a wild-type light chain polypeptide. In an embodiment, the second heavy chain polypeptide optionally comprises one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with (i) the lambda CL domain compared to the kappa CL domain, and / or (ii) the lambda light chain polypeptide compared to the kappa light chain polypeptide.

[0063] Also provided herein is an antibody or antibody fragment comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, wherein (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair, (b) the first heavy chain polypeptide comprises a first CH1 domain comprising a CH1 domain variant according to any one of the kappa-preferential CH1 domain variants described above, (c) the first light chain polypeptide comprises a kappa CL domain, and optionally is a kappa light chain polypeptide, (d) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair, (e) the second heavy chain polypeptide comprises a second CH1 domain comprising a CH1 domain variant according to any one of the lambda-preferential CH1 domain variants described above, and (f) the second light chain polypeptide comprises a lambda CL domain, and optionally is a lambda light chain polypeptide. In some embodiments, the first heavy chain polypeptide optionally comprises one or more amino acid substitutions outside of the CH1 domain that further promote preferential pairing of the heavy chain with (i) a kappa CL domain compared to a lambda CL domain, and / or (ii) a kappa light chain polypeptide compared to a lambda light chain polypeptide. The one or more amino acid substitutions outside of the CH1 domain can be, for example, within the VH. In some embodiments, the second heavy chain polypeptide optionally comprises one or more amino acid substitutions outside of the CH1 domain that further promote preferential pairing of the heavy chain with (i) a lambda CL domain compared to a kappa CL domain, and / or (ii) a lambda light chain polypeptide compared to a kappa light chain polypeptide.

[0064] Any of the antibodies or antibody fragments may be multispecific, and optionally bispecific. Optionally, the structure of such antibodies or antibody fragments is as depicted in any one of Figures 24-29.

[0065] In some embodiments, in the multispecific antibody or antibody fragment described above, the first and second CH1 domain variants reduce non-cognate heavy chain-light chain pairing by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, in the multispecific antibody or antibody fragment described above, the first and second CH1 domain variants increase cognate heavy chain-light chain pairing by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0066] In some embodiments, such as in Example 7 and Figures 23, 30, or 31, a decrease in non-cognate heavy chain-light chain pairing and / or an increase in cognate heavy chain-light chain pairing can be quantified by transfecting cells with a HC (or VH plus CH1) containing a CH1 of interest, a kappa LC and lambda in a defined ratio, such as HC:kappa LC:lambda LC=2:1:1, and measuring the light chain species by LCMS. In certain embodiments using such or similar quantification methods, an exemplary wild-type (WT) CH1 may result in HC-LC pairs, of which 60% are cognate pairs and 40% are non-cognate pairs, and in CH1 variants according to the present disclosure, the proportion of cognate pairs may be increased to at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, and the proportion of non-cognate pairs may be decreased to at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, or 0%. In certain embodiments using such or similar quantification methods, the proportion of cognate pairs may be increased to at least 85%, at least 90%, at least 95%, or 100%, while the proportion of non-cognate pairs may be decreased to at least 15%, at least 10%, at least 5%, or 0%.

[0067] In some embodiments, in the multispecific antibody or antibody fragment as described above, the first and second CH1 domain variants reduce non-cognate heavy chain-light chain pairing by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold. In some embodiments, in the multispecific antibody or antibody fragment as described above, the first and second CH1 domain variants increase cognate heavy chain-light chain pairing by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold.

[0068] In some embodiments, as in Figures 2-5, 8-13, and 19-22, a reduction in non-cognate heavy-light chain pairs and / or an increase in cognate heavy-light chain pairs can be quantified by co-expressing the HC (or VH plus CH1), kappa LC, and lambda LC comprising the CH1 of interest in a cell (e.g., yeast cell) in a predetermined ratio that allows for the presentation of heavy-light chain pairs, staining the cells with anti-kappa and anti-lambda antibodies, and quantifying the presence of kappa and lambda by FACS, e.g., comparing MFI values. To compare the kappa selectivity of a CH1, the ratio of MFI of cells stained with anti-kappa antibody:MFI of cells stained with anti-lambda antibody can be calculated and divided by such ratio for wild-type (WT) CH1 to obtain a fold-over-parent (FOP) value. To compare the lambda selectivity of a given CH1, the ratio of MFI of cells stained with anti-lambda antibody to MFI of cells stained with anti-kappa antibody can be calculated and divided by such ratio for wild-type (WT) CH1.

[0069] In certain embodiments using such or similar quantification methods, the kappa-preferring CH1 variants of the present disclosure may be used to increase FOP values ​​(calculated in terms of kappa selectivity, i.e., MFI of kappa:lambda) by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold. In certain embodiments using such or similar quantification methods, lambda-preferring CH1 variants of the disclosure may be used to increase FOP values ​​(calculated in terms of lambda selectivity, i.e., MFI of lambda:kappa) by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold.

[0070] In some embodiments, the second CH1 domain variant comprises a substitution at position 141 and reduces non-cognate heavy chain-light chain pairing by at least 50%. In some embodiments, the second CH1 domain variant comprises a substitution at position 141 and the first CH1 domain variant comprises substitutions at positions 183 and optionally 147, or vice versa, and reduces non-cognate heavy chain-light chain pairing by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises 141D or 141E and the second CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and reduces non-cognate heavy chain-light chain pairing by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises one or more of 141D or 141E, 170E, 171E, 181K, 185R, 187R and 218P, and the first CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and reduces non-cognate heavy chain-light chain pairing by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises a combination of 141D, 171E and 185R, a combination of 141D, 171E and 187R, or a combination of 141D, 181K and 218P, and the second CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and reduces non-cognate heavy chain-light chain pairing by at least 50% to at least 75%.

[0071] In some embodiments, the first and second CH1 domain variants provide for at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% formation of the desired first and second cognate pairs. In some embodiments, the first and second CH1 domain variants provide for about 85% to about 95% formation of the desired first and second cognate pairs. In some embodiments, the second CH1 domain variant comprises a substitution at position 141 and the first CH1 domain variant comprises a substitution at position 183 and optionally at position 147, and provides for about 85% to at least about 95% formation of the desired first and second cognate pairs. In some embodiments, the second CH1 domain variant comprises 141D or 141E and the first CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and provides for about 85% to at least about 95% formation of the desired first and second cognate pairs. In some embodiments, the first and second CH1 domain variants provide a reduction in non-cognate heavy chain-light chain pairing of less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the second CH1 domain variant comprises a substitution at position 141, 170, 171, 181, 185, 187, and / or 218, and the first CH1 domain variant comprises a substitution at position 183 and optionally at position 147, or vice versa, and provides a reduction in non-cognate heavy chain-light chain pairing of less than about 15%, less than about 10%, or less than about 5%.In some embodiments, the second CH1 domain variant comprises one or more of 141D or 141E, 170E, 171E, 181K, 185R, 187R and 218P, and the first CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and provides a reduction in non-cognate heavy chain-light chain pairing of less than about 15%, less than about 10%, or less than about 5%.

[0072] In yet another aspect, further provided herein are pharmaceutical and diagnostic compositions comprising (i) the CH1 domain variant polypeptides described above, (ii) the antibody heavy chain polypeptides described above, and / or (iii) the antibodies or antibody fragments described above.

[0073] In another aspect, further provided herein are therapeutic and diagnostic uses of antibodies and pharmaceutical compositions comprising (i) the above-described CH1 domain variant polypeptides, (ii) the described antibody heavy chain polypeptides, and / or (iii) the above-described antibodies or antibody fragments.

[0074] In yet another aspect, further provided herein is a nucleic acid encoding (i) an aforementioned CH1 domain variant polypeptide, (ii) an aforementioned antibody heavy chain polypeptide, and / or (iii) an aforementioned antibody or antibody fragment.

[0075] In yet another aspect, further provided herein is a vector comprising a nucleic acid encoding (i) the above-described CH1 domain variant polypeptide, (ii) the described antibody heavy chain polypeptide, and / or (iii) the above-described antibody or antibody fragment, or a cell transfected with said nucleic acid, and the use thereof for producing the foregoing.

[0076] In another aspect, the disclosure provides a method for generating a CH1 variant domain library, the method comprising the steps of: (a) selecting one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a kappa CL domain (a "Cκ set"); (ii) one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain (a "Cλ set"); and / or (iii) selecting one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain (a "Cλ set"); κ Set and / or C λ (b) selecting one or more amino acid positions in the CH1 domain that are in contact with one or more amino acid positions in a kappa CL domain in the Cκ set and / or one or more amino acid positions in a lambda CL domain in the Cλ set, and (c) generating a library of CH1 domain variant polypeptides or a library of CH1 domain variant-encoding constructs, wherein one or more of the one or more amino acid positions selected in step (b) are substituted with any non-wild type amino acid. Optionally, the polypeptide comprising a CH1 domain further comprises a heavy chain variable region (VH), and optionally the polypeptide comprising a kappa or lambda CL domain further comprises a light chain variable region (VL).

[0077] Optionally, (I) in step (a), said CH1 domain, said kappa CL domain, and said lambda CL domain are wild type and / or human; (II) in step (a), both of said polypeptides comprising (i) a CH1 domain paired with a polypeptide comprising a kappa CL domain and (ii) a CH1 domain paired with a polypeptide comprising a lambda CL domain are intact antibodies or antigen-binding fragments ("Fab"); and (III) in step (b), an amino acid residue at said one or more amino acid positions in the CH1 domain is selected from (i) said one or more amino acid residues in the kappa CL domain and (ii) said one or more amino acid residues in the lambda CL domain and (iii) in step (b), or a side chain atom of an amino acid residue at said one or more amino acid positions in the VH, (ii) a side chain atom of an amino acid residue at said one or more amino acid positions in the lambda CL domain, and / or (iii) a side chain atom of an amino acid residue at said one or more amino acid positions in the VH, and / or (IV) said generating in step (c) is via degenerate codons, optionally via degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues.

[0078] In some embodiments, the one or more CH1 amino acid positions selected in step (b) are: (i) at an interface with a kappa CL domain in at least 10% of a representative set of the Cκ set and have a fractional solvent accessible surface area of ​​greater than 10% in at least 90% of a representative set of the Cκ set; (ii) at an interface with a lambda CL domain in at least 10% of a representative set of the Cλ set and have a fractional solvent accessible surface area of ​​greater than 10% in at least 90% of a representative set of the Cλ set; and / or (iii) at an interface with a lambda CL domain in at least 10% of a representative set of the Cλ set and have a fractional solvent accessible surface area of ​​greater than 10% in at least 90% of a representative set of the Cλ set. κ and / or C λ At least 10% of the representative sets of the set are at the interface with VH, andκ and / or C. λ At least 90% of representatives of the set have a solvent accessible surface area greater than 10%.

[0079] In some embodiments, the amino acid positions selected in step (b) include one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering. Optionally, certain CH1 domain variants may be excluded as described above, and CH1 domain variants according to the present invention may fulfill criteria (a)-(o) above.

[0080] In some embodiments, the synthetic polypeptide encoding the CH1 variant domain or library of CH1 domain variants in step (c) is expressed in a yeast strain. In some embodiments, the yeast strain is Saccharomyces cerevisiae. In some embodiments, a cell line such as a yeast strain co-expresses (i) one or more polypeptides comprising a kappa CL domain, such as a kappa light chain, and (ii) one or more polypeptides comprising a lambda CL domain, such as a lambda light chain. Optionally, the kappa and / or lambda CL domains are wild type. Further optionally, the kappa and / or lambda CL domains are human.

[0081] In some embodiments, the methods of the present disclosure further include verifying that the one or more substituted CH1 amino acid residues drive preferential pairing to a kappa or lambda light chain. In some embodiments, fluorescence activated cell sorting is used to verify that the one or more substituted CH1 amino acid residues drive preferential pairing to a kappa or lambda light chain.

[0082] In some embodiments, one or more of the kappa constant (Cκ) domains, one or more of the lambda constant (Cλ) domains, and one or more of the CH1 domains are wild type. In some embodiments, one or more of the kappa constant (Cκ) domains, one or more of the lambda constant (Cλ) domains, and one or more of the CH1 domains are human.

[0083] In some embodiments, the method for generating a CH1 domain library comprises the following steps (a)-(c): (a) selecting one or more of the following CH1 amino acid positions: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, according to EU numbering; (b) selecting one or more of the following CH1 amino acid positions: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, according to EU numbering; and (c) generating a library of CH1 domain variant polypeptides or libraries of constructs encoding CH1 domain variants, wherein one or more of the one or more amino acid positions selected in steps (a) and (b) are substituted with any non-wild type amino acid. In certain embodiments, the amino acid positions selected in (a) may include positions 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof. In certain embodiments, said generating in step (c) is via degenerate codons, optionally via degenerate RMW codons representing the six natural amino acids (D, T, A, E, K, and N), or degenerate NNK codons representing all 20 natural amino acid residues. In certain embodiments, in step (c), the amino acid positions selected in step (a) may be substituted with predetermined amino acids, and the amino acid positions selected in (b) are substituted via degenerate codons. Optionally, the substitutions with predetermined amino acids may include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P, or any combination thereof.

[0084] In yet another aspect, the disclosure provides a method for identifying one or more CH1 domain variant polypeptides that preferentially pair (A) with a polypeptide comprising a kappa CL domain compared to a polypeptide comprising a lambda CL domain, or (B) with a polypeptide comprising a lambda CL domain compared to a polypeptide comprising a kappa CL domain, the method comprising the steps of: (a) co-expressing one or more candidate CH1 domain variant polypeptides with (i) one or more polypeptides comprising a kappa CL domain and (ii) one or more polypeptides comprising a lambda CL domain, (b) comparing the amount of the candidate CH1 domain variant polypeptide that is paired with (i) the polypeptide comprising a kappa CL domain and (ii) the polypeptide comprising a lambda CL domain, and (c) selecting, based on the comparison in step (b), one or more CH1 domain variants that provide preferential pairing with (A) the polypeptide comprising a kappa CL domain compared to a polypeptide comprising a lambda CL domain, or (B) the polypeptide comprising a lambda CL domain compared to a polypeptide comprising a kappa CL domain. In step (a), generally, the total amount of candidate CH1 domain variant polypeptides expressed and the total amount of polypeptides comprising (kappa and lambda) CL domains expressed may be about the same. Optionally, in step (a), the candidate CH1 domain variant polypeptides, the polypeptides comprising kappa CL domains, and the polypeptides comprising lambda CL domains are expressed in a ratio of about 2:1:1.

[0085] In some embodiments, in step (a), the (i) one or more polypeptides comprising a kappa CL domain and (ii) one or more polypeptides comprising a lambda CL domain are wild-type and / or human.

[0086] In some embodiments, in step (b), the amount is determined via fluorescence activated cell sorting or via liquid chromatography-mass spectrometry.

[0087] In some embodiments, the method further comprises the step (d): (d) co-expressing one or more control CH1 domain variants with (i) one or more polypeptides comprising a kappa CL domain, and (ii) one or more polypeptides comprising a lambda CL domain, optionally wherein one or more of the one or more control CH1 domain variants are according to any of the CH1 domain variants described above. [Brief description of the drawings]

[0088] [Figure 1] 1A-C are schematic diagrams of the binding of CH1 domain variants to Cλ or Cκ domains. FIG. 1A shows the heterodimerization of wild-type CH1 domain with Cλ and Cκ (wild-type or unmodified CH1 domain is referred to as CH1WT). FIG. 1B shows a CH1 domain variant that preferentially pairs with Cκ (this CH1 domain variant that preferentially pairs with Cκ is referred to as CH1κ). FIG. 1C shows a CH1 domain variant that preferentially pairs with Cλ (this CH1 domain variant that preferentially pairs with Cλ is referred to as CH1λ).

[0089] [Diagram 2] Figures 2A and 2B show exemplary FACS plots across multiple rounds of selection identifying CH1 domain variants with lambda CL domain selectivity (Figure 2A) or kappa CL domain selectivity (Figure 2B). R1 ​​= first round of selection, R2 = second round of selection, R3 = third round of selection. The x-axis shows lambda light chains labeled with PE and the y-axis shows kappa light chains labeled with FITC.

[0090] [Diagram 3]Figure 3 shows individual unique clones expressing CH1 domain variants with lambda or kappa CL domain preference. Clones were scored for the ratio of anti-kappa median fluorescence intensity (MFI) to anti-lambda MFI (kappa:lambda ratio). The kappa:lambda ratio for any individual clone was compared to the corresponding strain with the wild-type CH1 sequence ("parent"). FOP means fold-over-parent.

[0091] [Figure 4] Figure 4 shows individual unique clones expressing CH1 domain variants with amino acid substitutions at positions 141, 147, or 183 (EU numbering). Clones were scored for the ratio of anti-kappa to anti-lambda MFI and compared to the parent to determine FOP. CH1 positions 147 and 183 were identified as the two positions that confer kappa CL domain selectivity. CH1 position 141 was identified as the position that conferred lambda CL domain selectivity.

[0092] [Diagram 5]Figure 5 shows specific amino acid substitutions at positions 141, 147 and / or 183 (EU numbering) in the CH1 domain with lambda CL domain selectivity (A141T, Q, D, or R) or kappa CL domain selectivity (K147V, A, F, Y, or M; S183K, Y, E, R, W, Q) as measured by the ratio of anti-kappa to anti-lambda MFI. Amino acid substitutions shown as white dots (V134, T141, V147, A151, and K183) were identified after an initial round of selection from a library diversified at multiple positions, and amino acid substitutions shown as black dots were identified after further rounds of selection from a library diversified targeting positions 141, 147, and 183. Parental kappa:lambda ratios (wild type signal): GAL1 Ckappa; GAL10 Clamma: 3.58, and GAL1 Clamma; GAL10 Ckappa: 0.3. Parental ratios are averages over experimental replicates. For CH1 variants with substitutions at both positions 147 and 183, the first amino acid listed is the variant at position 147, and the second amino acid listed is the variant at position 183 (e.g., YxF means a CH1 variant with substitutions K147Y and S183F).

[0093] [Figure 6A] Figures 6A-E show representative binding data demonstrating that the CH1 domain variants did not alter target binding of multispecific antibodies (BsAb2-BsAb14) compared to the wild-type CH1 domain (BsAb1 and BsAb15). Figure 6A shows IL12B and EGFR binding data for BsAb1-3. [Figure 6B] FIG. 6B shows IL12B and EGFR binding data for BsAbs 5, 7 and 4. [Figure 6C] FIG. 6C shows IL12B and EGFR binding data for BsAbs 9, 10 and 6. [Figure 6D] FIG. 6D shows IL12B and EGFR binding data for BsAbs 11, 12 and 8. [Figure 6E]Figure 6E shows IL12B and EGFR binding data for BsAbs 13-15. Pani = Panitumumab; Uste = Ustekinumab.

[0094] [Figure 7] FIG. 7 shows an increase in correct heavy-light chain pairing (HC1-LC1 or HC2-LC2) and a concomitant decrease in heavy-light chain mispairings (HC1-LC2 and HC2-LC1) in bispecific antibodies containing CH1 domain variants (BsAb2-BsAb14) compared to a bispecific antibody containing a wild-type CH1 domain (BsAb1).

[0095] [Figure 8] 8 shows the lambda-selective FOP values ​​of the wild-type (WT) clone, the A141D clone, and individual clones with different amino acid substitutions at positions 141, 181, and 218 of the CH1 domain obtained from the outcome of the 141×181×218 library selection of Example 5. The 13 data points marked within the rectangle represent the clones with the highest FOP values, and the amino acid residues at positions 141, 181, and 218 of CH1, as well as the FOP value of each clone, are provided in Table 8.

[0096] [Figure 9] 9 shows the lambda-selective FOP values ​​of wild-type (WT) clones, A141D clones, and individual clones with D at position 141, K at position 181, and various amino acids at position 218 of the CH1 domain in the outcome of the 141×181×218 library selection of Example 5. The open circle data points represent the FOP of individual clones with the same CH1 sequence, and the closed circle data points represent the average FOP value.

[0097] [Figure 10]FIG. 10 shows the lambda-selective FOP values ​​measured in the recloned clones and in wild-type (WT) and A141D clones, confirming the maintenance of lambda selectivity.

[0098] [Figure 11] 11 shows exemplary scatter plots of IgG produced in HEK293 with CH1 of one of the nine 141x181x218 leads selected in Example 5, and wild type (WT) and A14D, stained for kappa and lambda CL. Scatter plots of individual clones are overlaid with the WT plot. The x-axis shows lambda light chain labeled with PE, and the y-axis shows kappa light chain labeled with FITC.

[0099] [Figure 12] Figure 12 shows the lambda-selective FOP values ​​for the nine leads from Example 5, along with wild type (WT) and A141D. The three CH1 variants with the highest FOP values ​​(D_K_WT, D_K_P, and D_K_A) were selected for subsequent two-chain (kappa or lambda) transfection in HEK293.

[0100] [Figure 13] Figure 13 compares the FOP values ​​for lambda selectivity between CH1 variants with the same amino acid at position 141. When position 141 is D, additional amino acid substitutions at position 181, or at positions 181 and 218, further increase the FOP value.

[0101] [Figure 14] Figure 14 shows the % light chain species (comparing kappa and lambda) of full-length IgG produced in HEK293 of the nine leads as measured by liquid chromatography-mass spectrometry ("LCMS"). The three CH1 variants with the highest FOP values ​​(D_K_WT, D_K_P, and D_K_A) were selected for subsequent transfection in HEK293.

[0102] [Figure 15] FIG. 15 shows exemplary process yields of three leads (D_K_WT, D_K_P, and D_K_A) and A141D relative to the process yield of the wild type (WT), shown as fold-over-parent ("FOP") values.

[0103]

[0104] [Figure 16] FIG. 16 shows the Tm (° C.) of kappa- and lambda-paired Fabs with one of the three lead CH1 variants (D_K_WT, D_K_P, and D_K_A), or A141D, or wild type (WT).

[0105] [Figure 17] FIG. 17 shows the relative lambda Tm (° C.), defined as: [Tm change of lambda-paired variant Fab relative to lambda-paired WT Fab ("Δlambda Tm"))]-[Tm change of kappa-paired variant Fab relative to kappa-paired WT Fab ("Δkappa Tm")].

[0106] [Figure 18] FIG. 18 provides the sequencing results from the recloning outcomes in Example 6, visualizing the frequent amino acid substitutions observed among the resulting clones.

[0107] [Figure 19] Figure 19 shows lambda-selective FOP values ​​(lambda MFI: kappa MFI) for the reads from the recloning results of Example 6, expressed as IgG in yeast, and a portion of the 141x181x218 reads (DKP, DKA, KKE, KKP, and EKK) from Example 5. At least seven reads, marked with arrows, have FOP values ​​that are comparable to or higher than the values ​​of the 141x181x218 reads tested.

[0108] [Figure 20] Figure 20 shows the lambda-selective FOP values ​​for DKP, A141D, and wild type identified in Example 5, as well as the 14 reads from Example 7. Two reads marked with arrows, "A414D_P171E_V185R" and "A141D_F170E_T187R", showed higher FOP values ​​than DKP. All 14 reads showed higher FOP values ​​than wild type.

[0109] [Figure 21] Figure 21 shows exemplary FACS plots comparing lambda selectivity for 14 CH1 domain variants in Example 7 and three controls (DKP, A141D, and wild type identified in Example 5). The x-axis shows PE-labeled lambda light chain, and the y-axis shows FITC-labeled kappa light chain. The numbering of each plot is the Rank number shown in Table 14. For example, the first two plots numbered "1" and "2" are the plots of "A414D_P171E_V185R" and "A141D_F170E_T187R", respectively.

[0110] [Figure 22] FIG. 22 shows an exemplary overlay of FACS plots of individual plots (marked as "a"), the wild-type plot (marked as "b"), and the DKP plot (marked as "c") from FIG. 21.

[0111] [Figure 23] Figure 23 shows the % light chain species (comparing kappa and lambda) of full length IgG of 14 leads and three controls produced in HEK293 as measured by LCMS in Example 7. The three controls are indicated with white arrows. "A414D_P171E_V185R" and "A141D_F170E_T187R" (filled arrow) showed higher % lambda chains and lower % kappa chains compared to the positive control "DKP".

[0112] [Figure 24A]Figures 24-29 provide exemplary, non-limiting embodiments of various multispecific antibody structures in which the CH1 domain variants disclosed herein may be used. In Figures 24-29, unless otherwise indicated, the following applies: (1) each domain is presented as a rectangle with text therein indicating the domain name (e.g., CH1, VH1, etc.); (2) the black and dotted rectangles are CH1 domain variants with kappa or lambda selectivity, which may be the CH1 domain variants disclosed herein; (3) "CH1κ" is a CH1 domain variant with kappa CL selectivity, "CH1λ" is a CH1 domain variant with lambda selectivity, and "CH1" without a "κ" or "λ" designation is any CH1 domain, wild type, or variant with or without light chain isotype selectivity; (4) "Cκ" is a kappa CL domain, "Cλ" is a lambda CL domain, and "CL" without a "κ" or "λ" designation, when shown paired with a black or dotted CH1 domain, is an isotype for which the paired black or dotted CH1 domain has selectivity. (5) when multiple black and / or dotted CH1 domains are present in a multispecific structure, at least one is a CH1 domain variant disclosed herein, and the others may or may not be a CH1 domain variant disclosed herein; (6) when both black and dotted CH1 domains are present in a multispecific structure, the black and dotted ones represent CH1 domains with different light chain isotype selectivities (i.e., when black represents a CH1 domain with kappa selectivity, the dotted ones represent a CH1 domain with lambda selectivity, and vice versa); (7) VH1 and VL1 form an antigen-binding site for a first epitope, VH2 and VL2 form an antigen-binding site for a second epitope, VH3 and VL3 form an antigen-binding site for a third epitope, VH4 and VL4 form an antigen-binding site for a fourth epitope, and VH5 and VL5 form an antigen-binding site for a fifth epitope;VH6 and VL6 also form an antigen-binding site for a sixth epitope; (8) all of the first through sixth epitopes may or may not be different from each other, so long as the overall combination of specificities results in multispecificity for the presented structure; (9) a set of multiple domains linked together represents a polypeptide (e.g., a heavy chain polypeptide, a light chain polypeptide, etc.); (10) the orientation of the domains within a polypeptide follows the direction of the text indicating the domain name, from the N-terminus to the C-terminus; (11) even if a linker, hinge, or disulfide bond is not explicitly shown in the figure, a linker or hinge can be used between the domains as needed, and disulfide bonds can be used to link the domains together, possibly to allow for accurate formation of the antigen-binding site. (12) the CH2 and / or CH3 domains shown in the figures may be omitted where possible and replaced with hinges where appropriate; (13) the CH1, CH2, and CH3 domains may independently be wild-type or variant and independently be of any (heavy chain) isotype; and (14) if multiple CH1 domains are present in the structure, the CH1 domains may or may not be of the same isotype, if multiple CH2 domains are present in the structure, the CH2 domains may or may not be of the same isotype, and if multiple CH3 domains are present in the structure, the CH3 domains may or may not be of the same isotype.

[0113] Figures 24A-24C provide exemplary and non-limiting embodiments of various multispecific antibody structures in which the CH1 domain variants disclosed herein may be used. In Figure 24A, a kappa-preferential CH1 domain ("CH1κ") is used in one polypeptide. The other CH1 domain may or may not have a preference for a lambda CL domain and may or may not be a CH1 domain variant disclosed herein. In Figure 24B, a lambda-preferential CH1 domain ("CH1λ") is used in one polypeptide. The other CH1 domain may or may not have a preference for a kappa CL domain and may or may not be a CH1 domain variant disclosed herein. In Figure 24C, a CH1κ is used in one polypeptide and a CH1λ is used in one polypeptide. This general structure allows for the production of bispecific compounds without or with minimal or less effort to remove mismatched compounds. At least one of the CH1κ and CH1λ domains is a CH1 domain variant disclosed herein. As described above in (10), the orientation of domains within a polypeptide follows the N-terminal to C-terminal textual orientation of the domain names. Thus, for the compound in the upper left of Figure 24A, from the N-terminal to C-terminal orientation, the first polypeptide comprises VH1-CH1k-CH2-CH3, the second polypeptide comprises VL1-Ck, the third polypeptide comprises VH2-CH1-CH2-CH3, and the fourth polypeptide comprises VL2-CL. The triangles in the upper center and upper right compounds in Figures 24A-24C (and all other applicable figures) represent mechanisms that promote heterodimerization of two non-identical polypeptides, such as "knobs-in-holes" engineering. The compound in the lower center of Figures 24A-24C (and all other applicable figures) shows a hinge structure connecting the CH1κ-containing and CH1λ-containing polypeptides.Two bonds (e.g., disulfide bonds) are shown connecting the two polypeptides, although the number of bonds and the exact location / position of the bonds may vary and may be selected appropriately. The "+" in the lower right of Figure 24C indicates a mixture of two different Fab fragments. [Figure 24B] Same as above. [Figure 24C] Same as above.

[0114] [Figure 25A] Figures 25A-25B provide exemplary, non-limiting embodiments of various multispecific antibody structures in which the CH1 domain variants disclosed herein may be used. The structures are similar to those in Figures 24A-23C, but the order of the domains is different. In Figure 25A, CH1κ is in the same polypeptide with the VL, and CH1λ is in the same polypeptide with the VL. In Figure 25B, Cλ is in the same polypeptide with CH2 (top three and bottom left), and Cλ is in a heavy chain-like polypeptide (hinge-containing polypeptide) (bottom right). [Figure 25B] Same as above.

[0115] [Figure 26A] 26A-26C provide further exemplary, non-limiting embodiments of various multispecific antibody structures, which contain two sets of two antigen binding sites in tandem and are therefore tetravalent. The structures can be bispecific, trispecific, or tetraspecific, depending on what the first, second, third, and fourth epitopes are. For example, if the first, second, and epitopes are different from each other, and if the fourth epitope is the same as the first, second, or third epitope, the structure will be a tetravalent trispecific structure. [Figure 26B] Same as above. [Figure 26C] Same as above.

[0116] [Figure 27A]Figures 27A-27C provide further illustrative and non-limiting embodiments of various multispecific antibody structures, which are similar to the structures in Figures 26A-26C, but with different domain order. As described above in (10), the orientation of the domains within a polypeptide follows the N-terminal to C-terminal direction of the text showing the domain name. Thus, for the structure in the upper left of Figure 27A, from the N-terminal to the C-terminal direction, the first polypeptide comprises VH3-VH1-CH1(black)-CH2-CH3, the second polypeptide comprises VL1-VL3-CL, the third polypeptide comprises VH4-VH2-CH1(polka dot)-CH2-CH3, and the fourth polypeptide comprises VL2-VL4-CL. In any of the structures in Figures 27A-27C, appropriate interdomains may be used to allow proper formation of the antigen binding site. [Figure 27B] Same as above. [Figure 27C] Same as above.

[0117] [Figure 28A] Figures 28A-28D provide further exemplary and non-limiting embodiments of various multispecific antibody structures, which contain at least one scFv. Any of the structures provided in Figures 24-29 may additionally contain, or be modified to contain, one or more scFv-containing moieties, e.g., conjugated to any of the heavy chain constant domain, light chain constant domain, and / or antigen binding domain. As an example, Figures 28A-28C provide a structure in which the upper left structure of Figure 24A is conjugated with two scFvs, allowing specificity for up to four epitopes. In Figure 28A, the scFv is conjugated to the CH3 domain. In Figure 28B, the scFv is conjugated to the CL domain. In Figure 28C, the scFv is conjugated to the VH domain. In certain examples, more than one scFv may be conjugated. As an example, Figures 28A-28C provide a structure in which the structure in the upper left of Figure 24A is conjugated with four scFvs, allowing specificity for up to six epitopes. [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 28D] Same as above.

[0118] [Figure 29A]Figures 29A-29D provide further exemplary and non-limiting embodiments of various multispecific antibody structures, which contain two additional Fab fragments. Note that although two Fab fragments are conjugated to the CH3 domain, Fab fragments may be conjugated to other parts of the structure, and one (or more) Fab fragments may be conjugated instead of two. In Figure 29A, the two CH1 domains are in the same polypeptide with the CH2 and CH3 domains. In the middle structure, a kappa-preferred CH1 domain and a lambda-preferred CH1 domain are in the same polypeptide (for both of the two CH1-containing polypeptides). When the two CH1-containing polypeptides are the same, this structure facilitates the production of tetravalent bispecific compounds without the need for mechanisms to facilitate heterodimerization of two non-identical polypeptides (e.g., "knobs-in-holes" engineering, etc.), simply by using, for example, the three-chain transfection system utilized in the examples. In Figure 29B, the two polypeptides do not contain any CH1 domains. In the middle structure, when the two non-CH1-containing polypeptides are the same, this structure facilitates the production of tetravalent bispecific compounds without the need for mechanisms to promote heterodimerization of two non-identical polypeptides (e.g., "knobs-in-holes" engineering), for example, by simply using the three-chain transfection system utilized in the examples. In Figures 29C and 29D, each polypeptide contains a CH1 domain. In the middle structure of Figures 29C and 29D, when the first epitope and the third epitope are the same epitope, and the second epitope and the fourth epitope are the same epitope but different from the first epitope and the third epitope, the structure is bispecific. In such a structure, when the two CH2 / CH3-containing polypeptides are the same, this structure facilitates the production of tetravalent bispecific compounds without the need for mechanisms to promote heterodimerization of two non-identical polypeptides (e.g., "knobs-in-holes" engineering), for example, by simply using the three-chain transfection system utilized in the examples. [Figure 29B] Same as above. [Figure 29C] Same as above. [Figure 29D] Same as above.

[0119] [Diagram 30] FIG. 30 shows exemplary process yields for each full IgG containing one of the top two lambda-preferred CH1 variants identified in Example 7 ("A141D P171E V185R" or "A141D F170E T187R"), or the kappa-preferred CH1 variant identified in Example 4 ("K147F S183R"), or the wild-type (WT) CH1, normalized to the process yield of wild-type (WT). The hatched bars (paired with kappa) and the black bars (paired with lambda) represent process yields normalized to the corresponding yields of wild-type (WT). The open diamonds (paired with kappa) and black triangles (paired with lambda) represent the raw process yields (mg / L).

[0120] [Diagram 31] FIG. 31 shows exemplary process yields for each Fab containing one of the top two lambda-preferred CH1 variants identified in Example 7 ("A141D P171E V185R" or "A141D F170E T187R"), or the lambda-preferred CH1 variants identified in Examples 4 and 5 ("A141D" or "A141D S181K K218P"), or the kappa-preferred CH1 variant identified in Example 4 ("K147F S183R"), or the wild-type (WT) CH1. Normalized to the process yield of the wild-type (WT). Yields are normalized to the corresponding yield of the wild-type (WT). The shaded bars represent Fabs containing a kappa LC and the solid bars represent Fabs containing a lambda LC.

[0121] [Diagram 32]Figure 32 shows the wild-type CH1-Cλ interface in its electron density. Representative electron density of the region of interest for the panitumumab variable fragment (Fv) and wild-type IgG1-CH1 Fab crystal structures paired with a wild-type lambda constant domain (Cλ). Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the lambda light chain (λLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Proteins are represented as sticks. The electron density map of 2Fo-Fc is shown as a gray mesh drawn at 1σ at 1.6 Å resolution. Data for this crystal structure is scaled to an atomic resolution of 1.09 Å.

[0122] [Diagram 33] Figure 33 shows the A141D CH1-Cλ interface in its electron density. Representative electron density of the region of interest for the panitumumab variable fragment (Fv) and A141D substituted IgG1-CH1 Fab crystal structures paired with a wild-type lambda constant domain (Cλ). Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the lambda light chain (λLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Proteins are represented as sticks. The electron density map of 2Fo-Fc is shown as a gray mesh drawn at 1σ at 2.0 Å resolution. Data for this crystal structure is extended to an atomic resolution of 1.2 Å.

[0123] [Diagram 34] Figure 34 shows the wild-type CH1-Cκ interface in its electron density. Representative electron density of the region of interest for the panitumumab variable fragment (Fv) and wild-type IgG1-CH1 Fab crystal structures paired with a wild-type kappa constant domain (Cκ). Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the kappa light chain (κLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Proteins are represented as sticks. The electron density map of 2Fo-Fc is shown as a gray mesh drawn at 0.9σ at 1.6 Å resolution. Data for this crystal structure is extended to an atomic resolution of 2.6 Å.

[0124] [Diagram 35] Figure 35 shows the K147F-S183R CH1-Cκ interface in its electron density. Representative electron density of the region of interest for the crystal structure of panitumumab variable fragment (Fv) and K147F-S183R substituted IgG1-CH1 paired with a wild-type kappa constant domain (Cκ). Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the kappa light chain (κLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Proteins are represented as sticks. The electron density map of 2Fo-Fc is shown as a gray mesh drawn at 0.9σ at 1.6 Å resolution. Data for this crystal structure is extended to near atomic resolution of 2.1 Å.

[0125] [Diagram 36] Figures 36A-36D show the HC-A141D substitution, which allows hydrogen bonding with λLC while at the same time destabilizing kappa pairing due to steric clash with κLC. Figures 36A-36D provide illustrations of the pairing interface surrounding the HC-Ala141 position between wild-type (WT) CH1 and λLC (Figure 36A), between wild-type (WT) CH1 and κLC (Figure 36B), between A141D CH1 and λLC (Figure 36C), or between A141D CH1 and κLC (Figure 36D). The interface of the kappa light chain constant domain (κLC) contains three hydrophobic residues Phe116, Phe118, and Leu135, and is illustrated in Figure 36B. The presence of Thr116 in λLC at the structurally equivalent κLC-Phe116 position allows hydrogen bonding to the carboxyl group of HC-Asp141, shown as a black dotted line (Figure 36C). In Figure 36D, HC alignment of A141D CH1 constant lambda (Cλ) and wild-type (WT) CH1-Cκ reveals a steric clash of the HC-Asp141 side chain with the side chain of κLC-Phe116. Heavy chain (HC) carbon atoms are colored light gray, light chain (LC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are represented as sticks with transparent molecular surfaces, and main chain atoms are represented as cartoons.

[0126] [Figure 37] Figures 37A and 37B show that the wild-type CH1 sequence traps HC-Gln175 in an intrachain hydrogen bond network that can be disrupted by the substitution of K147F, allowing freedom for HC-Gln175 to interact with κLC-Gln160. Figures 37A and 37B provide illustrations of the intra-HC ternary hydrogen bonds involving Lys147, Asp148 and Gln175 in the panitumumab wild-type CH1 constant kappa (Cκ) structure (Figure 37A) and the panitumumab K147F-S183R-CH1-Cκ structure (Figure 37B). Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the kappa light chain (κLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are represented as sticks and main chain atoms are represented as cartoons. Hydrogen bonds are shown as dotted lines.

[0127] [Figure 38]Figures 38A-38D show that hydrogen bonds between HC-Arg183 and κLC-Thr178 can drive kappa pairing, while steric clashes of HC-Arg183 with λLC-Tyr178 reduce selectivity for lambda pairing. Figures 38A-38D provide illustrations of the region surrounding the S183R substitution in IgG1-CH1, along with hydrogen bonds between HC-Ser183 and λLC-Thr178 in the panitumumab wild-type CH1 constant lambda (Cλ) structure (Figure 38B), and between HC-Arg183 and κLC-Thr178 in the panitumumab K147F-S183R-CH1 constant kappa (Cκ) structure (Figure 38C). Figure 38A shows that HC-Ser183 and κLC-Thr178 are too far apart for hydrogen bonding to occur. The carbon atoms of the heavy chain (HC) are colored light gray, the carbon atoms of the light chain (LC) are colored white, the nitrogen atoms are colored dark gray, and the oxygen atoms are colored black. The side chains are represented as sticks. Also shown is the side chain of λLC-Tyr178 as a transparent molecular surface. Hydrogen bonds are shown as black dotted lines. Figure 38D provides a model of the superposition of the HC of the panitumumab K147F-S183R-CH1-Cκ structure with the HC of the panitumumab wild-type CH1-Cλ structure. The resulting model shows a clear steric clash between HC-Arg183 and λLC-Tyr178. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0128] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value can vary by 1% or less from the recited value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0129] It should be understood that the aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0130] Provided herein is an engineered CH1 domain that contains at least one amino acid substitution that prevents heavy-light chain mispairing by promoting preferential pairing of a CH1 domain-containing heavy chain with either a kappa CL domain (or kappa light chain) or a lambda CL domain (or lambda light chain). The term "preferential pairing" refers to the pairing of a heavy chain (or CH1 domain) with a light chain (or CL domain) in a polypeptide, such as an antibody, such as a bispecific antibody. When a heavy chain (H1) is co-expressed with two different light chains (L1 and L2), H1 will pair with each of L1 and L2, resulting in a mixture of H1:L1 and H1:L2. In some cases, H1 can pair equally well with both L1 and L2, resulting in a mixture of approximately 50:50 H1:L1 to H1:L2. As an example, "preferential pairing" between H1 and L1 occurs if the amount of H1:L1 heterodimer formed exceeds the amount of H1:L2 heterodimer formed when H1 is co-expressed with L1 and L2. In this example, H1 preferentially pairs with L1 compared to L2. If H1 has an inherent tendency to pair with L1 over L2 (e.g., the ratio of H1:L1 to H1:L2 is not 50:50, but is 60:40 or 70:30, in which case formation of H1:L2 is still undesirable), preferential pairing between the desired pair, i.e., H1:L1, occurs when there is an improved (increased) amount of pairing between H1:L1 compared to H1:L2. As used herein, the term "preferential pairing" encompasses not only heavy chain and light chain pairing (as described above), but also CH1 and CL domain pairing. For example, if the amount of CH1:Cκ formed exceeds the amount of CH1:Cλ formed when CH1 is co-expressed with Cκ and Cλ, then "preferential pairing" between the CH1 domain and the kappa CL domain has occurred. Similarly, if the amount of CH1:Cλ formed exceeds the amount of CH1:Cκ formed when CH1 is co-expressed with Cλ and Cκ, then "preferential pairing" between the CH1 domain and the lambda CL domain has occurred.

[0131] It has been discovered that certain positions in the CH1 domain, identified as part of the CH1-CL interface (for both Cκ and Cλ), affect the binding of the heavy chain to the light chain. In addition, it has also been shown that positions in the CH1 domain at the CH1:VH interface affect the binding of the heavy chain to the light chain. The heavy chain pairs with the light chain through two sets of domain interfaces, one between the VH and VL domains and the other between the CH1 and CL domains, and the place where the chains pair or face or contact is called the "interface". Furthermore, in the heavy chain, the CH1 domain also contacts a portion of the VH, and the space where the CH1 domain and the VH are in close proximity is also encompassed by the term "interface". An interface includes amino acid residues in the heavy chain and amino acid residues in the light chain, or alternatively amino acid residues in the CH1 domain and amino acid residues in the VH, that contact each other in three-dimensional space. In some embodiments, the interface includes the CH1 domain of the heavy chain and the CL domain of the light chain. In other embodiments, the interface comprises the CH1 and VH domains of the heavy chain. Preferably, the "interface" is derived from an IgG antibody or its Fab.

[0132] The CH1 variant domains described herein contain amino acid substitutions at one or more CH1:CL interface (CH1:kappa CL, or CH1:lambda CL) positions, such as 141, 147, 170, 171, 175, 181, 183, 184, 185, 187, and / or 218 positions, or at one or more CH1:VH interface positions, such as 151 positions, compared to the parent. The term "parent" refers to a polypeptide (and the amino acid sequence encoding it) that is subsequently modified to generate a variant. A parent polypeptide may be a wild-type or naturally occurring polypeptide or a variant or engineered version thereof. Thus, a "parent CH1 domain" refers to a CH1 domain polypeptide (and the amino acid sequence encoding the CH1 domain polypeptide) that is subsequently modified to generate a CH1 domain variant. Such a parent CH1 domain may be a wild-type or naturally occurring CH1 domain, or a variant or engineered version thereof, such as a wild-type CH1 domain modified to conjugate a toxin or a small molecule drug. Such a parent CH1 domain may be isolated from or be part of a larger construct, e.g., a Fab, F(ab')2, or IgG, which may optionally contain further modifications, such as, for example, CH3 modifications that promote heterodimer formation, CH2 and / or CH3 modifications that alter Fc receptor binding, extend half-life, and / or link additional binding domains.

[0133] The resulting CH1 variant domain preferentially pairs with either a kappa CL (Cκ) domain or a lambda CL (Cλ) domain, where the Cκ and Cλ domains may be part of a light chain. Mutation of the amino acid at one or both of positions 147 and 183 (EU numbering) of the CH1 domain promotes binding to Cκ (and at the same time inhibits pairing with Cλ), while mutation of the amino acid at position 141 of the CH1 domain promotes binding to Cλ (and at the same time inhibits pairing with Cκ). The kappa and lambda CL domains can be present in any number of formats, including, but not limited to, Fab or IgG, wild-type or chimeric, such as Fab or IgG containing Vκ and Cκ, Vκ and Cλ, Vλ and Cκ, or Vλ and Cλ. Such CH1 variant domains may be useful in engineering multispecific antibodies by improving the precision of heavy-light chain pairing while maintaining the native IgG structure of the bispecific antibody, and are preferred based on their well-established properties as therapeutic molecules, including their long half-life in vivo and ability to elicit effector functions.

[0134] The term "CH1 domain" refers to the first constant domain of the heavy chain of an antibody, the C-terminus of the variable domain of the heavy chain, and the N-terminus of the hinge region. According to IMGT, the CH1 domain is the amino acid sequence of positions 118-215 (EU numbering) and the hinge region is the amino acid sequence of positions 216-230 (EU numbering). As used herein, the term "CH1 domain variant" refers to an amino acid sequence comprising the entire CH1 domain (positions 118-215 according to EU numbering) or a fragment thereof comprising at least seven of the CH1 residues 118-215 (according to EU numbering), such fragment comprising one or more of the modifications disclosed herein, as well as a portion of the hinge region (positions 216-218). The library screened to identify the described CH1 domain variants contained variegation within the hinge region, for example at positions 216 and 218.

[0135] The CH1 domain pairs with the CL domain of the light chain. In some embodiments, the light chain is a kappa chain. In some embodiments, the light chain is a lambda chain. The term "kappa constant domain", "kappa CL domain", or "Cκ" refers to the constant domain of the kappa light chain. The term "lambda constant domain", "lambda CL domain", or "Cλ" refers to the constant domain of the lambda light chain. A single disulfide bond covalently links CH1 to the CL domain. As used herein, the CH1 domain refers to all antibody isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE.

[0136] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments exhibiting the desired antigen-binding activity, also referred to as "antigen-binding antibody fragments").

[0137] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for potential variant antibodies (e.g., containing natural mutations and / or substitutions or arising during production of the monoclonal antibody preparation). Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0138] A "multispecific antibody," which may also be referred to herein as a "multispecific compound," refers to an antibody that comprises at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens or at least two different epitopes. In some embodiments, a multispecific antibody contains (1) a first heavy chain and a first light chain that form a cognate pair and bind to a first antigen, and (2) a second heavy chain and a second light chain that form a cognate pair and bind to a second antigen.

[0139] A "bispecific antibody", also referred to herein as a "bispecific compound", is a type of multispecific antibody and refers to an antibody that contains two different antigen-binding domains that recognize and specifically bind to at least two different antigens or at least two epitopes. The at least two epitopes may or may not be within the same antigen. A bispecific antibody may target, for example, two different surface receptors, two different cytokines / chemokines, receptors and ligands on the same or different (e.g., immune and cancer) cells. Combinations of antigens that can be targeted by bispecific antibodies include CD3 and Her2, CD3 and Her3, CD3 and EGFR, CD3 and CD19, CD3 and CD20, CD3 and EpCAM, CD3 and CD33, CD3 and PSMA, CD3 and CEA, CD3 and gp100, CD3 and gpA33, CD3 and B7-H3, CD64 and EGFR, CEA and HSG, TRAIL-R2 and LTbetaR, EGFR and IGFR, VEGFR2 and VEGFR3, VEGFR2 and PDGFR alpha, PDGFRalpha and PDGFR beta, EGFR and MET, EGFR and EDV-miR16, EGFR and CD64, EGFR and Her2, EGFR and Her3, Her2 domain ECD2 and Her2 domain ECD4, Her2 and Her3, IGF-1R and HER3, CD19 and CD22, CD20 and CD22, CD30 and CD16A, FceRI and CD32B, CD32B and CD79B, MP65 and SAP-2, IL-17A and IL-23, IL-1alpha and IL-1beta, IL-12 and IL-18, VEGF and osteopontin, VEGF and Ang-2, VEGF and PDGFRbeta, VEGF and Her2, VEGF and DLL4, FAP and DR5, FcgRII and IgE, CEA and DTPA, CEA and IMP288, and LukS-PV and LukF-PV.

[0140] "Different antigens" can refer not only to different and / or distinct proteins, polypeptides, or molecules, but also to different and / or distinct epitopes, which may be contained within one protein, polypeptide, or other molecule. As a result, a bispecific antibody may bind to two epitopes on the same polypeptide.

[0141] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells react. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be structurally or functionally defined. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational; that is, they may be composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, epitopes may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0142] In some examples, an antibody comprises four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a variable region, such as a heavy chain variable region ("VH"), and a heavy chain constant region ("CH"). In the case of an intact antibody, the CH comprises domains CH1, CH2, and CH3. In the case of an antibody fragment, the CH may comprise CH1, CH2, and / or CH3 domains, and in some preferred embodiments, the CH comprises at least a CH1 domain. The CH1 domain variants disclosed herein may be used in combination with wild-type CH2 and / or CH3 domains, or with CH2 and / or CH3 domains that comprise one or more amino acid substitutions, e.g., that alter or improve the stability and / or effector function of the antibody. Each light chain comprises a variable region, such as a light chain variable region ("VL"), and a light chain constant region ("CL"). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each of the VH and VL comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally occurring or artificially modified. A consensus sequence of amino acids may be defined based on the alignment and analysis of two or more CDRs. Thus, the CDRs in the heavy chain are denoted as "CDRH1", "CDRH2", and "CDRH3", respectively, and the CDRs in the light chain are denoted as "CDRL1", "CDRL2", and "CDRL3", respectively. In other examples, the antibody may include a multimer thereof (e.g., IgM) or an antigen-binding fragment thereof.

[0143] In some instances, VH and CL may be present in one polypeptide. In some instances, VL domain and CH1 domain, CH2 domain, and / or CH3 domain may be present in one polypeptide. For example, in a certain antibody or antibody fragment, a first polypeptide comprises VH1 and CH1, and a second polypeptide comprises VL1 and CL (VH1 and VL form an antigen binding site for a first epitope), a third polypeptide comprises VH2 and CL, and a fourth polypeptide comprises VL2 and CH1 (VH2 and VL2 form an antigen binding site for a second epitope). In another certain antibody or antibody fragment, a first polypeptide comprises VH1 and CH1, and a second polypeptide comprises VL1 and CL (VH1 and VL form an antigen binding site for a first epitope), while a third polypeptide comprises VL2, CL, and one or more of CH2 and / or CH3 domains, and a fourth polypeptide comprises VH and CH1. Any antibody or antibody fragment comprising any of the CH1 variants disclosed herein, which provides preferential pairing with a kappa CL or preferential pairing with a lambda CL, regardless of whether the CH1 domain is in the heavy chain or the light chain, is encompassed by the present invention.

[0144] As used herein, the term "cognate pair" or "cognate pairing" refers to a pairing or association of two antibody chains (e.g., heavy and light chains) each of which contains a variable region (e.g., VH and VL, respectively) and the combination of the variable regions provides the intended binding specificity for an epitope or antigen. As used herein, the term "non-cognate pair" or "non-cognate pairing" refers to a pairing or association of two antibody chains (e.g., heavy and light chains) each of which contains a variable region (e.g., VH and VL, respectively) and the combination of the variable regions does not provide the intended binding specificity for an epitope or antigen.

[0145] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0146] Unless otherwise indicated, as used herein, the term "antibody" includes a molecule comprising two heavy immunoglobulin chains and two light immunoglobulin chains (which may also be referred to as a "full-length antibody" or "intact antibody" or "whole antibody", etc., and in all instances refers to an antibody having substantially the same structure as a naturally occurring antibody), as well as antigen-binding antibody fragments thereof. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody, or a combination of portions derived from an intact antibody, that binds to the antigen bound by the intact antibody.

[0147] Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fv, fragments of antigen-binding fragments ("Fab"), Fab' fragments, Fab' containing a free sulfhydryl group ("Fab'-SH"), F(ab')2 fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain variable fragments ("scFv"), nanobodies or VHH, or only VH or VL domains), and monospecific or multispecific compounds formed from one or more of the antibody fragments described above. In some embodiments, the antigen-binding fragment of the bispecific antibody described herein is an scFv. In a preferred embodiment, the antigen-binding fragment comprises a CH1 domain that preferentially pairs with a kappa CL or with a lambda CL.

[0148] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). A multispecific antigen-binding fragment of an antibody may contain at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to different epitopes on the same antigen.

[0149] The present disclosure provides CH1 domain variants that preferentially pair (or bind) with kappa or lambda light chain CL domains. In one embodiment, the CH1 domain variants exhibit no or reduced binding to kappa or lambda class light chains, while exhibiting exclusivity or increased selectivity for binding to the other class of light chain (in this example, lambda or kappa, respectively). These CH1 domain variants can be used to completely or partially resolve mispairing of heavy and light chains in the generation of multispecific antibodies, such as bispecific antibodies, by promoting proper heavy and light chain pairing. In one embodiment, the CH1 domain variants may be used, optionally in combination with other variants outside the CH1 domain, to further promote preferential pairing with the kappa or lambda light chain CL domains (e.g. VH:VL substitutions, such as Q39E / K:Q38K / E (Dillon et al., MAbs 2017 9(2):213-230), or Q39K+R62E:Q38D+D1R or Q39Y+Q105R:Q38R+K42D (Brinkmann et al., MAbs 2017 9(2):182-212, etc.). More specifically, bispecific antibodies comprising these CH1 variant domains will form fewer undesired product-associated contaminants, i.e. molecules containing mispaired domains, which may be difficult to remove during further processing. For example, a bispecific antibody comprising (i) a heavy chain and a light chain from antibody A (wherein the light chain is a kappa light chain) and (ii) a heavy chain and a light chain from antibody B (wherein the light chain is a lambda light chain) may be produced more efficiently, i.e., free of many undesirable production-related contaminants, by engineering the heavy chain CH1 domain of antibody A into a CH1 domain variant that is kappa-preferring (e.g., Phe at 147 and / or Arg, Lys, Tyr at 183, etc.), and engineering the heavy chain CH1 domain of antibody B into a CH1 domain variant that is lambda-preferring (e.g., Asp at 141, etc.).As a result, the heavy chain of antibody A will readily bind to the light chain of antibody A (and will dislike binding to the light chain of antibody B), while the heavy chain of antibody B will readily bind to the light chain of antibody B (and will dislike binding to the light chain of antibody A). See Figures 1 and 7, and Table 6.

[0150] In some embodiments, the CH1 domain variant reduces mismatching, i.e., non-cognate HC1-LC2 and / or HC2-LC1 pairing, by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, the CH1 domain variant containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 147F+183R, 147F+183K, 147F+183Y, reduces mismatching, i.e., non-cognate HC1-LC2 and / or HC2-LC1 pairing, by at least 25% to at least 80%. In some embodiments, a CH1 domain variant containing a substitution at position 141, such as 141D, alone or in combination with other substitutions, such as 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduces mismatching, i.e., non-cognate HC1-LC2 and / or HC2-LC1 pairing, by at least 50%. In some embodiments, a CH1 domain variant containing a substitution at position 141, such as 141D, alone or in combination with other substitutions, such as 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduces mismatching, i.e., non-cognate HC1-LC2 and / or HC2-LC1 pairing, by at least 75%.

[0151] In some embodiments, the CH1 domain variant preferentially pairs (binds) with a cognate CL domain (either Cκ or Cλ) or with a cognate light chain containing a corresponding CL domain (either Cκ or Cλ), resulting in formation of at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the desired first and second cognate pairs, i.e., HC1-LC1 and / or HC2-LC2. In some embodiments, the CH1 domain variant preferentially pairs (binds) with a cognate CL domain (either Cκ or Cλ) or with a cognate light chain containing the corresponding CL domain (either Cκ or Cλ), resulting in about 80% to about 99%, or more specifically at least about 85% to at least about 95% formation of the desired first and second cognate pairs, i.e., HC1-LC1 and / or HC2-LC2. In some embodiments, a CH1 domain variant containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, provides for about 85% to at least about 95% formation of the desired first and second cognate pairs, i.e., HC1-LC1 and / or HC2-LC2.

[0152] In some embodiments, the CH1 domain variant reduces the formation of mismatched heavy chain-light chain heterodimers, i.e., HC1-LC2 and / or HC2-LC1 pairs, by less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the CH1 domain variant containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduces the formation of mismatched heavy chain-light chain heterodimers by less than about 15%, less than about 10%, or less than about 5%.

[0153] Several CH1 domain positions have been identified as influencing light chain binding selectivity, i.e., preferential pairing with kappa or lambda CL domains, including positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 (EU numbering). Substitution of the wild-type amino acid residue at any one or more of these positions in the CH1 domain with a variant (non-wild-type) amino acid residue results in heavy chains that preferentially pair with light chains containing either kappa or lambda CL domains. For example, positions 147 and 183 were each identified as having preference for pairing with kappa CL domains, and positions 141, 170, 171, 175, 181, 184, 185, 187, and 218 were identified as having preference for pairing with lambda CL domains.

[0154] Substitution of the wild type amino acid residue (Ala) at position 141 of the CH1 domain with Thr, Asp, Lys, Glu, Arg, Met, Val, or Gln showed increased selectivity of the heavy chain for binding to light chains containing lambda CL domains. Substitution of the wild type amino acid residue (Phe) at position 170 of the CH1 domain with Glu, Gly, Ser, Asn, or Thr; substitution of the wild type amino acid residue (Pro) at position 171 of the CH1 domain with Glu, Gly, Ser, Asn, Asp, or Ala; substitution of the wild type amino acid residue (Met) at position 175 of the CH1 domain with Asp or Met; substitution of the wild type amino acid residue (Ser) at position 181 of the CH1 domain with Val, Leu, Ala, Lys, or Thr; substitution of the wild type amino acid residue (Ser) at position 184 of the CH1 domain with Arg substitution of the wild type amino acid residue at position 185 of the CH1 domain (Val) with Met, Leu, Ser, Arg, Thr; substitution of the wild type amino acid residue at position 187 of the CH1 domain (Thr) with Arg, Asp, Glu, Tyr, or Ser; and / or substitution of the wild type amino acid residue at position 218 of the CH1 domain (Lys) with Leu, Glu, Asp, Pro, Ala, His, Ser, Gln, Asn, Thr, Ile, Met, Gly, Cys, Lys, or Trp also contribute to increased pairing of heavy chains with light chains containing a lambda CL domain.

[0155] Substitution of the wild type amino acid residue (Lys) at position 147 of the CH1 domain with Val, Ala, Phe, Ile, Thr, Ser, Tyr, Leu, Arg, Asn, Glu, His, Met, or Gln showed increased selectivity of the heavy chain for binding to light chains containing kappa CL domains. Substitution of the wild type amino acid residue (Ser) at position 183 of the CH1 domain with Arg, Lys, Tyr, Trp, Glu, Phe, Ile, Leu, Asn, or Gln showed increased selectivity of the heavy chain for binding to light chains containing kappa CL domains (see FIG. 5). The effect of a given variant amino acid residue at a particular position may vary, but all variants show improved preferential pairing with Cκ or Cλ based on the amino acid position containing the variant residue. Furthermore, given the high degree of similarity in the CH1 regions of IgG1, IgG2, IgG3, and IgG4, it is expected that the CH1 domain variants described herein will exhibit similar preferential pairing properties in each isotype.

[0156] Initial rounds of selection identified Thr at position 141 as promoting preferential pairing with Cλ compared to the wild-type CH1 domain sequence (Ala at position 141), while further rounds of selection identified Asp, Arg, and Gln as conferring increased preferential pairing compared to Thr (see FIG. 5). Further screening strategies identified Lys and Glu as also conferring increased lambda selectivity (see Example 5, FIGS. 10-14). Glu at position 170, Glu at position 171, Met at position 175, Lys at position 181, Arg at position 184, Arg at position 185, Arg at position 187, and / or Pro, Ala, or Glu at position 218 were also found to increase lambda selectivity (see Examples 5-7). Further, the applicant indicates that specific combinations of substitutions that increase lambda selectivity include Asp at position 141 and Lys at position 181; Asp at position 141, Lys at position 181, and Ala at position 218; Asp at position 141, Lys at position 181, and Pro at position 218; Glu at position 141, Glu at position 170, Val at position 181, and Arg at position 187; Glu at position 141, Asp at position 171, and Arg at position 185; , Glu at position 171, and Arg at position 185, Glu at position 141, Gly at position 171, Arg at position 185, and Arg at position 187, Glu at position 141, Arg at position 185, and Arg at position 187, Glu at position 141, Ser at position 171, and Lys at position 181, Glu at position 141, Gly at position 170, Met at position 175, Val at position 181, Arg at position 184, and Arg at position 187, but are not limited thereto. In the results of further screening, "Asp at position 141, Glu at position 171, and Arg at position 185" and "Asp at position 141, Glu at position 170, and Arg at position 187" were identified as particularly lambda-preferential CH1 domain substitution combinations (see Figures 20, 23, 30, and 31).

[0157] Similarly, initial rounds of selection identified Val or Ala at position 147 and Lys at position 183 as promoting preferential pairing with Cκ compared to the wild-type CH1 domain sequence, while further rounds of selection identified Phe, Ile, Thr, Tyr, Leu, Arg, Asn, Glu, His, Met, or Gln at position 147 and / or Arg, Tyr, Trp, Glu, Phe, or Gln at position 183 as increasing preferential pairing compared to 147Val or Ala or 183Lys, respectively. These CH1 domain variants, alone or in combination with other amino acid substitutions, can improve preferential pairing of heavy chains containing such CH1 domain variants with light chains containing Cκ or Cλ.

[0158] Provided herein are variant CH1 domains comprising amino acid substitutions at one or more of the following positions, such that the CH1 domain variants exhibit preferential pairing with either Cκ or Cλ (or light chains comprising such domains): positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218 according to EU numbering. As demonstrated herein, substitution of different amino acid residues at one or more of these positions can result in a CH1 domain that preferentially pairs with either Cκ or Cλ (see Tables 3 and 4). In some embodiments, the amino acid substitution at position 147 (EU numbering) is not a cysteine. In some embodiments, the amino acid substitution at position 183 (EU numbering) is not a cysteine ​​or a threonine. In some embodiments, the amino acid substitution at position 147 (EU numbering) is not a cysteine ​​and the amino acid substitution at position 183 (EU numbering) is not a cysteine ​​or a threonine.

[0159] In some embodiments, the CH1 domain variant comprises an amino acid substitution at one or more of the following positions that drive preferential pairing of the CH1 domain variant (or a heavy chain comprising such domain) to Cκ (or a light chain comprising such domain): 118, 124, 126-129, 131-132, 134, 136, 139, 143, 145, 147-151, 153-154, 170, 172, 175-176, 181, 183, 185, 190-191, 197, 201, 203-206, 210, 212-214 and 218 (EU numbering). In some embodiments, the amino acid substitutions are one or more of the following: position 118 is substituted with G, position 124 is substituted with H, R, E, L, or V, position 126 is substituted with A, T, or L, position 127 is substituted with V or L, position 128 is substituted with H, position 129 is substituted with P, position 131 is substituted with A, position 132 is substituted with P, and position 134 is substituted with G. , position 136 is substituted with E, position 139 is substituted with I, position 143 is substituted with V or S, position 145 is substituted with F, I, N, or T, position 147 is substituted with F, I, L, R, T, S, M, V, E, H, Y, or Q, position 148 is substituted with I, Q, Y, or G, position 149 is substituted with C, S, or H, position 150 is substituted with L or S, position 151 is substituted with A, or position 153 is substituted with S; position 154 is substituted with M or G; position 170 is substituted with G or L; position 172 is substituted with V; position 175 is substituted with G, L, E, A; position 176 is substituted with P; position 181 is substituted with Y, Q, or G; position 183 is substituted with I, W, F, E, Y, L, K, Q, N, or R; position 185 is substituted with W; position 190 is Position 191 is substituted with I, position 197 is substituted with A, position 201 is substituted with S, position 203 is substituted with S, position 204 is substituted with Y, position 205 is substituted with Q, position 206 is substituted with S, position 210 is substituted with R, position 212 is substituted with G, position 213 is substituted with E or R, position 214 is substituted with R, and position 218 is substituted with Q.In some embodiments, the CH1 domain variant comprises amino acid substitutions at positions 147 and 183 to drive preferential pairing (binding) with kappa light chains. In some embodiments, the amino acid substituted at position 147 is selected from the group consisting of F, I, L, R, T, S, M, V, E, H, Y, and Q, and wherein the amino acid substituted at position 183 is selected from the group consisting of I, W, F, E, Y, L, K, Q, N, and R. In certain embodiments, the CH1 domain variant comprises R or K or Y at position 183, alone or in combination with F at position 147. Non-limiting examples of kappa-preferential CH1 domain variants may comprise the amino acid sequences of SEQ ID NOs: 137, 138, 139, 60, 41, or 136.

[0160] In some embodiments, the CH1 domain variant comprises an amino acid substitution at one or more of the following positions that drive preferential pairing of the CH1 domain variant (or a heavy chain comprising such a domain) to a Cλ (or a light chain comprising such a domain): 119, 124, 126-127, 130-131, 133-134, 138-142, 152, 163, 170-171, 175, 181, 183-185, 187, 197, 203, 208, 210-214, 216 and 218 (EU numbering).In some embodiments, the amino acid substitutions are one or more of the following: position 119 is substituted with R, position 124 is substituted with V, position 126 is substituted with V, position 127 is substituted with G, position 130 is substituted with H or S, position 131 is substituted with Q, T, N, R, V, or D, position 133 is substituted with D, T, L, E, S, or P, position 134 is substituted with A, H, I, P, V, N, or L, position 135 is substituted with H, I, P, V, N, or L, position 136 is substituted with H, I, P, V, N, or L, position 137 is substituted with H, I, P, V, N, or L, position 139 is substituted with H, I, P, V, N, or L, position 140 is substituted with H, I, P, V, N, or L, position 141 is substituted with H, I, P, V, N, or L, position 142 is substituted with H, I, P, V, N, or L, position 143 is substituted with H, I, P, V, N, or L, position 144 is substituted with H, I, P, V, N, or L, position 145 is substituted with H, I, P, V, N, or L, position 146 is substituted with H, I, P, V, N, or L, position 147 is substituted with H, I, P, V, N, or L, position 148 is substituted with H, I, P, V, N, or L, position 149 is substituted with H, I, P, V, N, or L, position 150 is substituted with H, I, P, V, N, or L, position 151 is substituted with H, I, P, V, N, or L, position 152 is substituted with H, I, P, V, Position 8 is substituted with R, position 139 is substituted with A, position 140 is substituted with I, V, D, Y, K, S, W, R, L, or P, position 141 is substituted with D, T, R, E, K, Q, V, or M, preferably D, E, or K, position 142 is substituted with M, position 152 is substituted with G, position 163 is substituted with M, position 168 is substituted with F, I, or V, position 170 is substituted with N, G, E, S, or T, preferably E, or or G, position 171 is substituted with N, E, G, S, A, D, preferably with D, E, G, or S, position 175 is substituted with D or M, preferably with M, position 181 is substituted with V, L, A, K, or T, preferably with K or V, position 183 is substituted with L or V, position 184 is substituted with R, position 185 is substituted with M, L, S, R, or T, preferably with R, position 187 is substituted with R, D, E, Y, or Position 197 is substituted with S, position 203 is substituted with D, position 208 is substituted with I, position 210 is substituted with T, position 211 is substituted with A, position 212 is substituted with N, position 213 is substituted with E, position 214 is substituted with R, position 216 is substituted with G, and position 218 is substituted with P, A, L, E, D, H, S, Q, N, T, I, M, G, C, K, or W, preferably P or A. In some embodiments, the CH1 domain comprises an amino acid substitution at residue 141 to drive preferential pairing to lambda light chains. In some embodiments, the amino acid substituted at residue 141 is selected from the group consisting of T, R, E, K, V, D, and M. In certain embodiments, the CH1 domain variant comprises Asp or Glu at position 141.In some embodiments, the amino acid substitution at position 141 may be combined with one or more substitutions in CH1, such as Lys at position 181, or Lys at position 181 and Ala or Pro at position 218. Asp or Glu at position 141 may be combined with one or more substitutions at positions 170, 171, 175, 181, 184, 185, and / or 187, such as Glu or Gly at position 170, Asp, Glu, Gly, or Ser at position 171, met at position 175, Val or Lys at position 181, Arg at position 184, Arg at position 185, and / or Arg at position 187. Non-limiting examples of lambda-preferring CH1 domain variants can include the amino acid sequence of SEQ ID NO: 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 155, 157, 159, 162, 163, 164, 165, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or 189.

[0161] In certain embodiments, the CH1 domain variant comprises a combination of 141D, 181K, and 218P, a combination of 141D, 171E, and 185R, or a combination of 141D, 170E, and 187R. In further embodiments, the CH1 domain variant comprises the amino acid sequence of SEQ ID NO: 188, 186, or 143.

[0162] The present disclosure also contemplates polypeptides, such as antibodies, that comprise CH1 domain variants. Such polypeptides may be multispecific antibodies that comprise a first heavy chain that contains a first CH1 domain variant and a second heavy chain that contains a second CH1 domain variant. The first heavy chain and the second heavy chain can bind to different epitopes. In some embodiments, the antibody comprises a first heavy chain that contains a first CH1 domain. In some embodiments, the antibody further comprises a second heavy chain that contains a second CH1 domain that contains an amino acid sequence different from the first heavy chain CH1 domain.

[0163] In some embodiments, the first CH1 domain variant is capable of preferentially pairing (or binding) with Cκ and the second CH1 domain variant is capable of preferentially binding with Cλ, where the first light chain comprises a Cκ domain and the second light chain comprises a Cλ domain. In some embodiments, the first light chain is a kappa light chain (Cκ and Vκ) or a chimeric light chain (Cκ and Vλ) and the second light chain is a lambda light chain (Cλ and Vλ) or a chimeric light chain (Cλ and Vκ).

[0164] In some embodiments, the first CH1 domain variant can preferentially pair (bind) with Cλ and the second CH1 domain can preferentially pair (bind) with Cκ, where the first light chain comprises a Cλ domain and the second light chain comprises a Cκ domain. In some embodiments, the first light chain is a lambda light chain (Cλ and Vλ) or a chimeric light chain (Cλ and Vκ) and the second light chain is a kappa light chain (Cκ and Vκ) or a chimeric light chain (Cκ and Vλ).

[0165] The first light chain and the second light chain may (or may not) include amino acid substitutions that drive preferential pairing to the CH1 domain. In some embodiments, the CL domain of the light chain is not modified to alter binding to the heavy chain, e.g., to the CH1 domain. In some embodiments, the first light chain contains a wild-type CL domain, e.g., a wild-type Cκ domain or a wild-type Cλ domain. In some embodiments, the second light chain contains a wild-type CL domain, e.g., a wild-type Cκ domain or a wild-type Cλ domain. The wild-type kappa light chain or Cκ domain may be encoded by IGKC. The wild-type lambda light chain or Cλ domain may be encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0166] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. Such multispecific and bispecific antibodies can include any format that contains a CH1 domain, including but not limited to the structures shown in Figures 24-29. See also, e.g., Table 2, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017), which is incorporated by reference in its entirety.

[0167] The multispecific antibody may comprise one or more of the CH1 domain variants having an amino acid sequence listed in Tables 3, 4, 7, 9, 12, or 13. In some embodiments, the antibody comprises a first heavy chain and a first light chain containing a first CH1 domain variant, wherein the first heavy chain and the first light chain form a first cognate pair. The first CH1 domain variant may comprise an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218 according to EU numbering. Such a first CH1 domain variant preferentially binds to the first light chain. The CL domain of the first light chain may or may not be modified to alter binding to the first heavy chain.

[0168] In some embodiments, the antibody comprises a second heavy chain containing a second CH1 domain variant and a second light chain, the second heavy chain and the second light chain forming a second cognate pair. The second CH1 domain variant may include an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218 according to EU numbering. Such second CH1 domain variant preferentially binds to the second light chain. The CL domain of the second light chain may or may not be modified to alter binding to the second heavy chain.

[0169] In some embodiments of the multispecific antibody or antibody fragment, the antibody or antibody fragment may comprise a kappa-preferred CH1 domain variant and a lambda-preferred CH1 domain variant. In some examples, the kappa-preferred CH1 domain variant may be a kappa-preferred CH1 domain variant disclosed herein, and the lambda-preferred CH1 domain may be a lambda-preferred CH1 domain that may or may not be described herein. In some examples, the lambda-preferred CH1 domain variant may be a lambda-preferred CH1 domain variant disclosed herein, and the kappa-preferred CH1 domain may be a kappa-preferred CH1 domain that may or may not be described herein. In some examples, both the kappa-preferred CH1 domain variant and the lambda-preferred CH1 domain variant are variants disclosed herein.

[0170] Any of the CH1 domain variants disclosed herein can be used to provide selectivity for pairing to a kappa or lambda CL domain, and the CL domain can be wild-type or non-wild-type. Additionally, any of the CH1 domain variants disclosed herein can be used to provide selectivity for kappa / lambda pairing within the structure of an antibody or antibody fragment, with or without the introduction of additional amino acid modifications into the remainder of the antibody structure, for example into the CH2, CH3, VH, VL, or CL domains. For example, the CH1 domain variants disclosed herein can be used with VH substitutions that can further enhance the selectivity of light chain pairing (e.g., VH:VL substitutions such as Q39E / K:Q38K / E (Dillon et al., MAbs 2017 9(2):213-230), or Q39K+R62E:Q38D+D1R or Q39Y+Q105R:Q38R+K42D (Brinkmann et al., MAbs 2017 9(2):182-212, etc.)).

[0171] Without intending to affect the scope of the invention, it is emphasized that while the CH1 domain variants provided herein provided selectivity for kappa / lambda pairing in the context of a wild-type light chain (or a polypeptide comprising a wild-type CL domain) without the need for further modifications in the CH2, CH3, or variable domains, such non-CH1 modifications may optionally be used in combination with novel CH1 domain variants discovered by the inventors herein. This is particularly unexpected in light of the many reported failures in the field of antibody production, particularly in multispecific antibodies, to modify the CH1 domain alone to provide significant kappa or lambda selectivity.

[0172] In some embodiments, the antibody is part of a pharmaceutical composition. Such a composition may contain multiple polypeptides, such as antibodies, that contain a CH1 domain variant described herein.

[0173] The present disclosure also contemplates methods of obtaining such CH1 domain variants. The variant CH1 domains described herein may be identified by rational design (in silico) or randomly, e.g., using ePCR or other mutagenic techniques known in the art. In one embodiment, a rational design approach is used to design variant CH1 domains. For such an approach, a set of structures, e.g., experimentally derived protein structures, e.g., Fab crystal structures, may be constructed and analyzed to identify solvent-exposed positions involved in contact across the CH1-CL domain interface (also referred to as CH1-CL domain interface positions). The set may be curated by selecting structures with certain properties, e.g., high identity to reference (wild-type) CH1, Cκ, and Cλ. In some embodiments, a position is described or defined as being in contact (or "in contact") with another residue if a pair of side chain atoms is within a cutoff distance of 5 Å. A "CH1 interface residue" may be defined as a residue in the CH1 domain that contacts a residue in the Cκ domain or the Cλ domain. The terms "residue" and "position" can be used interchangeably in this context. It is also an unexpected discovery by the inventors that amino acid substitutions at CH1 positions (e.g., CH1 position 151) in the CH1-VH interface change the selectivity of light chain isotypes. Therefore, in some embodiments, CH1 positions that contact with residues of VH (e.g., a pair of side chain atoms are within a cutoff distance of 5 Å) can also be selected for the identification of rational CH1 domain variants.

[0174] The choice of which amino acid positions to vary, whether alone or in combination (e.g., singlets, doublets, triplets, etc.), can depend on a variety of different parameters, such as, for example, the consistent role of that position in forming the interface between CH1 and CL or CH1 and VH in different structures, the accessibility of that position across structures, the relationship of that position to positions that affect antigen binding, or the potential for the residue to affect the formation of the CH1:CL or CH1:VH interface in an allosteric manner without directly participating in intermolecular contacts across the interface. In some embodiments, an amino acid residue in the CH1 domain is selected from the set of amino acid residues if: 1) the residue is at the interface with the light chain constant domain in at least 10% of structures in the Cκ set and has a solvent accessible surface area (SASA) ratio of greater than 10% in at least 90% of structures in the Cκ set (see Example 1); or 2) the residue is at the interface with the light chain constant domain in at least 10% of structures in the Cλ set and has a SASA ratio of greater than 10% in at least 90% of structures in the Cλ set; or 3) the residue is at the interface with the light chain constant domain in at least 10% of structures in the Cλ set and has a SASA ratio of greater than 10% in at least 90% of structures in the Cλ set. κ Set and / or C λ At least 10% of the representatives of the set have a residue at the interface with VH of C κ Set and / or C λ has a solvent accessible surface area fraction of greater than 10% in at least 90% of representative sets of the set.

[0175] Additionally, for each unique amino acid substitution in the CH1 domain provided herein that confers kappa or lambda selectivity, the resulting amino acid can be further substituted with a conservative amino acid substitution to obtain another CH1 domain variant that provides the same kappa or lambda selectivity. Alternatively, for each CH1 domain variant, one or more amino acid positions that were not affected in the CH1 domain variant compared to the wild-type sequence can be modified with a conservative substitution to obtain another CH1 domain variant that provides the same kappa or lambda selectivity.

[0176] "Conservative amino acid substitutions" are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain for another nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another β-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain for another aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0177] A library may then be generated in which CH1 domain residues are varied. One or more CH1 domain residues may be varied within the library. In some embodiments, about one to six CH1 domain residues are varied within the library. Amino acid diversity at individual residue positions may be generated via degenerate codons, e.g., NNK, allowing for representation of all of at least 20 naturally occurring amino acids at a given CH1 domain position. Selected CH1 domain positions may be varied individually to generate point substitutions (also called singlets), or a subset of position combinations may be varied in combination to generate, for example, double and triple substitutions (also called doublets and triplets). In some embodiments, a combination of variants is generated that includes CH1 domain positions that are close in 3D space, e.g., positions 147 x [124, 126, 145, 148, 175 and 181].

[0178] In some embodiments, a method for generating a CH1 domain variant library includes: a) providing a set of structures containing one or more kappa constant (Cκ) domains, one or more lambda constant (Cλ) domains, and one or more CH1 domains; b) selecting one or more solvent exposed CH1 domain positions that contact one or more Cκ domain positions and / or one or more Cλ domain positions for substitution; c) substituting one or more CH1 domain positions identified in step b) with any amino acid other than the parent amino acid; and d) synthesizing polypeptides encoding the CH1 variant domains of step c) to construct a CH1 variant domain library.

[0179] In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are wild type. In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are human (including all allelic functional variants). In some embodiments, the Cκ amino acid sequence of step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence of step a) is encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In certain embodiments, the Cλ amino acid sequence of step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions across the CH1-CL interface or the CH1-VH interface.

[0180] In some embodiments, one or more CH1 amino acid residues selected for substitution are: (i) at the interface with the light chain constant domain in at least 10% of the representative set of CH1:Cκ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of the representative set of CH1:Cκ structures; (ii) at the interface with the light chain constant domain in at least 10% of the representative set of CH1:Cλ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of the representative set of CH1:Cλ structures; or (iii) at the interface with the light chain constant domain in at least 10% of the representative set of CH1:Cλ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of the representative set of CH1:Cλ structures. κ and / or C λ At the interface with VH in at least 10% of the representative set of structures, and κ and / or C λ At least 90% of a representative set of structures have a solvent accessible surface area greater than 10%.

[0181] In some embodiments, libraries are generated by altering one or more CH1 positions disclosed herein that alter light chain isotype selectivity (e.g., positions 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof), and optionally one or more additional CH1 positions of interest. In an embodiment, libraries can be generated by combining predetermined substitutions at one or more CH1 positions disclosed herein that alter light chain isotype selectivity (e.g., positions 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof) with one or more additional CH1 positions of interest that are altered. In certain embodiments, the predetermined substitutions may include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P, or any combination thereof.

[0182] In some embodiments, the library is screened to identify CH1 domain variants that exhibit preferential binding to kappa light chain or lambda light chain. Such screening can be initiated by expressing the library in a suitable host cell, such as, for example, a eukaryotic cell, for example, a yeast cell, for example, Saccharomyces cerevisiae. After expressing the CH1 variant domains contained in the library in the host cell, the library of variants can be screened to identify those variants that have desired binding properties, for example, via FACS or MACS.

[0183] In some embodiments, a method for identifying a CH1 domain variant that preferentially binds to a Cκ or Cλ domain comprises: a) providing a set of structures containing one or more kappa constant (Cκ) domains, one or more lambda constant (Cλ) domains, and one or more CH1 domains; b) selecting one or more solvent exposed CH1 domain positions that contact one or more Cκ domain positions and / or one or more Cλ domain positions for replacement; c) replacing one or more CH1 domain positions identified in step b) with any amino acid other than the parent amino acid; d) synthesizing polypeptides encoding the CH1 variant domains of step c) to construct a CH1 variant domain library; and e) screening the library of d) to identify a CH1 domain variant that preferentially binds to a Cκ or Cλ domain.

[0184] In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are wild type. In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are human (including all allelic functional variants). In some embodiments, the Cκ amino acid sequence of step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence of step a) is encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In certain embodiments, the Cλ amino acid sequence of step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions across the CH1-CL interface or at the CH1-VH interface.

[0185] In some embodiments, one or more CH1 amino acid residues selected for substitution are: (i) at an interface with a light chain constant domain in at least 10% of a representative set of CH1:Cκ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of a representative set of CH1:Cκ structures; (ii) at an interface with a light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of a representative set of CH1:Cλ structures; or (iii) at an interface with a light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a solvent accessible surface area fraction greater than 10% in at least 90% of a representative set of CH1:Cλ structures. κ and / or CH1:C λ At the interface with VH in at least 10% of the representative set of structures, and κ and / or CH1:C λ At least 90% of a representative set of structures have a solvent accessible surface area greater than 10%.

[0186] The method described herein may further include verifying that the one or more substituted CH1 amino acid residues drive preferential heavy chain pairing to a kappa CL domain (or a light chain containing a kappa CL domain) compared to a lambda CL domain (or a light chain containing a lambda CL domain), or vice versa. A variety of methods can be used to assess preferential light chain pairing, including but not limited to, fluorescence activated cell sorting (FACS), LC-MS, alpha LISA, and SDS-PAGE. In some embodiments, the one or more CH1 domain positions selected for replacement in step c) occur at the interface with the light chain with a predetermined frequency, e.g., in any given set of wild-type antibody structures, the selected CH1 domain positions contact the CL domain in at least 10% of the structures. In some embodiments, the one or more CH1 domain positions selected for replacement in step c) have a solvent accessible surface area fraction of greater than about 10% in at least about 90% or more of the structures of any given set of Cκ or Cλ. In some embodiments, the one or more CH1 domain positions selected for replacement in step c) occur at the interface with the VH region with a predetermined frequency, e.g., in any given set of wild-type antibody structures, the selected CH1 domain position contacts the VH in at least 10% of the structures.

[0187] By employing the methods described herein to identify CH1 domain variants, the following CH1 domain positions were selected for substitution: 114, 116, 118, 119, 121-124, 124-143, 147-154, 160, 162-165, 167, 168, 170-172, 174, 175, 176, 178, 180, 181, 183-185, 187, 190, 191, 197, 201, 203-208, 210-214, 216, and / or 128 (according to EU numbering). Substitution of any one or combination of these CH1 domain positions can result in a CH1 domain that preferentially pairs with a particular CL domain. As a result, heavy chains containing such CH1 domain variants and light chains containing certain CL domains are more likely to form cognate pairs, i.e., there is preferential pairing between heavy and light chains to form cognate pairs that is driven, at least in part, by one or more CH1 domain substitutions.

[0188] In one embodiment, the CH1 domain variant preferentially pairs with Cκ, thereby driving preferential pairing of light chains containing Cκ domains with heavy chains containing the CH1 domain variant. In another embodiment, the CH1 domain variant preferentially pairs with Cλ domains, thereby driving preferential pairing of light chains containing Cλ domains with heavy chains containing the CH1 domain variant. Certain exemplary CH1 domain substitutions have been identified that promote preferential heavy chain pairing with kappa light chains, e.g., 147F and / or 183R, 183K or 183Y, while other CH1 domain substitutions have been identified that promote preferential heavy chain pairing with lambda light chains, e.g., 141D, 141E, 141K, 170E, 170G, 171E, 171D, 171G, 171S, 175M, 181K, 181B, 184R, 185R, 187R, 218A or 218P. Thus, bispecific antibodies comprising such CH1 domain variants can be generated with improved precision in heavy chain-light chain pairing. In some embodiments, the bispecific antibody contains a first heavy chain comprising CH1λ (e.g., 141D, 141E or 141K in combination with 170E, 170G, 171E, 171D, 171G, 171S, or 175M and / or 181K, 181B, 184R, 185R, 187R, 218A and / or 218P) and a second heavy chain comprising CH1κ (e.g., 147F and / or 183R, 183K, or 183Y), each of which preferentially pairs to its cognate light chain. In some embodiments, the bispecific antibody contains a first heavy chain comprising CH1κ (such as, for example, 147F and / or 183R, 183K or 183Y) and a second heavy chain comprising CH1λ (such as, for example, 141D, 141E or 141K in combination with 170E, 170G, 171E, 171D, 171G, 171S or 175M and / or 181K, 181B, 184R, 185R, 187R, 218A and / or 218P), e.g., "141D, 171E and 185R" or "141D, 170E and 187R", each of which preferentially pairs to its cognate light chain.

[0189] The polypeptide encoding the CH1 variant domain obtained by using the method described herein may be recombinantly expressed in a host cell, such as a eukaryotic cell. In some embodiments, the CH1 variant domain is expressed in yeast. In some embodiments, the yeast strain is Saccharomyces cerevisiae. In some embodiments, the yeast strain co-expresses one or more wild-type kappa light chains and one or more wild-type lambda light chains.

[0190] Examples are provided below to demonstrate the invention and are not intended to limit the invention to any particular application or theory of operation. EXAMPLES

[0191] Example 1: In silico selection of CH1 domain positions for diversification in the library A set of Fab crystal structures was assembled from the Protein Data Bank (PDB) and used in a structure-guided approach to identify CH1-CL interface residues for diversification.

[0192] The initial set of 2,367 Fab crystal structures was narrowed by selecting structures with high identity to the reference (wild type) CH1, Cκ and Cλ sequences (shown below). The reference sequence for CH1 alignment spans the CH1 proper (EU residues 118-215) plus a portion of the IgG1 hinge (EU residues 216-229). The Cκ and Cλ reference sequences span EU residue numbers 108-214 and 107A-215, respectively.

[0193] Reference for CH1 (plus middle hinge from above): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (SEQ ID NO: 1).

[0194] Reference for Cκ: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0195] Reference for Cλ: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3).

[0196] Residues were defined as being in "contact" if a pair of side chain atoms was within a cutoff distance of 5 Å. Residues in the CH1 interface were defined as those that contacted one or more Cκ or Cλ residues in a particular structure.

[0197] The solvent accessible surface area (SASA) of individual heavy and light chain residues was calculated in the "free state", i.e., unpaired with the light and heavy chains, respectively. The SASA ratio was defined as the ratio of the SASA of the residue in question to the SASA of the residue of an isolated Gly-X-Gly tripeptide model incorporating the same amino acid as the residue (i.e., X). Solvent-exposed residues were defined as those with a SASA ratio of greater than 10%.

[0198] Narrowing the initial set of crystal structures by high identity resulted in the identification of a set of 183 CH1:Cκ structures (the "Cκ set") and 43 CH1:Cλ structures (the "Cλ set"). After accounting for gaps in the alignment due to missing amino acids in the structures, all entries in the Cκ set were 100% identical to the CH1 and Cκ reference sequences, while entries in the Cλ set were >99% identical to the reference sequences.

[0199] A structure-based sequence alignment between Cκ and Cλ is shown below. CH1 forms stable interfaces with both Cκ and Cλ despite the low sequence identity between the latter domains. Conservative and semi-conservative substitutions according to the BLOSUM62 score are indicated using "|" and ":", respectively. The sequence identity between the domains is 38.3% (41 matches to 107 Cκ residues). [ka] The underlined amino acids represent the Cκ and Cλ residues that contact the CH1 domain. This determination is based on the consensus on Fab structures in the Cκ and Cλ sets. There are 25 Cκ interface residues and 26 Cλ interface residues. A 2×2 matrix was constructed focusing on positions that are at the interface in either Cκ or Cλ (N=28) and according to (1) whether the residue at a given position contacts CH1 and (2) whether the amino acid at that position is identical between Cκ and Cλ (see Table 1). [Table 1] Table 1 highlights that there is a set of 14 Cκ and Cλ positions that contact the CH1 domain that are structurally conserved, i.e., have identical EU residue numbering, but differ in amino acid identity. Table 2 lists the 14 amino acid positions (EU numbering) and indicates the amino acids present in kappa and lambda light chains. These differences in the identity of the Cκ and Cλ interface residues can be exploited to generate mutant CH1 domains that specifically bind only to Cκ or Cλ, but not both. [Table 2]

[0200] As an initial threshold for selection of mutations in the library, individual CH1 domain positions had to meet the following criteria: 1) the position is at the interface with the light chain constant domain in at least 10% of the structures in the Cκ set and the residue at that position has a SASA ratio of greater than 10% in at least 90% of the structures in the Cκ set, or 2) the position is at the interface with the light chain constant domain in at least 10% of the structures in the Cλ set and the residue at that position has a SASA ratio of greater than 10% in at least 90% of the structures in the Cλ set, or 3) the position is at the interface with the VH region in at least 10% of the structures in the CH1:Cκ set (Cκ set) or CH1:Cλ set (Cλ set) and has a SASA ratio of greater than 10% in at least 90% of the structures in the Cκ and / or Cλ set. The definition of the interface considers contacts of the CH1 residue with any CL domain residue, including but not limited to the set of fourteen CL domain residues listed in Table 2, or contacts of the CH1 residue with any VH residue.

[0201] Based on this threshold criterion, a set of 30 CH1 amino acid positions was identified for potential inclusion (after removing Cys220 from consideration). From this larger set, a group of 25 CH1 positions was selected to be varied in the library. Amino acid diversity at each position was generated via degenerate NNK codons representing all 20 natural amino acids (Stemmer et al., Proc Natl Acad Sci USA 1994 Oct 25;91(22):10747-51). Amino acid substitutions were made individually at each of the 25 CH1 positions, and a subset of single substitutions were selectively combined to generate, for example, double and triple mutants. The final library design consisted of 89 CH1 oligonucleotides representing 25 singlets (diversification of the NNK codon at a single CH1 position), 48 doublet mutants (diversification of the NNK codon at two CH1 positions), and 16 triplet mutants (diversification of the NNK codon at three CH1 positions). Example 2: Cκ and Cλ light chain CH1 domain variant library co-expressed in yeast

[0202] A library of human CH1 domain variants was constructed and expressed in an engineered yeast strain co-expressing wild-type human IgG Cκ and Cλ light chains (at various expression levels allowing subsequent selection of Cλ-preferential and Cκ-preferential CH1 substitutions).

[0203] Bidirectional expression plasmids (pAD7064 and pAD4800) were constructed, each of which contained wild-type human IgG light chain kappa and lambda constant domains and the yeast (Saccharomyces cerevisiae) Gal1 / Gal10 promoter region adjacent to the yeast (S. cerevisiae) URA3 gene (selection marker). Plasmids pAD7064 and pAD4800 differed in the orientation of the kappa and lambda constant domains relative to the Gal1 / 10 promoter region. Unique restriction enzyme sites (PME-I and SFI-I) were placed upstream of the kappa and lambda constant domains in each plasmid. pAD7064 and pAD4800 were digested separately with PME-I and SFI-I ​​and then transformed into the engineered yeast strains along with the PCR amplified DNA inserts (ADI-26140 light chain region; Gal1 / 10 promoter region; and differentially encoded ("modified") ADI-26140 light chain variable region (IDT gblock), with 5' and 3' ends directing assembly via homologous recombination into each plasmid). Transformed yeast were plated onto solid agar plates lacking URA3+ and grown at 30°C for 48 hours, after which clones were picked and DNA extracted and purified. After sequencing, two dual light chain DNA constructs were identified: (1) Gal10::ADI-26140-VL-Cκ Gal1::ADI-26140-VL-Cλ (human Cλ, allowing subsequent selection of CH1 substitutions in favor of Cκ under the control of this dominant promoter); and (2) Gal10::ADI-26140-VL-Cλ Gal1::ADI-26140-VL-Cκ (human Cκ, allowing subsequent selection of CH1 substitutions in favor of Cλ under the control of this dominant promoter). ADI-26140 is an anti-hen egg lysozyme (HEL) IgG.

[0204] For heavy chain expression, a DNA vector (pAD4466) was constructed containing the Gal1 promoter, an SFI-I ​​restriction site, the CH2-CH3 domains of the human IgG heavy chain (IgG1(N297A)), and TRP1 (selection marker).

[0205] In parallel, two independent pools of CH1 domain variant DNA fragments were generated for insertion into pAD4466. The first pool was generated using the in silico design approach described in Example 1. The second pool was generated via error-prone PCR (ePCR). Briefly, the mutagenic nucleotide analogs dPTP (0.01 mM) and 8-oxo-DGTP (0.01 mM) were included in the PCR reaction at (a) dilutions of 1:100 and 1:100, respectively, or (b) dilutions of 1:100 and 1:10, respectively.

[0206] pAD4466 was digested with SFI-I ​​and introduced into yeast strains expressing Cκ and Cλ along with PCR amplified DNA encoding the ADI-26140 HC variable region and CH1 domain variant DNA from rational design efforts or ePCR. Each DNA fragment carried the appropriate DNA sequences at the 5' and 3' ends to direct assembly (via homologous recombination) with the digested plasmid or PCR fragment (ADI-26140 heavy chain variable region or CH1 protein domain).

[0207] The assembly of the individual libraries was carried out via the natural yeast (Saccharomyces cerevisiae) process of homologous recombination. Dilutions of transformants from each library were plated on media lacking uracil and tryptophan to quantitate the number of members of each library. 7 To select for the presence of each (HC and double LC) plasmid, the remainder of the transformed cells were cultured in liquid medium lacking uracil and tryptophan. Example 3: Identification of CH1 domain positions that influence light chain binding

[0208] Libraries were grown as previously described (see, e.g., WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70). Briefly, after IgG induction and presentation, yeast cells (approximately 10^7-10^8) were stained with 1:100 dilution of goat anti-human F(ab')2 kappa-FITC (Southern Biotech, Birmingham, AL, Cat. No. 2062-02) and 1:100 dilution of goat anti-human F(ab')2 lambda-PE (Southern Biotech, Birmingham, AL, Cat. No. 2072-09) in PBSF for 15 min at 4°C. After washing twice with ice-cold wash buffer, cell pellets were resuspended in 0.4 mL PBSF and transferred to strainer-capped sorting tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sorting gates for (1) enriching for lambda light chains with a corresponding lack of kappa light chains (Figure 2A) or (2) enriching for kappa light chains with a corresponding lack of lambda light chains (Figure 2B). After three rounds of selection, yeast were plated on media lacking uracil and tryptophan to generate single isolates for sequence identification.

[0209] Individual clones representing unique sequences were cultured in 96-well plates. After IgG induction and presentation, approximately 2 × 10 6Yeast cells were stained with goat anti-human F(ab')2 kappa-FITC (Southern Biotech, Birmingham, AL, Cat. No. 2062-02) diluted 1:100 and goat anti-human F(ab')2 lambda-PE (Southern Biotech, Birmingham, AL, Cat. No. 2072-09) diluted 1:100 in PBSF for 15 min at 4°C. After two washes with ice-cold wash buffer, cell pellets were resuspended in 0.1 mL wash buffer and evaluated on a BD FACS Canto instrument equipped with a 96-well plate handler. Individual unique clones were scored for the ratio of median anti-kappa fluorescence intensity (MFI) to anti-lambda MFI (kappa:lambda ratio) (Figure 3) and then compared to the corresponding strain with the wild-type CH1 sequence ("parent") to calculate FOP.

[0210] The following CH1 domain positions (EU numbering) were identified as influencing light chain binding selectivity, i.e., preferential binding to either kappa CL domains (or light chains containing a kappa CL domain) or lambda CL domains (or light chains containing a lambda CL domain): positions 118, 119, 124, 126-134, 136, 139-141, 143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183, 185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218. Table 3 provides a list of CH1 sequences identified from the selection that have a preference for kappa light chains. Bold amino acid residues in the sequence column indicate substituted positions, i.e., amino acid substitutions that differ from the parent (SEQ ID NO: 1). Table 4 provides a list of CH1 sequences identified from the selection that are preferential for lambda light chains. Bold amino acid residues in the sequence column indicate the positions that are substituted. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 4-1] [Table 4-2] [Table 4-3]

[0211] Unexpectedly, several CH1 amino acid substitutions located at the VH:CH1 interface, but not at the CH1:light chain interface, were found to result in kappa binding preference in the three-chain system. In particular, the mutation sets K147V+P151A and P151L+N201S (SEQ ID NOs: 36 and 70, Table 3) yielded kappa FOP values ​​of 18.1 and 10.4, respectively. Position CH1:147 is at the CH1:LC interface, whereas CH1:201 is not (fully solvent exposed and not part of any interdomain interface). Thus, the appearance of the P151 substitution in both of these high FOP clones suggests a potential role for this position in determining kappa selectivity over lambda. Without intending to be bound by theory, it is believed that such distal mutations affect HC:LC pairing for reasons discussed below, and thus it may be possible to exploit mutations at the VH:CH1 interface for preferential kappa over lambda pairing.

[0212] First, P151 is part of the so-called "ball-and-socket joint" between the VH and CH1 domains (Lesk AM et al., Nature. 1988 Sep 8; 335(6186):188-90; Landolfi NF et al., J Immunol. 2001 Feb 1; 166(3):1748-54). This joint is hypothesized to regulate intradomain flexibility by influencing the "elbow angle" between the variable and constant domains of the antibody (Stanfield RL et al., J Mol Biol. 2006 Apr 14; 357(5):1566-74). Substitutions within the ball-and-socket joint may have functional consequences, as in the case of anti-IFN-gamma monoclonal antibodies with reduced neutralizing activity due to a single amino acid substitution in this region (Landolfi NF et al., J Immunol. 2001 Feb 1; 166(3): 1748-54). This effect is not due to a direct change in the antigen-binding interface, but rather to altered flexibility and allosteric mechanisms. Secondly, it is also known that Fabs with lambda constant domains have a larger range of elbow angles compared to Fabs with kappa domains (Stanfield RL et al., J Mol Biol. 2006 Apr 14; 357(5): 1566-74. doi: 10.1016 / j.jmb.2006.01.023. Epub 2006 Jan 25.). This high flexibility is due to the insertion of a single residue in the so-called switch region between the VL and CL domains. Third, further analysis of the Fab crystal structures (Adimab unpublished data) reveals differences in atomic packing in the ball-and-socket joint region between kappa and lambda Fabs, suggesting a novel mechanism by which modulation of Fab flexibility by the ball-and-socket joint, together with the inherent differences between Fabs with kappa and lambda light chains, may derive differential selectivity for kappa over lambda through mutations at the VH:CH1 interface. Example 4: Identification and characterization of CH1 domain variants with kappa- or lambda-preferred light chain pairing

[0213] Clones derived from selection for increased Cκ and Cλ selectivity were selected for further characterization based on the MFI ratio between kappa and lambda (see FIG. 4). For each of the 20 amino acids (NNK), pools of DNA with a single mutation at each position of interest (141, 147, or 183) were isolated and amplified. Libraries targeting these single positions were constructed in the manner described above using the appropriate light chain base stock. Four libraries were constructed containing mutations present at positions 141, 147, 183, or 147+183, respectively, of the CH1 domain. Selection for kappa or lambda selectivity was performed as described above. Results were sequenced as described above, and FACS-based quantification of kappa or lambda selectivity compared to the appropriate parent was performed to determine the amino acid substitutions that conferred preferential pairing to light chain kappa or lambda.

[0214] Several CH1 domain variants with amino acid residue substitutions at each of positions 141, 147, and 183 were identified as having preference for pairing with either kappa CL domains (or light chains containing kappa CL domains) or lambda CL domains (or light chains containing lambda CL domains). Substitutions with D, R, or Q (compared to wild-type A) at position 141 of the CH1 domain increase preferential pairing with lambda CL domains (or light chains containing lambda CL domains) (i.e., decrease the MFI ratio of kappa:lambda) (see FIG. 5). Substitutions with F, I, T, Y, L, R, N, E, H, M, or Q (compared to wild-type K) at position 147 of the CH1 domain increase preferential pairing with kappa CL domains (or light chains containing kappa CL domains) (i.e., increase the MFI ratio of kappa:lambda) (see FIG. 5). Substitutions at position 183 in the CH1 domain with R, K, Y, W, E, F or Q (compared to wild type S) increase preferential pairing with kappa CL domains (or light chains containing kappa CL domains) (i.e., increase the kappa:lambda MFI ratio) (see FIG. 5). Table 5 shows the number of observed CH1 domain variants with specific amino acid substitutions that drive pairing selectivity. [Table 5]

[0215] We then evaluated the impact of the identified CH1 domain variants on a control bispecific antibody (2 heavy chains × 2 light chains) in an IgG-like format (two Fab regions N-terminally linked to a dimeric Fc molecule). VH-CH1 sequences derived from two approved clinical therapeutic antibodies ustekinumab and panitumumab were used. To promote the desired heterodimeric pairing of the heavy chains, "knob" mutations (S354C, T366W) and "hole" mutations (Y349C, T366S, L368A, Y407V) were introduced. DNA plasmids were verified by Sanger sequencing before transfection into HEK293 cells by standard protocols.

[0216] The transfected HEK cells were cultured in CD optiCHO medium (Invitrogen), and the supernatant was collected 6 days after transfection and subjected to Protein A-based affinity purification. The purified IgG was treated with GingisKHAN® (Genovis AB) to enzymatically cleave the Fab region from the Fc portion.

[0217] LCMS was performed on the purified Fab to confirm the sequence of each IgG component (2 heavy chains x 2 light chains) and to determine the relative proportion of each component (see Figure 7). Briefly, purified IgG was digested with GingisKHAN to enzymatically cleave the Fab region from the Fc portion. The Fab sample was injected into an Agilent 1100 series HPLC using an Applied Biosystems POROS® R2 10 μm column (2.1 x 30 mm, 0.1 mL) maintained at 65°C. After injection, the sample was eluted from the column with a 0.21 min gradient of 2-95% acetonitrile at a flow rate of 2 mL / min (mobile phase A: 0.1% formic acid in H2O, mobile phase B: 0.1% formic acid in acetonitrile). A diverter valve was used to load a total flow rate of 150 μL / min into a Bruker maXis 4G mass spectrometer. The mass spectrometer was run in positive ion mode with a m / z range of 700-2500. Other source parameters were set as follows: capillary at 5500 V, nebulizer at 4.0 Bar, drying gas at 4.0 L / min, and drying temperature at 200 °C. Acquired MS spectra were analyzed using Bruker Compass Data Analysis version 4.1. Detection of intact Fab species was confirmed based on mass measurements compared to the theoretical sequence. Relative quantification of each species was calculated by comparing the intensity of the peak per species to the sum of all peak intensities.

[0218] When both heavy chains are wild type, incorrect pairing occurs about 30% of the time. However, when the heavy chain contains the CH1 variant domain described herein, the correct pairing between the heavy chain and the light chain is significantly improved (see FIG. 7 and Table 6). The pani light chain is wild type. The uste light chain is a lambda fusion. HC1 is pani, LC1 is pani kappa, HC2 is uste, and LC2 is uste lambda. For example, when the first heavy chain (HC1) contains K147F and S183R / K / Y and the second heavy chain contains A141D (BsAb10, 12, and 14, respectively), the mispairing is alleviated by at least half, i.e., occurs only 6.8%, 10.5%, or 11% of the time. Indeed, a single substitution at position 141 (141D) results in a 50% reduction in mismatches, i.e., 6.1% vs. 3.1% for HC1-LC2 and 22.8% vs. 9.9% for HC2-LC1 (BsAb2). Based thereon, Applicants provide in Table 7 exemplary CH1 domain sequences with kappa or lambda light chain / CL domain preferences. [Table 6] [Table 7]

[0219] The expression and quality of the purified antibodies were assessed by size-exclusion chromatography (SEC). Briefly, column chromatography (TSKgel Super SW3000 column) was observed using an Agilent 1 100 HPLC. Prior to use, the column was preconditioned with hyperglycosylated and aggregated IgG to minimize potential antibody-column interactions and equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride pH 6.8). Approximately 2-5 μg of protein sample was injected onto the column and the flow rate was adjusted to 0.400 mL / min. Protein migration was observed at a wavelength of 280 nm. The total assay time was approximately 11 min. Data were analyzed using ChemStation software. SEC profiles confirmed that the CH1 domain substitutions did not affect the variant profile compared to the wild type (data not shown). The binding affinity and binding kinetics of the purified bispecific antibodies to human IL-12B (Uste) and human EGFR (Pani) were measured to confirm that the CH1 variant domain did not affect target binding (see Figures 6A-6E). Bispecific IgG samples were captured on an anti-hIgG Fc sensor chip using an Octet® QKe instrument (ForteBio) containing 100 nM antigen, and binding kinetics to IL12B or EGFR were measured (onset rate: 180 s and dissociation rate: 180 s). BLI analysis was performed at 29°C using 1x kinetics buffer (ForteBio) as assay buffer. Anti-human IgG Fc capture (AHC) biosensors (ForteBio) were first presoaked in assay buffer for at least five minutes. Bispecific IgG samples (5 μg / mL) were captured on the sensor for 300 seconds. The sensor was then soaked in assay buffer for 120 seconds to establish a baseline before measuring binding to IL12B or EGFR protein (100 nM concentration). Dissociation of IL12B or EGFR was measured by running the sensor in assay buffer for 180 seconds. Agitation was at 1000 rpm in all steps. Octet® Data Analysis software version 8.2.0.7 was used to generate kinetic parameters using reference subtraction, dissociation-based stepwise correction, one-to-one binding model, and global fit (Rmax unbound by sensor). The association rate constant (ka), dissociation rate constant (kd), and equilibrium constant (K D ) values ​​were assigned separately for each measurement. Example 5: Construction and selection of the 141x181x218 library

[0220] Additional CH1 amino acid substitutions that result in preferential pairing with the lambda CL domain were also identified. Based on previous selection data as well as structural analysis, a set of three CH1 positions (141, 181, and 218) was selected for further variegation. Amino acid diversity at position 141 was generated via the degenerate codon RMW, which represents the six natural amino acids (D, T, A, E, K, and N). Amino acid diversity at positions 181 and 218 was generated via the degenerate codon NNK, which represents all 20 natural amino acids. The library design included all possible combinations of amino acids at these three positions, resulting in a diversity of 2,400. The library was constructed as previously described using a light chain strain with a lambda light chain under the GAL10 promoter (GAL1::ADI-26140 VL-Ck x GAL10::ADI-26140 VL-Cl). Selection for lambda selectivity was performed via staining with anti-human kappa-FITC and anti-human lambda-PE antibodies followed by multiple rounds of cell sorting as previously described. The results (96 clones) were sequenced as previously described and FACS-based quantification of lambda selectivity over the parental strain was quantified. Wild type ("WT") and previously identified lead clone A141D were included in the analysis. Based on these data, the amino acid combinations that provided the greatest improvement in preferential light chain lambda pairing over the parent and A141D were identified.

[0221] Figure 8 shows that most of the resulting clones have higher selectivity in pairing with the lambda chain as determined by the FOP values. Table 8 provides the FOP values ​​of CH1 domain substitutions and lambda:kappa MFI ratios for the top 13 clones marked in Figure 8. [Table 8]

[0222] Analysis showed that substitutions of D, K or E at position 141 paired with substitutions of K at position 181 and L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W at position 218 were frequent among the resulting clones and increased lambda light chain selectivity (increased lambda:kappa MFI ratio) over A141D. Figure 9 shows individual and average FOP values ​​measured within clones with D at position 141, K at position 181 and various amino acids at position 218 of CH1. Lead CH1 sequences were cloned back to LC staining (this process was subsequently employed in all assays) and clones and lambda selectivity was confirmed by calculating FOP values ​​in triplicates (Figure 10).

[0223] Additional analysis generated nine unique candidate CH1 sequences for mammalian IgG production (see Table 9). [Table 9]

[0224] The nine candidate CH1 sequences, along with wild type (WT) (i.e., "ASK") and A141D (i.e., "DSK"), were cloned into mammalian expression vectors via standard methods. To determine lambda selectivity, plasmids representing the desired heavy chain, lambda light chain, and kappa light chain were transfected into HEK293 cells at a plasmid ratio of 2:1:1. The transfected HEK cells were cultured and IgG was purified using the protocol described above. Without intending to be bound by theory, it is clear that by expressing approximately equal amounts of total heavy and light chain polypeptides (HC:kappa LC:lambda LC="2:1:1", where total HC:total LC=1:1) (i.e., no excess of HC and no excess of LC), the inventors were able to avoid various biases, thereby visualizing the true kappa or lambda selectivity of the CH1 domain variants.

[0225] FACS-based quantification of lambda selectivity was performed on this mammalian-produced IgG. Figure 11 provides the FACS plots, and Figure 12 and Table 10 provide the FOP values ​​(lambda:kappa MFI) for the nine CH1 variants and wild type (WT) and A141D (i.e., "DSK"). Figure 13 shows that when CH1 is D at position 141, additional substitutions at positions 181 or 181 and 218 further improve lambda selectivity (based on lambda:kappa MFI ratio). [Table 10]

[0226] Additionally, LCMS data of reduced full-length IgG was used to determine the relative amounts of lambda and kappa light chains in purified IgG samples. Figure 14 compares the % of species paired with kappa light chains (LC) and lambda light chains.

[0227] Analysis of these data yielded three CH1 sequences (SEQ ID NOs: 143, 142, and 141, with DKP, DKA, and DKK substitutions, respectively) that exhibit improved lambda selectivity over the parent and previously identified lead A141D.

[0228] To determine whether these CH1 sequences pair with kappa light chains, candidate CH1 heavy chain plasmids were transfected into HE293 cells with either 1) kappa light chains, or 2) lambda light chains. K147F S183R as CH1 with kappa selection, and wild type (WT), A141D, were also included as controls. Transfected HEK cells were cultured and purified by standard methods. Linked heavy and light chain Fabs were generated from purified IgG using methods previously described. Process yields were determined using standard methods and normalized to wild type (WT) process yields to calculate "FOP" process yields. Based on the process yield FOP, when only kappa LC was present (but not lambda LC), A141D, A141D S181K, A141D S181K K218A, and A141D S181K K218P all still bound to kappa LC, but more binding occurred to lambda LC than to kappa LC (Figure 15). The Fab Tm of kappa-Fab and lambda-Fab were measured by differential scanning fluorimetry using a BioRad CFX96 RT PCR (Figure 16). For each CH1 variant, the relative gain of Tm of lambda-paired Fab ("relative lambda Tm gain" or "net lambda Tm gain"), defined as [Tm change of lambda-paired variant Fab to lambda-paired WT Fab ("Δlambda Tm") minus [Tm change of kappa-paired variant Fab to kappa-paired WT Fab ("Δkappa Tm")], was calculated (Figure 17). As shown in Figure 17, the relative lambda Tm gain increased with further substitutions at S181 or S181 and K218. Without intending to be bound by theory, based on Figures 16 and 17, it appears that destabilization of kappa LC pairing contributed to the relative lambda Tm gain and increased pairing with lambda CL. Example 6: Construction and selection of the 141xALL library

[0229] Additional libraries were constructed to sample additional residues within CH1 that drive lambda-preferential binding when paired with a substitution at position 141. Six new libraries (LAD11522-LAD11527) were designed with up to three substitutions across three regions of CH1 (DOR1, DOR2, and DOR3) (Table 11). Together, the six libraries represent the set of all possible substitutions, including two substitutions in the three domains of interest paired with position 141. In all libraries, amino acid diversity at position 141 was generated via the degenerate codon RMW, and amino acid diversity at the other two varied positions was generated via the degenerate codon NNK. Libraries were constructed using methods previously described. Selections for lambda selectivity were performed as previously described. [Table 11]

[0230] After the second round of FACS selection, the diversity CH1 of the selection result was isolated and recloned into the appropriate two-chain light chain strain to recover the reduced kappa light chain expression in the library. This diversity CH1 was isolated using PCR amplification with appropriate primers and standard DNA purification. This pool of DNA fragments was then electroporated with the ADI-26140 heavy chain variable region and the plasmid was digested into the appropriate two-chain light chain strain.

[0231] Results were sequenced as described above ( FIG. 18 ) and FACS-based quantification of lambda selectivity over the parental strain was quantified. The previously identified lead clone A141D S181K K218P was included in the analysis. Based on these data, the amino acid combinations that provided the greatest improvement in light chain lambda-preferential pairing over the parent were identified.

[0232] The top 46 clones (Table 12), containing 28 unique CH1 sequences, were expressed as IgG in yeast. The novel CH1 sequences, along with wild type (WT), A141D (or "DSK"), and a portion of the reads from the 141x181x218 series in Example 5 (DKP, DKA, KKE, KKP, and EKK), were compared in terms of FOP values ​​determined by flow cytometry (lambda MFI: kappa MFI) (Figure 19). At least seven of the strains with CH1 sequences of SEQ ID NOs: 155, 157, 159, 162, 163, 164, or 165, corresponding to the data points marked with arrows in Figure 19, showed FOP values ​​comparable to or higher than those of the 141x181x218 reads tested. [Table 12-1] [Table 12-2] Example 7: Construction and screening of the 141 x (170 / 171) x (185 / 187) series

[0233] Analysis of the results of Example 6 provided four new positions / residues of interest, including F170, P171, V185, and T187. Based on the frequently observed amino acids at positions 170, 171, 185, and 187, along with 141, which yielded high FOP values ​​in previous studies (e.g., frequent E and D at position 141, frequent E at positions 170 or 171 in 141×ALL results, and frequent R at positions 185 and / or 187 when position 141 was substituted and independent of position 171), 14 unique CH1 domain variants (Table 13) with up to three amino acid substitutions per CH1 domain were rationally designed as candidates for a set of lead lambda-preferential substitutions. The 14 leads in Table 13 include "A141E;V185R;T187R" (SEQ ID NO: 163) and "A141E;P171E;V185R (SEQ ID NO: 159)," which were tested in Example 6. [Table 13]

[0234] Heavy chains containing one of the 14 CH1 domain variant sequences were cloned into mammalian (HEK) cells co-expressing kappa and lambda light chains as described above (heavy chain (HC):lambda light chain (LC):kappa LC ratio = 2:1:1, i.e., HC:LC ratio was always 1:1). Wild type (ADI-26140 heavy chain), "A141D" variant, and "A141D_S181K_K218P" variant were also included as controls. Lambda selectivity was determined using the same assay as described above.

[0235] The ratio of lambda MFI to kappa MFI was evaluated by flow cytometry. The FOP values ​​and individual FACS plots of the 14 reads are provided in Table 14 and Figures 20-22 (the numbers of each plot are the Rank numbers shown in Table 14). Among the 14 reads, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed even higher FOP values ​​than "A141D_S181K_K218P", the read identified in Example 5. Many other variants among the 14 reads also showed higher FOP values ​​compared to "A141D", and all 14 reads showed higher FOP values ​​compared to the wild type. [Table 14]

[0236] The amount of kappa and lambda LC per sample was quantified using LCMS (Table 15 and Figure 23). Similar to the findings from the FACS-based lambda selectivity assessment, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed higher lambda chain% and lower kappa chain% compared to "A141D_S181K_K218P", the lead identified in Example 6. Many other variants of the 14 reads also showed higher lambda% and lower kappa% compared to "A141D", and all 14 reads showed higher lambda% and lower kappa% compared to wild type. [Table 15]

[0237] To determine whether the top two lambda-preferring CH1 variants ("A141D_P171E_V185R" and "A141D_F170E_T187R") pair with kappa light chains, the CH1 variant heavy chain plasmids were transfected into HEK293 cells with either 1) kappa light chain or 2) lambda light chain (heavy:light ratio = 1:1). K147F S183R as the CH1 with kappa preference and wild type (WT) were also included as controls. Transfected HEK cells were grown and purified by standard methods using a Protein A column. Process yields (mg / L) were determined using standard methods and normalized to the process yield of wild type (WT). Based on normalized process yields, when only kappa LC was present (but not lambda LC), both "A141D_P171E_V185R" and "A141D_F170E_T187R" still bound to kappa LC, but more binding occurred to lambda LC than to kappa LC (Figure 30).

[0238] The process yield of the Fab format was also evaluated. IgG with CH1 variant heavy chains were produced and purified using the same method. Wild type (WT), A141D, and A141D S181K K218P as CH1 with kappa selectivity were also included as controls. Linked heavy and light chain Fabs were generated from IgG purified via papain enzyme digestion and CH1 column purification using standard methods. Normalized Fab digests were calculated for each light chain as the % recovery of Fab from the IgG digest normalized to the % recovery of the parent (amount of Fab recovered / amount of IgG in digest). Process yields were determined using standard methods and normalized to the process yield of wild type (WT). Consistent with the data in Figure 15, "A141D" and "A141D S181K K218P" had higher process yields with lambda LC than with kappa LC, and "K147F S183R" showed very high kappa selectivity (Figure 31). When only kappa CH1 was present (but not lambda CH1), both "A141D_P171E_V185R" and "A141D_F170E_T187R" still bound to kappa CH1, but gave significantly higher yields with lambda LC than with kappa LC (Figure 31). Mutation of "P171E_V185R" or "F170E_T187R" added to "A141D" further enhanced the lambda selectivity of "A141D". Example 8: Structural analysis of the "A141D" and "K147F S183R" variants method

[0239] Crystallization and structure determination of panitumumab wild-type CH1-Cλ

[0240] 6.5 mg / ml of panitumumab wild-type CH1-constant lambda (Cλ) Fab protein was centrifuged at 14,000×g for 5 minutes at 4° C. 305 nL of protein was mixed with 150 nL of reservoir droplet and 50 nL of seed solution and equilibrated with 40 ul of reservoir solution at 20° C. in an MRC 3-well plate. Seed crystals identified from the BCS screen (Molecular Dimensions) were used for crystallization experiments in microseed matrix screening (MMS) (D'Arcy, A., Villard, F., and Marsh, M. (2007) “An automated microseed matrix-screening method for protein crystallization” Acta Crystallogr D Biol Crystallogr 63, 550-554.) and were grown in 0.1 M phosphate / citric acid pH 5.5 and 36% (v / v) PEG Smear Low, transferred to 0.1 M phosphate / citric acid pH 5.5, 38% PEG Smear Low and 4% glycerol to obtain crystals that were then flash frozen in liquid nitrogen. Diffraction data were collected at 100 K at 1.09 Å at station I03 of Diamond Light Source, Didcot, UK, equipped with an Eiger2 XE 16M detector (DECTRIS).Data sets were integrated in autoPROC (Vonrhein,C.et al.(2011)“Data processing and analysis with the autoPROC toolbox” Acta Crystallogr.D67,293-302.) using XDS (Kabsch W.(2010)“XDS” Acta Crystallogr.D Biol.Cryst.D66,125-132.) and integrated using Aimless (Evans PRand Murshudov,GN(2013)“How good are my data and what is the resolution” Acta Crystallogr.D67,235-242.) in the CCP4 software package (Winn MDet al.(2011)“Overview of the CCP4 suite and current developments” Acta Crystallogr.D Biol.Cryst.D67,235-242.). The crystals were scaled using the NMR spectroscopy (Biol. Crystallogr. 69, 1204-1214.) The crystals consisted of two molecules per asymmetric unit (ASU) in the space group P1211.The structure was solved by the automated molecular replacement system MoRDA (Vagin A. and Lebedev A. (2015) “MoRDa, an automatic molecular replacement pipeline” Acta Cryst A. A71, s19.) (incorporating MOLREP (Vagin A., Teplyakov A. (1997) “MOLREP: an automated program for molecular replacement” J. Appl. Cryst. 30, 1022-1025.) and Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.)) which used the Protein Data Bank (Berman Biol. Crystallogr. 67, 355-367.) as an initial search model. Entries 5N7W and 5SX4 from HMet al. (2000) The “Protein Data Bank” Nucleic Acids Research, 28.) were selected. Automated model building was performed using BUCCANEER software (Cowtan K. (2006) “The Buccaneer software for automated model building. 1. Tracing protein chains” Acta Crystallographica D62, 1002-1011.).The model was refined using manual refinements in Coot (Emsley P., Lohkamp, ​​B., Scott, WG and Cowtan K. (2010) “Features and development of Coot” Acta Crystallogr. D Biol. Crystallogr. 66, 486-501.), as well as the refinements in Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.) and Buster (Bricogne G, Blanc E, Brandl M, Flensburg C, Keller P, Paciorek W, Roversi P, Sharff A, Smart O, Vonrhein Final R and R were adjusted in BUSTER version 2.11.7. Global Phasing Ltd, Cambridge, United Kingdom. free further improved to 14.5% and 16.9%, respectively (Figure 32).

[0241] Crystallization and structure determination of panitumumab A141D CH1-Cλ, wild-type CH1-Cκ and K147F-S183R CH1-Cκ

[0242] Fabs of panitumumab A141D CH1-Cλ, panitumumab wild-type CH1-constant kappa (Cκ), and panitumumab K147F-S183R CH1-Cκ were centrifuged at 14,000×g for 5 min at 4°C. For panitumumab A141D CH1-Cλ and K147F-S183R CH1-Cκ, 200 nL of 10.0 mg / mL Fab was mixed with 150 nL of reservoir droplet and 50 nL of seed solution and equilibrated with 40 ul of reservoir solution. Seed crystals identified from the BCS screen were used in MMS experiments to search for optimal crystallization conditions. 0.1M phosphate / citrate buffer pH 5.5 and 36% (v / v) PEG Smear Low were used for panitumumab A141D CH1-Cλ, and 0.1M sodium acetate pH 4.5 with 30% v / v PEG Smear Low was used for panitumumab K147F-S183R CH1-Cκ. 150nL of 19.2mg / ml wild type CH1-Cκ was mixed with 150nL of reservoir droplet, added to 40ul of reservoir solution, and screened using PACT Suite (Molecular Dimensions). Final crystallization conditions consisted of 0.1M MES pH 6.0 with 20% w / v PEG 6000 and 0.2M calcium chloride dihydrate. Crystals were transferred to cryo-solutions consisting of 0.1M phosphate / citrate buffer pH 5.5, 38% PEG Smear Low, 4% glycerol; 0.07M MES, pH 6.0, 21% PEG 6000, 0.2M CaCl2, 23.5% glycerol; and 0.1M NaAc pH 4.5, 32.5% PEG Smear Low, 25% glycerol for panitumumab A141D CH1-Cλ, wild-type CH1-Cκ, and K147F-S183R CH1-Cκ, respectively. All crystals were flash frozen in liquid nitrogen, and crystallographic data were collected at 100K at 1.2–2.6 Å resolution at Diamond Light Source I03 station, Didcot, UK, equipped with an Eiger2 XE 16M detector (DECTRIS).Data were indexed and integrated in iMOSFLM (Battye, TGG, Kontogiannis, L., Johnson, O., Powell, HR, & Leslie, AG (2011). iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallographica Section D: Biological Crystallography, 67(4), 271-281.) and scaled and merged using AIMLESS (Evans PR and Murshudov, GN (2013) "How good are my data and what is the resolution" Acta Crystallogr D Biol. Crystallogr. 69, 1204-1214.) via the CCP4 suite (Winn MD et al. (2011) "Overview of the CCP4 suite and current developments" Acta Crystallogr. D Biol. Crystallogr. 67, 235-242. 235-242.).

[0243] The structure of panitumumab A141D-CH1-Cλ was solved by molecular replacement using the crystal structure of wild-type CH1-Cλ as a search model. Several rounds of anisotropic B-factor and simple restricted refinement were performed in Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.) applying a blurring factor in the final round of refinement. Position occupancies of A141D CH1-Cλ were assigned based on the occupancies of wild-type CH1-Cλ and were also manually adjusted in Coot (Emsley P., Lohkamp, ​​B., Scott, WG and Cowtan K. (2010) “Features and development of Coot” Acta Crystallogr. D Biol. Crystallogr. 66, 486-501.) during iterative refinement. The final structure is solved in P1211 with two molecules per ASU, but R and R free The values ​​were 15.2% and 17.0%, respectively (Figure 33).

[0244] The structures of panitumumab wild-type CH1-Cκ and K147F-S183R-CH1-Cκ were determined by molecular replacement in Phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Stroni, LC, & Read, RJ (2007). Phaser crystallographic software. Journal of Applied Crystallography, 40(4), 658-674.) using the coordinates of the panitumumab Fab fragment in complex with EGFR (PDB code 5SX4) and in complex with the wild-type CH1-Cκ structure to be solved, respectively, followed by Coot (Emsley P., Lohkamp, ​​B., Scott, WGand Cowtan K. (2010) “Features and development of Coot” Acta Crystallogr. D The structure was solved by manual model building using Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.) and iterative rounds of automated refinement in Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.). As non-crystallographic translational symmetry was observed for the wild-type CH1-Cκ structure, the structure was solved in a lower space group (P1211) using six Fab molecules at ASU. The structure had a final R and R of 19.8% and 23.2%, respectively. free The K147F-S183R CH1-Cκ structure was adjusted to final R and R values ​​of 19.8% and 23.3%, respectively, in the P31 space group using one molecule per ASU. free This was clarified by the values ​​(Figure 35).

[0245] Structural analysis and interpretation

[0246] HC-A141D-mediated lambda LC selectivity

[0247] Without intending to be bound by theory, the enhanced lambda selectivity of panitumumab A141D CH1-Cλ is potentially mediated by interchain hydrogen bonds formed between the carboxyl group of the side chain of HC-Asp141 and the hydroxyl group of the side chain of λLC-Thr116 (FIG. 36C), which cannot be formed by HC-Ala141 in panitumumab wild-type CH1-Cλ (FIG. 36A). The κLC region surrounding HC-Ala141 consists of hydrophobic residues Phe116, Phe118 and Leu135, while κLC-Phe116 is replaced by the polar residue Thr116 in λLC (FIG. 36B). Thus, charge introduction via the A141D mutation may reduce kappa selectivity by disrupting the hydrophobicity of the CH1-κLC interface while stabilizing the CH1-λLC pairing via hydrogen bonds with λLC-Thr116. Furthermore, without intending to be bound by theory, a steric clash of HC-Asp141 with κLC-Phe116 may further reduce kappa selectivity, as shown by alignment of panitumumab A141D CH1-Cλ and wild-type CH1-κLC (Figure 36D).

[0248] In the hydrogen bond between HC-Asp141 and λLC-Thr116, a bond is formed between the hydrogen acceptor atom (O) in the side chain of Asp141 and the hydrogen donor atom (H) in the side chain of Thr116. Therefore, another amino acid with a hydrogen acceptor atom in its side chain can also form a hydrogen bond with Thr116 of λLC to provide lambda selectivity. Based on the fact that the side chain of glutamic acid also has a hydrogen acceptor atom (O) and glutamic acid is similar in size and shape to aspartic acid, glutamic acid will probably form a hydrogen bond with Thr116 of λLC while causing a steric clash with κLC as shown in Figure 36D, thus providing overall lambda selectivity. Indeed, the A141E substitution provides strong lambda selectivity as demonstrated in the above examples, and the structural analysis was confirmed by the applicant.

[0249] HC-K147F-S183R-mediated kappa LC selectivity

[0250] The observed kappa selectivity of panitumumab K147F-S183R CH1-Cκ may be mediated by two new hydrogen bonds at the CH1 and Cκ interface. In the panitumumab wild-type CH1-Cκ structure, a hydrogen bond network coordinated by HC-Lys147 and HC-Asp148 traps HC-Gln175, which contributes to the baseline kappa pairing selectivity (Figure 37A). One explanation is that substitution of CH1 HC-Lys147 with phenylalanine at this position disrupts this network, freeing the HC-Gln175 side chain to interact with κLC via a hydrogen bond with the formamide oxygen of κLC-Gln160, thereby increasing kappa selectivity (Figure 37B). Furthermore, without intending to be bound by theory, the HC-S183R substitution results in an additional hydrogen bond between the guanidinium group of the HC-Arg183 side chain and the hydroxyl group of κLC-Thr178 (Figure 37B, Figure 38C). Conversely, without intending to be bound by theory, the hydrogen bond observed at position 183 of the HC between HC-Ser183 and λLC-Tyr178 in panitumumab wild-type CH1-Cλ is abolished by the severe steric clash of the side chains of HC-Arg183 and λLC-Tyr178 in the model pairing of K147F-S183R CH1 with λLC, destabilizing the lambda pairing in favor of κLC (Figures 38B and 38D).

[0251] In the hydrogen bond between HC-Arg183 and κLC-Thr178, a bond is formed between the hydrogen donor atom (H) in the side chain of Arg183 and the hydrogen acceptor atom (O) in the side chain of Thr178. Therefore, another amino acid with a hydrogen donor atom in its side chain may also form a hydrogen bond with Thr178 of κLC, providing kappa selectivity. A larger side chain, such as the side chain of Arg, may create a steric clash with Tyr178 of λLC, helping to provide further kappa selectivity. For example, both lysine and tryptophan have large side chains that contain a hydrogen donor atom (H). Thus, lysine and tryptophan likely form a hydrogen bond with Thr178 of κLC, and also likely result in a steric clash with λLC, as shown in Figure 38D, providing overall kappa selectivity. The side chain of threonine can also function as a hydrogen donor through the H atom of -OH. Thus, Applicants further envision that amino acids with relatively large side chains that can function as hydrogen acceptors may form hydrogen bonds with Thr178 of κLC to provide kappa selectivity. For example, glutamic acid, glutamine, histidine, or tyrosine, which also have relatively large side chains with hydrogen acceptor atoms, may provide kappa selectivity when placed at residue 183 of HC. Indeed, most of these amino acid substitutions at the newly proposed residue 183 were indeed identified in Example 3 as being kappa-preferential (see Table 3).

[0252] As mentioned above, substitution of Lys147 with Phe disrupted the hydrogen bond between Lys147 and Gln175, thereby freeing Gln175 to form a hydrogen bond with Gln160 of κLC, thereby contributing to kappa selectivity. Thus, substitution of Lys147 with another amino acid with a side chain that does not contain a hydrogen donor or hydrogen acceptor atom, such as alanine, glycine, isoleucine, leucine, or valine, may also aid in kappa selectivity. Indeed, most of these amino acid substitutions at the newly proposed residue 147 were indeed identified as kappa-preferential in Example 3 (see Table 3).

Claims

1. a heavy chain constant region 1 ("CH1") domain variant polypeptide comprising an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering; Optionally, whereby the CH1 domain variant polypeptide is (i) with a kappa light chain constant region ("CL") domain compared to a lambda CL domain, and / or with a kappa light chain polypeptide compared to a lambda light chain polypeptide, or (ii) preferentially pairs with a lambda CL domain relative to a kappa CL domain, and / or with a lambda light chain polypeptide relative to a kappa light chain polypeptide; With the proviso that, optionally, one or more of the following combinations of substitutions are excluded: (a) if residue 141 on CH1 is substituted with C or L, if residue 166 is substituted with D or K, if residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or if residue 147 is substituted with D, then the CL does not contain any amino acid substitutions; (b) the CL does not contain an amino acid substitution where position 126 or 220 on CH1 is substituted with valine or alanine, where the non-cysteines at positions 128, 141, or 168 are substituted with cysteines, or where the CH1 substitution is L145F, K147A, F170V, S183F, or V185W / F; (c) if residue 172 on CH1 is substituted with 172R, if residue 174 is mutated to 174G, or if residue 190 is substituted with 190M or 190I, these are not the only CH1 substitutions; (d) when said CH1 substitutions consist of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, CL is unaltered; (e) if said CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S and / or 193F / L, these are not the only CH1 substitutions and / or are of the same human immunoglobulin subtype or allotype in a bispecific antibody containing a CH1 domain; (f) if said CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), then there is no LC substitution corresponding to 129D / E / R / H / K (IMGT position 18); (g) if the CH1 substitution consists of 124K / E / R / D, then there is no corresponding LC substitution at 176; (h) when said CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, the corresponding LC substitution is not present; (i) if said CH1 substitutions consist of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L and / or 190S / A / G / Y, then the corresponding LC substitutions are not present; (j) when said CH1 substitutions consist of 143A / E / R / K / D and 145T / L, then the corresponding LC substitutions are not present; (k) when said CH1 substitutions consist of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, and 139W / G / C, 179E and / or 186R, the corresponding LC substitutions are not present; (l) when the CH1 substitution consists of a substitution with a cysteine ​​at position 126, 127, 128, 134, 141, 171, or 173, the corresponding LC position is not modified to form a disulfide bond; (m) when said CH1 substitutions consist of L145Q, H168A, F170G, S183V and / or T187E, there are no corresponding kappa or lambda LC substitutions; (n) when said CH1 substitutions consist of 143D / E, 145T, 190E / D and / or 124R, the corresponding CL substitutions are not present; or (o) CH1 substitutions consist of A140C, K147C and / or S183C with corresponding CL substitutions present.

2. the CH1 domain variant polypeptide comprises an amino acid substitution at one or more of positions 118, 124, 126-129, 131, 132, 134, 136, 139, 143, 145, 147-151, 153, 154, 170, 172, 175, 176, 181, 183, 185, 190, 191, 197, 201, 203-206, 210, 212-214, and 218 according to EU numbering; Optionally, whereby said CH1 domain variant polypeptide comprises: (i) a kappa CL domain, as compared to a lambda CL domain; and / or (ii) the CH1 domain variant polypeptide of claim 1 which preferentially pairs with kappa light chain polypeptides as compared to lambda light chain polypeptides.

3. 3. The CH1 domain variant polypeptide of claim 2, comprising an amino acid substitution at positions 147, 183, or 147 and 183.

4. 4. The CH1 domain variant polypeptide of claim 2 or 3, comprising one or more of the following amino acid substitutions: a. position 118 is substituted with G; b. position 124 is substituted with H, R, E, L, or V; c. position 126 is substituted with A, T, or L; d. position 127 is substituted with V or L; e. position 128 is substituted with H; f. position 129 is substituted with P; g. position 131 is substituted with A; h. position 132 is substituted with P; i. position 134 is substituted with G; j. position 136 is substituted with E; k. position 139 is substituted with I; l. Position 143 is substituted with V or S; m. position 145 is substituted with F, I, N, or T; n. Position 147 is substituted with F, I, L, R, T, S, M, V, N, E, H, Y, Q, A, or G; o. position 148 is substituted with I, Q, Y, or G; p. Position 149 is substituted with C, S, or H; q. position 150 is substituted with L or S; r. position 151 is substituted with A or L; s. position 153 is substituted with S; t. position 154 is substituted with M or G; u. position 170 is substituted with G or L; v. position 172 is substituted with V; w. position 175 is substituted with G, L, E, A; x. position 176 is substituted with P; y. position 181 is substituted with Y, Q, or G; z. Position 183 is substituted with I, W, F, E, Y, L, K, Q, N, R, or H; aa. Position 185 is substituted with W; bb. position 190 is substituted with P; cc. position 191 is substituted with I; dd. position 197 is substituted with A; ee. position 201 is substituted with S; ff. position 203 is substituted with S; gg. position 204 is substituted with Y; hh. position 205 is substituted with Q; ii. position 206 is substituted with S; jj. position 210 is substituted with R; kk. position 212 is substituted with G; ll. position 213 is substituted with E or R; mm. position 214 is substituted with R, and Position nn. 218 is substituted with Q.

5. (i) the amino acid residue F, I, L, R, T, S, M, V, N, E, H, Y, or Q at position 147; and / or (ii) the CH1 domain variant polypeptide of any one of claims 2 to 4, comprising amino acid residue I, W, F, E, Y, L, K, Q, N, or R at position 183.

6. (i) amino acid residue R, K, or Y at position 183; and / or (ii) comprising an amino acid residue F at position 147.

7. (i) an amino acid residue F at position 147 and an amino acid residue R at position 183; (ii) an amino acid residue F at position 147 and an amino acid residue K at position 183; (iii) an amino acid residue F at position 147 and an amino acid residue Y at position 183, (iv) an amino acid residue R at position 183, (v) an amino acid residue K at position 183, or (vi) containing an amino acid residue Y at position 183, Optionally, (i) SEQ ID NO: 137, (ii) SEQ ID NO: 138, (iii) SEQ ID NO: 139, (iv) SEQ ID NO: 60, (v) SEQ ID NO: 41, or (vi) A CH1 domain variant polypeptide according to any one of claims 2 to 6, comprising the amino acid sequence of SEQ ID NO:

136.

8. 8. The CH1 domain variant polypeptide of any one of claims 2 to 7, comprising an amino acid substitution at a CH1 amino acid position within the interface between CH1 and VH, optionally wherein said CH1 amino acid position is position 151, and further optionally comprising amino acid residue A or L at position 151.

9. (i) an amino acid residue F at position 147 and an amino acid residue R at position 183; (ii) an amino acid residue F at position 147 and an amino acid residue K at position 183; (iii) an amino acid residue F at position 147 and an amino acid residue Y at position 183, (iv) an amino acid residue R at position 183, (v) an amino acid residue K at position 183, or (vi) a CH1 domain variant polypeptide comprising an amino acid residue Y at position 183.

10. (i) an amino acid residue F at position 147 and an amino acid residue R at position 183; (ii) an amino acid residue F at position 147 and an amino acid residue K at position 183; (iii) an amino acid residue F at position 147 and an amino acid residue Y at position 183, (iv) an amino acid residue R at position 183, (v) an amino acid residue K at position 183, or (vi) an amino acid substitution at position 183 consisting of amino acid residue Y.

11. (i) SEQ ID NO: 137, (ii) SEQ ID NO: 138, (iii) SEQ ID NO: 139, (iv) SEQ ID NO: 60, (v) SEQ ID NO: 41, or (vi) The CH1 domain variant polypeptide of claim 10, comprising the amino acid sequence of SEQ ID NO:

136.

12. (i) with a kappa CL domain, as compared to a lambda CL domain; and / or (ii) a CH1 domain variant polypeptide according to any one of claims 2 to 11, further comprising one or more amino acid substitutions which increase pairing of the CH1 domain with a kappa light chain polypeptide as compared to a lambda light chain polypeptide.

13. (i) of pairing with a kappa CL domain, as compared to a lambda CL domain; and / or (ii) of association with a kappa light chain polypeptide, as compared to a lambda light chain polypeptide; or an increase of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, optionally as measured by liquid chromatography-mass spectrometry (LCMS); or 12. The CH1 domain variant polypeptide of any one of claims 2 to 11, which provides an increase of at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 21 fold, at least 22 fold, at least 23 fold, at least 24 fold, or at least 25 fold, optionally measured by flow cytometry, and optionally by comparing the ratio of mean fluorescence intensity (MFI) of kappa CL staining to lambda CL staining.

14. the CH1 domain variant polypeptide comprises an amino acid substitution at one or more of positions 119, 124, 126, 127, 130, 131, 133, 134, 138-142, 152, 163, 168, 170, 171, 175, 176, 181, 183-185, 187, 197, 203, 208, 210-214, 216, and 218 according to EU numbering; Optionally, the CH1 domain variant comprises: (i) a lambda CL domain compared to a kappa CL domain; and / or (ii) a CH1 domain variant polypeptide of claim 1 that preferentially pairs with lambda light chain polypeptides as compared to kappa light chain polypeptides.

15. 15. The CH1 domain variant polypeptide of claim 14, comprising an amino acid substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218.

16. 16. The CH1 domain variant polypeptide of claim 14 or 15, comprising one or more of the following amino acid substitutions: a. position 119 is substituted with R; b. position 124 is substituted with V; c. position 126 is substituted with V; d. position 127 is substituted with G; e. position 130 is substituted with H or S; f. position 131 is substituted with Q, T, N, R, V, or D; g. Position 133 is substituted with D, T, L, E, S, or P; h. position 134 is substituted with A, H, I, P, V, N, or L; i. position 138 is substituted with R; j. position 139 is substituted with A; k. position 140 is substituted with I, V, D, Y, K, S, W, R, L, or P; l. Position 141 is substituted with D, K, E, T, R, Q, V, or M; m. position 142 is substituted with M; n. Position 152 is substituted with G; o. Position 163 is substituted with M; p. Position 168 is substituted with F, I, or V; q. position 170 is substituted with N, G, E, S, or T; r. position 171 is substituted with N, E, G, S, A, or D; s. position 175 is substituted with D or M; t. position 176 is substituted with R or M; u. Position 181 is substituted with V, L, A, K, or T; v. position 183 is substituted with L or V; w. position 184 is substituted with R; x. position 185 is substituted with M, L, S, R, or T; y. position 187 is substituted with R, D, E, Y, or S; z. Position 197 is substituted with S; aa. Position 203 is substituted with D; bb. position 208 is substituted with I; cc. position 210 is substituted with T; dd. position 211 is substituted with A; ee. position 212 is substituted with N; ff. position 213 is substituted with E; gg. position 214 is substituted with R; hh. position 216 is substituted with G; and ii. Position 218 is substituted with L, E, D, P, A, H, S, Q, N, T, I, M, G, C, K, or W.

17. 17. The CH1 domain variant polypeptide of any one of claims 14 to 16, comprising any one or more of the following (i) to (xvii): (i) an amino acid residue V at position 126; (ii) an amino acid residue G at position 127; (iii) an amino acid residue V at position 131; (iv) an amino acid residue S at position 133; (v) an amino acid residue R at position 138; (vi) amino acid residue I or V at position 140, (vii) an amino acid residue D, K, E, or T at position 141; (viii) an amino acid residue M at position 142, (ix) amino acid residue I at position 168, (x) an amino acid residue E, G, or S at position 170; (xi) an amino acid residue E, D, G, S, or A at position 171; (xii) an amino acid residue M at position 175, (xiii) an amino acid residue R at position 176, (xiv) an amino acid residue K, V, A, or L at position 181; (xv) an amino acid residue R at position 184, (xvi) an amino acid residue R at position 185, (xvii) an amino acid residue R at position 187, and (xviii) the amino acid residue L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W at position 218.

18. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, wherein the CH1 substitutions comprise or consist of one or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

19. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, wherein the CH1 substitutions comprise or consist of two or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

20. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, wherein the CH1 substitutions comprise or consist of three or more of the following substitutions: 141D, 141E, 171E, 170E, 185R and 187R.

21. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, wherein the CH1 substitutions comprise or consist of the following substitutions: (i) 141E and 185R, (ii) 141E and 187R, (iii) 141E, 170E or 171E, and 185R, (iv) 141E, 170E or 171E, and 187R, (v) 141D and 185R, (vi) 141D and 187R, (vii) 141D, 170E or 171E, and 185R, (viii) 141D, 170E or 171E, and 187R, (ix) 141E, 185R, and 187R, or (x) 141D, 185R, and 187R.

22. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, comprising a substitution at position 141 to D, K or E, optionally paired with a substitution at position 181 to K, and further optionally with a substitution at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

23. 18. A CH1 domain variant polypeptide according to any one of claims 14 to 17, comprising a substitution at position 141 to D, K or E, which is paired with a substitution at position 181 to K and / or a substitution at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

24. 18. The CH1 domain variant polypeptide of any one of claims 14 to 17, comprising any one or more of the following (i) to (ix): (i) an amino acid residue D, E, or K at position 141; (ii) an amino acid residue E at position 170; (iii) an amino acid residue E at position 171; (iv) an amino acid residue M at position 175; (v) the amino acid residue K at position 181; (vi) an amino acid residue R at position 184; (vii) an amino acid residue R at position 185, (viii) an amino acid residue R at position 187, and / or (ix) amino acid residue P, A, or E at position 218.

25. (i) an amino acid residue D at position 141; (ii) an amino acid residue D at position 141 and an amino acid residue K at position 181; (iii) an amino acid residue D at position 141, an amino acid residue K at position 181, and an amino acid residue A at position 218; (iv) an amino acid residue D at position 141, an amino acid residue K at position 181, and an amino acid residue P at position 218; (v) the amino acid residue E at position 141, (vi) an amino acid residue E at position 141, and an amino acid residue K at position 181; (vii) an amino acid residue K at position 141; (viii) an amino acid residue K at position 141 and an amino acid residue K at position 181; (ix) an amino acid residue K at position 141, an amino acid residue K at position 181, and an amino acid residue E at position 218; (x) an amino acid residue K at position 141, an amino acid residue K at position 181, and an amino acid residue P at position 218; (xi) an amino acid residue E at position 141, an amino acid residue E at position 170, an amino acid residue V at position 181, and an amino acid residue R at position 187; (xii) an amino acid residue E at position 141, an amino acid residue D at position 171, and an amino acid residue R at position 185; (xiii) an amino acid residue E at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 185; (xiv) an amino acid residue E at position 141, an amino acid residue G at position 171, an amino acid residue R at position 185, and an amino acid residue R at position 187; (xv) an amino acid residue E at position 141, an amino acid residue R at position 185, and an amino acid residue R at position 187; (xvi) an amino acid residue E at position 141, an amino acid residue S at position 171, and an amino acid residue K at position 181; (xvii) an amino acid residue E at position 141, an amino acid residue G at position 170, an amino acid residue M at position 175, an amino acid residue V at position 181, an amino acid residue R at position 184, and an amino acid residue R at position 187; (xviii) an amino acid residue E at position 141, and an amino acid residue R at position 185; (xix) an amino acid residue E at position 141, and an amino acid residue R at position 187; (xx) an amino acid residue E at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 185; (xxi) an amino acid residue E at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 187; (xxii) an amino acid residue D at position 141, and an amino acid residue R at position 185; (xxiii) an amino acid residue D at position 141, and an amino acid residue R at position 187; (xxiv) an amino acid residue D at position 141, an amino acid residue R at position 185, and an amino acid residue R at position 187; (xxv) an amino acid residue D at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 185; (xxvi) an amino acid residue D at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 187; (xxvii) an amino acid residue E at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 187; (xxiii) an amino acid residue D at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 185; or (xxix) comprising an amino acid residue D at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 187; Optionally, (i) SEQ ID NO: 140, (ii) SEQ ID NO: 141, (iii) SEQ ID NO: 142, (iv) SEQ ID NO: 143, (v) SEQ ID NO: 144, (vi) SEQ ID NO: 145, (vii) SEQ ID NO: 146, (viii) SEQ ID NO: 147, (ix) SEQ ID NO: 148, (x) SEQ ID NO: 149, (xi) SEQ ID NO: 155, (xii) SEQ ID NO: 157, (xiii) SEQ ID NO: 159, (xiv) SEQ ID NO: 162, (xv) SEQ ID NO: 163, (xvi) SEQ ID NO: 164, (xvii) SEQ ID NO: 165, (xviii) SEQ ID NO: 178, (xix) SEQ ID NO: 179, (xx) SEQ ID NO: 180, (xxi) SEQ ID NO: 181, (xxii) SEQ ID NO: 182, (xxiii) SEQ ID NO: 183, (xxiv) SEQ ID NO: 184, (xxv) SEQ ID NO: 185, (xxvi) SEQ ID NO: 186, (xxvii) SEQ ID NO: 187, (xxviii) SEQ ID NO: 188, or (xxix) A CH1 domain variant polypeptide according to any one of claims 14 to 17, comprising the amino acid sequence of SEQ ID NO:

189.

26. (i) an amino acid residue D at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 185; (ii) an amino acid residue D at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 187; or (iii) a heavy chain CH1 domain variant polypeptide comprising an amino acid residue D at position 141, an amino acid residue K at position 181, and an amino acid residue P at position 218.

27. (i) SEQ ID NO: 188, (ii) SEQ ID NO: 186, or (iii) The heavy chain CH1 domain variant polypeptide of claim 26, comprising SEQ ID NO:

143.

28. (i) with a lambda CL domain, as compared to a kappa CL domain; and / or (ii) a CH1 domain variant polypeptide according to any one of claims 14 to 27, further comprising one or more amino acid substitutions which increase pairing of the CH1 domain with a lambda light chain polypeptide as compared to a kappa light chain polypeptide.

29. (i) of pairing with a lambda CL domain, as compared to a kappa CL domain; and / or (ii) of association with a lambda light chain polypeptide, as compared to a kappa light chain polypeptide; or an increase of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, optionally as measured by liquid chromatography-mass spectrometry (LCMS); or 29. The CH1 domain variant polypeptide of any one of claims 14 to 28, which provides an increase of at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 21 fold, at least 22 fold, at least 23 fold, at least 24 fold, or at least 25 fold, optionally measured by flow cytometry, and optionally by comparing the ratio of MFI values ​​of lambda CL staining to kappa CL staining.

30. 30. An antibody heavy chain polypeptide comprising a variable region and a constant region, said constant region comprising a CH1 domain variant according to any one of claims 1 to 29, and optionally (I) (i) with a kappa CL domain, as compared to a lambda CL domain; and / or (ii) with a kappa light chain polypeptide, as compared to a lambda light chain polypeptide; or (II) (i) with a lambda CL domain, as compared to a kappa CL domain; and / or (ii) an antibody heavy chain polypeptide further comprising one or more amino acid substitutions outside of the CH1 domain that further promote preferential pairing of the heavy chain with lambda light chain polypeptides relative to kappa light chain polypeptides.

31. The antibody heavy chain polypeptide of claim 30, wherein the CH1 domain variant is as defined in any one of claims 7 to 11 and 25 to 27.

32. 1. An antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair, and (b) the first heavy chain polypeptide comprises a first CH1 domain variant comprising an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering, such that the first CH1 domain variant preferentially binds the first light chain; Optionally, the first light chain polypeptide comprises a first CL domain that is a wild-type CL domain; Optionally, excluding one or more of the following combinations of substitutions in said first CH' domain: (a) if residue 141 on CH1 is substituted with C or L, if residue 166 is substituted with D or K, if residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or if residue 147 is substituted with D, then the CL does not contain any amino acid substitutions; (b) the CL does not contain an amino acid substitution where position 126 or 220 on CH1 is substituted with valine or alanine, where the non-cysteines at positions 128, 141, or 168 are substituted with cysteines, or where the CH1 substitution is L145F, K147A, F170V, S183F, or V185W / F; (c) when residue 172 is substituted with 172R, when residue 174 is mutated to 174G, or when residue 190 is substituted with 190M or 190I, these are not the only CH1 substitutions; (d) when said CH1 substitutions consist of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, CL is unaltered; (e) if said CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S and / or 193F / L, these are not the only CH1 substitutions and / or are of the same human immunoglobulin subtype or allotype in a bispecific antibody containing a CH1 domain; (f) if said CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), then there is no LC substitution corresponding to 129D / E / R / H / K (IMGT position 18); (g) if the CH1 substitution consists of 124K / E / R / D, then there is no corresponding LC substitution at 176; (h) when said CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, the corresponding LC substitution is not present; (i) if said CH1 substitutions consist of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L and / or 190S / A / G / Y, then the corresponding LC substitutions are not present; (j) when said CH1 substitutions consist of 143A / E / R / K / D and 145T / L, then the corresponding LC substitutions are not present; (k) when said CH1 substitutions consist of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, and 139W / G / C, 179E and / or 186R, the corresponding LC substitutions are not present; (l) when the CH1 substitution consists of a substitution with a cysteine ​​at positions 126, 127, 128, 134, 141, 171, and / or 173, the corresponding LC position is not modified to form a disulfide bond; (m) when said CH1 substitutions consist of L145Q, H168A, F170G, S183V and / or T187E, there are no corresponding kappa or lambda LC substitutions; (n) when said CH1 substitutions consist of 143D / E, 145T, 190E / D and / or 124R, the corresponding CL substitutions are not present; or (o) the CH1 substitutions consist of A140C, K147C and / or S183C with corresponding CL substitutions present;

33. The antibody or antibody fragment further comprising a second heavy chain polypeptide and a second light chain polypeptide, (a) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; and (b) the second heavy chain polypeptide comprises a second CH1 domain variant comprising an amino acid substitution at one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering, such that the second CH1 domain variant preferentially binds to the second light chain polypeptide comprising a second CL domain; with the proviso that, optionally, one or more of the following combinations of substitutions are excluded in the second CH1 domain: (a) if residue 141 on CH1 is substituted with C or L, if residue 166 is substituted with D or K, if residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or if residue 147 is substituted with D, then the CL does not contain any amino acid substitutions; (b) the CL does not contain an amino acid substitution where position 126 or 220 on CH1 is substituted with valine or alanine, where the non-cysteines at positions 128, 141, or 168 are substituted with cysteines, or where the CH1 substitution is L145F, K147A, F170V, S183F, or V185W / F; (c) when residue 172 is substituted with 172R, when residue 174 is mutated to 174G, or when residue 190 is substituted with 190M or 190I, these are not the only CH1 substitutions; (d) when said CH1 substitutions consist of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, CL is unaltered; (e) if said CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S and / or 193F / L, these are not the only CH1 substitutions and / or are of the same human immunoglobulin subtype or allotype in a bispecific antibody containing a CH1 domain; (f) if said CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), then there is no LC substitution corresponding to 129D / E / R / H / K (IMGT position 18); (g) if the CH1 substitution consists of 124K / E / R / D, then there is no corresponding LC substitution at 176; (h) when said CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, the corresponding LC substitution is not present; (i) if said CH1 substitutions consist of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L and / or 190S / A / G / Y, then the corresponding LC substitutions are not present; (j) when said CH1 substitutions consist of 143A / E / R / K / D and 145T / L, then the corresponding LC substitutions are not present; (k) when said CH1 substitutions consist of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, and 139W / G / C, 179E and / or 186R, the corresponding LC substitutions are not present; (l) when the CH1 substitution consists of a substitution with a cysteine ​​at position 126, 127, 128, 134, 141, 171, or 173, the corresponding LC position is not modified to form a disulfide bond; (m) when the CH1 substitutions consist of L145Q, H168A, F170G, S183V, and T187E, there are no corresponding kappa or lambda LC substitutions; (n) when said CH1 substitutions consist of 143D / E, 145T, 190E / D and / or 124R, the corresponding CL substitutions are not present; or (o) the CH1 substitutions consist of A140C, K147C, and / or S183C with corresponding CL substitutions present; Further optionally, the antibody or antibody fragment comprises: (i) the first CL domain is a wild-type CL domain; (ii) the second CL domain is a wild-type CL domain; (iii) the first CL domain is a kappa CL domain; (iv) the first CL domain is a lambda CL domain; (v) the second CL domain is a kappa CL domain; (vi) the second CL domain is a lambda CL domain; (vii) the first CH1 domain variant is the CH1 domain variant according to any one of claims 1 to 29; (viii) the second CH1 domain variant is a CH1 domain variant according to any one of claims 1 to 29; and / or (ix) the amino acid substitutions in the first CH1 domain variant are different from the amino acid substitutions in the second CH1 domain variant.

34. 1. An antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises a first CH1 domain variant according to any one of claims 2 to 13; and (c) the first light chain polypeptide comprises a kappa CL domain and is optionally a kappa light chain polypeptide; Optionally, (i) the kappa CL domain is a wild-type CL domain, and / or (ii) the first light chain polypeptide is a wild-type light chain polypeptide; Further optionally, the first heavy chain polypeptide comprises: (i) with a kappa CL domain, as compared to a lambda CL domain; and / or (ii) an antibody or antibody fragment which contains one or more amino acid substitutions outside the CH1 domain which further promote preferential pairing of the heavy chain with kappa light chain polypeptides compared to lambda light chain polypeptides.

35. An antibody or antibody fragment comprising a second heavy chain polypeptide and a second light chain polypeptide, (a) the second heavy chain polypeptide and the second light chain polypeptide form a first cognate pair; (b) the second heavy chain polypeptide comprises a second CH1 domain variant according to any one of claims 14 to 29; and (c) the second light chain polypeptide comprises a lambda CL domain, and is optionally a lambda light chain polypeptide; Optionally, (i) the lambda CL domain is a wild-type CL domain, and / or (ii) the second light chain polypeptide is a wild-type light chain polypeptide; Further optionally, the second heavy chain polypeptide comprises: (i) with a lambda CL domain, as compared to a kappa CL domain; and / or (ii) an antibody or antibody fragment which contains one or more amino acid substitutions outside the CH1 domain which further promote preferential pairing of the heavy chain with lambda light chain polypeptides relative to kappa light chain polypeptides.

36. 1. An antibody or antibody fragment comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises a first CH1 domain comprising a CH1 domain variant according to any one of claims 2 to 13; (c) the first light chain polypeptide comprises a kappa CL domain and is optionally a kappa light chain polypeptide; (d) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; (e) the second heavy chain polypeptide comprises a second CH1 domain comprising a CH1 domain variant according to any one of claims 14 to 29; and (f) the second light chain polypeptide comprises a lambda CL domain, and is optionally a lambda light chain polypeptide; Further optionally, the first heavy chain polypeptide comprises: (i) with a kappa CL domain, as compared to a lambda CL domain; and / or (ii) comprises one or more amino acid substitutions outside of the CH1 domain that further promote preferential pairing of the heavy chain with kappa light chain polypeptides as compared to lambda light chain polypeptides; Optionally, the second heavy chain polypeptide further comprises: (i) with a lambda CL domain, as compared to a kappa CL domain; and / or (ii) an antibody or antibody fragment comprising one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with lambda light chain polypeptides relative to kappa light chain polypeptides.

37. are multispecific, optionally bispecific; Further optionally, the structure of the antibody or antibody fragment is as depicted in any one of Figures 24 to 29. The antibody or antibody fragment of any one of claims 32 to 36.

38. multispecific, wherein the first CH1 domain variant and the second CH1 domain variant are (i) reducing non-cognate heavy chain-light chain pairing by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, or by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold , at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold (ii) providing for the formation of at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the desired first and second cognate pairs; (iii) providing about 85% to about 95% formation of the desired first and second cognate pairs; and / or (iv) providing a reduction in non-cognate heavy chain-light chain pairing of less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%; Optionally, the amount of cognate pairs and / or non-cognate pairs is determined by LCMS or flow cytometry.

39. The antibody or antibody fragment of claim 33, 36 or 38, which is multispecific and comprises one or more of the following features (i) to (iv): (i) the first CH1 domain variant comprises a substitution at position 147 and / or 183 and reduces noncognate heavy chain-light chain pairing by at least about 50%; (ii) the second CH1 domain variant comprises a substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218, and reduces noncognate heavy chain-light chain pairing by at least about 50%; (iii) the first CH1 domain variant comprises a substitution at position 147 and / or 183 and the second CH1 domain variant comprises a substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218, and reduces noncognate heavy chain-light chain pairing by at least about 50% to at least about 75%; or (iv) a first CH1 domain variant comprises a substitution at position 147 and / or 183 and a second CH1 domain variant comprises a substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218, and provides for formation of at least about 85% to at least about 95% of the desired first and second cognate pairs.

40. Multispecific and (a) the first CH1 domain variant comprises: (i) an amino acid residue F at position 147, and / or (ii) containing an amino acid residue R, K, or Y at position 183; and (b) the second CH1 domain variant is (i) an amino acid residue E or D at position 141; (ii) an amino acid residue E at position 170; (iii) an amino acid residue E at position 171; (iv) an amino acid residue K at position 181; (v) the amino acid residue R at position 185; (vi) an amino acid residue R at position 187; (vii) The antibody or antibody fragment of claim 33, 36, 38, or 39, comprising amino acid residue P, A, E or K at position 218.

41. Multispecific and (a) the first CH1 domain variant comprises: (i) an amino acid residue F at position 147, and / or (ii) an amino acid substitution at position 183 consisting of amino acid residues R, K, or Y; and (b) the second CH1 domain variant is (i) an amino acid residue D at position 141, an amino acid residue E at position 171, and an amino acid residue R at position 185; (ii) an amino acid residue D at position 141, an amino acid residue E at position 170, and an amino acid residue R at position 187; or (iii) comprising an amino acid substitution consisting of amino acid residue D at position 141, amino acid residue K at position 181, and amino acid residue P at position 218.

42. (a) the first CH1 domain variant comprises: (i) SEQ ID NO: 137, (ii) SEQ ID NO: 138, (iii) SEQ ID NO: 139, (iv) SEQ ID NO: 60, (v) SEQ ID NO: 41, or (vi) comprising the amino acid sequence of SEQ ID NO: 136; and (b) the second CH1 domain variant is (i) SEQ ID NO: 188, (ii) SEQ ID NO: 186, or (iii) The antibody or antibody fragment of claim 33 or 36, comprising the amino acid sequence of SEQ ID NO:

143.

43. the first CH1 variant and the second CH1 variant are (i) reducing non-cognate heavy chain-light chain pairing by at least 50% to at least 75%; and / or (ii) providing from about 85% to at least about 95% formation of the desired first cognate pair and second cognate pair.

44. (i) a CH1 domain variant polypeptide according to any one of claims 1 to 29; (ii) an antibody heavy chain polypeptide according to claim 30 or 31, and / or (iii) 44. A pharmaceutical composition comprising the antibody or antibody fragment according to any one of claims 32 to 43.

45. 1. A method for generating a CH1 domain variant library, comprising: (a) (i) one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a kappa CL domain ("C κ set"), and / or (ii) one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain ("C λ providing a polypeptide comprising a kappa or lambda CL domain and a light chain variable region (VL) comprising a CL domain; (b)(i) the C κ (ii) the C λ and / or (iii) within the lambda CL domain in the set κ In the set and / or the C λ selecting one or more amino acid positions in the CH1 domain that contact one or more amino acid positions in the VH in the set; (c) generating a library of CH1 domain variant polypeptides, or a library of constructs encoding CH1 domain variants, wherein one or more of the one or more amino acid positions selected in step (b) are substituted with any non-wild type amino acid; Optionally, (I) in step (a), the CH1 domain, the kappa CL domain, and the lambda CL domain are wild-type and / or human; (II) in step (a), (i) the polypeptide comprising a CH1 domain paired with a polypeptide comprising a kappa CL domain, and (ii) the polypeptide comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain, are intact antibodies or antigen-binding fragments ("Fab"); (III) in step (b), selecting one or more amino acid positions in the CH1 domain if the amino acid residue at the one or more amino acid positions in the CH1 domain has a side chain atom within a distance of 5 Å of (i) a side chain atom of the amino acid residue at the one or more amino acid positions in the kappa CL domain, (ii) a side chain atom of the amino acid residue at the one or more amino acid positions in the lambda CL domain, and / or (iii) a side chain atom of the amino acid residue at the one or more amino acid positions in the VH; and / or (IV) the generating in step (c) is via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues.

46. The one or more CH1 amino acid positions selected in step (b) are (i) Said C κ at the interface with the kappa CL domain in at least 10% of representative sets of the set, and κ having a fractional solvent accessible surface area of ​​greater than 10% in at least 90% of representatives of the set; (ii) the C λ at the interface with the lambda CL domain in at least 10% of representative sets of the set, and λ and / or having a solvent accessible surface area of ​​greater than 10% in at least 90% of representatives of the set; and / or (iii) the C κ Set and / or C λ At least 10% of representatives of a set are at the interface with the VH, and κ Set and / or C λ 46. ​​The method of claim 45, wherein at least 90% of representatives of the set have a solvent accessible surface area fraction of greater than 10%.

47. the amino acid positions selected in step (b) include one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering; 47. The method of claim 45 or 46, optionally excluding one or more of the following substitution combinations: (a) if residue 141 on CH1 is substituted with C or L, if residue 166 is substituted with D or K, if residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or if residue 147 is substituted with D, then the CL does not contain any amino acid substitutions; (b) the CL does not contain an amino acid substitution where position 126 or 220 on CH1 is substituted with valine or alanine, where the non-cysteines at positions 128, 141, or 168 are substituted with cysteines, or where the CH1 substitution is L145F, K147A, F170V, S183F, or V185W / F; (c) when residue 172 is substituted with 172R, when residue 174 is mutated to 174G, or when residue 190 is substituted with 190M or 190I, these are not the only CH1 substitutions; (d) when said CH1 substitutions consist of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, CL is unaltered; (e) if said CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S, and / or 193F / L, these are not the only CH1 substitutions and / or are of the same human immunoglobulin subtype or allotype in a bispecific antibody containing a CH1 domain; (f) if said CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), then there is no LC substitution corresponding to 129D / E / R / H / K (IMGT position 18); (g) if the CH1 substitution consists of 124K / E / R / D, then there is no corresponding LC substitution at 176; (h) when said CH1 substitution consists of 133V, 150A, 150D, 152D, 173D, and / or 188W, the corresponding LC substitution is not present; (i) if the CH1 substitution consists of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L, and / or 190S / A / G / Y, then the corresponding LC substitution is not present; (j) when said CH1 substitutions consist of 143A / E / R / K / D and 145T / L, then the corresponding LC substitutions are not present; (k) when said CH1 substitutions consist of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, and 139W / G / C, 179E and / or 186R, the corresponding LC substitutions are not present; (l) if the CH1 substitution consists of a substitution with a cysteine ​​at 126, 127, 128, 134, 141, 171, or 173, the corresponding LC position is not modified to form a disulfide bond; (m) when said CH1 substitutions consist of L145Q, H168A, F170G, S183V, and T187E, there are no corresponding kappa or lambda LC substitutions; (n) when said CH1 substitutions consist of 143D / E, 145T, 190E / D and / or 124R, the corresponding CL substitutions are not present; or (o) the CH1 substitutions consist of A140C, K147C and / or S183C with corresponding CL substitutions present;

48. The library of CH1 domain variants in step (c) (I) yeast strains, (II) yeast (Saccharomyces cerevisiae), and / or (III) A cell line co-expressing (i) one or more polypeptides comprising a kappa CL domain, and (ii) one or more polypeptides comprising a lambda CL domain, optionally wherein the kappa CL domain and / or the lambda CL domain are wild-type; 48. The method of any one of claims 45 to 47, further optionally wherein the kappa CL domain and / or the lambda CL domain are expressed in said cell line which is human.

49. 1. A method for generating a CH1 domain variant library, comprising: (a) selecting one or more of the following CH1 amino acid positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218; (b) selecting one or more CH1 amino acid positions of interest that are different from the positions selected in step (a); (c) generating a library of CH1 domain variant polypeptides, or a library of constructs encoding CH1 domain variants, wherein one or more of the one or more amino acid positions selected in steps (a) and (b) are substituted with any non-wild type amino acid; Optionally, (I) the amino acid positions selected in (a) include 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof; (II) the generating in step (c) is via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues; and / or (III) In step (c), the amino acid position selected in step (a) is substituted with a predetermined amino acid, and the amino acid position selected in (b) is substituted via a degenerate codon, and optionally, the substitution with the predetermined amino acid in step (a) comprises A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P, or any combination thereof.

50. 1. A method for identifying one or more CH1 domain variant polypeptides, the method comprising: (A) a polypeptide that comprises a kappa CL domain, as compared to a polypeptide that comprises a lambda CL domain; or (B) preferentially pairs with a polypeptide comprising a lambda CL domain relative to a polypeptide comprising a kappa CL domain; The method comprises: (a) co-expressing one or more candidate CH1 domain variant polypeptides from a CH1 domain variant library generated by the method of any one of claims 44 to 49 with (i) one or more polypeptides comprising a kappa CL domain, and (ii) one or more polypeptides comprising a lambda CL domain; (b) comparing (i) the amount of the candidate CH1 domain variant polypeptide associated with a polypeptide comprising a kappa CL domain and (ii) the amount of the candidate CH1 domain variant polypeptide associated with a polypeptide comprising a lambda CL domain; (c) based on said comparing in step (b), (A) with a polypeptide comprising a kappa CL domain, as compared to a polypeptide comprising a lambda CL domain; or (B) selecting one or more CH1 domain variants that provide preferential pairing with a polypeptide comprising a lambda CL domain compared to a polypeptide comprising a kappa CL domain; Optionally, in step (a), the total amount of said candidate CH1 domain variant polypeptide expressed and the total amount of said polypeptide comprising a (kappa and lambda) CL domain expressed are about the same; Optionally, in step (a), the candidate CH1 domain variant polypeptide, the polypeptide comprising a kappa CL domain, and the polypeptide comprising a lambda CL domain are expressed in a ratio of approximately 2:1:

1.

51. 51. The method of claim 50, wherein in step (a), the (i) one or more polypeptides comprising a kappa CL domain and (ii) one or more polypeptides comprising a lambda CL domain are wild-type and / or human.

52. 52. The method of claim 50 or 51, wherein in step (b) the amount is determined via fluorescence activated cell sorting or via liquid chromatography-mass spectrometry.

53. 53. The method of any one of claims 50 to 52, further comprising (d) co-expressing one or more control CH1 domain variants with (i) one or more polypeptides comprising a kappa CL domain, and (ii) one or more polypeptides comprising a lambda CL domain, optionally wherein one or more of the one or more control CH1 domain variants are according to the CH1 domain variant of any one of claims 1 to 29.

Images