CH1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising same

Abstract

The present invention relates to CH1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising the CH1 domain variants. Specifically, provided are CH1 domain variants engineered to preferentially bind to a [kappa] CL domain or [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 mismatch and promote homologous heavy chain-light chain pairing, thereby improving multispecificity, such as bispecificity, antibody production. Methods of making a library of CH1 domain variants and methods of identifying one or more CH1 domain variants are also provided.

Description

[0001] This application is a divisional application of PCT application PCT / US2020 / 053482, filed on September 30, 2020, with the invention title "CH1 Domain Variants Engineered for Preferential Light Chain Pairing and Multispecific Antibodies Comprising the CH1 Domain Variants", and the application number after entering the Chinese national phase is 202080068887.1.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 908,367, filed on September 30, 2019, entitled "CH1 Domain Variants Engineered for Preferential Light Chain Pairing and Multispecific Antibodies Comprising the Same", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0004] The present invention relates to CH1 domain variants, as well as antibody heavy chains and antibodies, particularly multispecific antibodies, comprising the CH1 domain variants, wherein the CH1 domain variants contain at least one amino acid substitution that promotes proper heavy chain-light chain pairing. The present invention further relates to compositions comprising such antibodies and their use, for example as therapeutic or diagnostic agents. The present invention further relates to methods of preparing libraries of CH1 domain variants and methods of identifying one or more CH1 domain variants. BACKGROUND OF THE INVENTION

[0005] Efforts are underway to develop antibody therapeutics with more than one antigen-binding specificity, such as 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 bring targets into close proximity and modulate protein complex formation or drive cell-to-cell contact. The generation 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 antibody was generated using hybridoma technology in the 1980s (Milstein et al., Nature 1983 305(5934):537-540). In the past decade, interest in bispecific antibodies has increased significantly due to their therapeutic potential, and bispecific antibodies are now used in the clinic; for example, blinatumomab and emicizumab have been approved for the treatment of specific cancers (see Sedykh et al., Drug Des Devel Ther 12:195-208 (2018) and Labrijn et al., Nature Reviews Drug Discovery 18:585-608 (2019) for recent reviews of bispecific antibody generation methods and the characteristics of bispecific antibodies approved for medical use).

[0006] Although bispecific antibodies have shown significant advantages over monospecific antibodies, the widespread commercial application of bispecific antibodies has been hindered by the lack of an effective / low-cost production method, the lack of stability of bispecific antibodies, and the lack of a long half-life in the human body. In the past few decades, a variety of methods have been developed to improve the production of bispecific antibodies. These include recombinant co-expression of two immunoglobulin heavy-chain-light-chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)); "milling structure" engineering (see, for example, U.S. Patent No. 5,731,168); immunoglobulin cross-technology (also known as Fab domain exchange or CrossMab format) (see, for example, WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)); engineered electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); leucine zippers (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); "diabody" technology (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0007] Despite these improvements, generating bispecific antibodies with the correct heavy-chain-light-chain pairing remains a challenge. Bispecific antibodies can be formed by co-expression of two different heavy chains and two different light chains. Appropriately forming the bispecific antibody in the desired form remains a challenge because the heavy chains have evolved to bind the light chains in a relatively non-proximate manner. Thus, co-expression of two heavy chains and two light chains can lead to disruption of heavy-chain-light-chain pairing—a complex mixture of sixteen possible combinations, only one of which represents the desired bispecific antibody out of ten different antibodies (the maximum yield in the mixture would be 12.5% if there were complete promiscuity). This mismatch (also known as the chain-association problem) remains a major challenge in generating bispecifics because uniform pairing is required for manufacturability and efficacy.

[0008] One strategy for alleviating the mismatch 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 κ and one λ) can be used (see, e.g., Fischer et al., Nature Commun. 6:6113 (2015)). However, this strategy requires identification of antibodies with common chains, which is difficult and tends to compromise the specificity of each binding arm and substantially reduce diversity (see, e.g., Wang et al., MABS 10(8):1226-1235 (2018)).

[0009] Other methods for improving correct heavy-chain-light-chain pairing include the CrossMab technology (Roche), in which the light chain or one of its subdomains of one fragment antigen-binding (Fab) arm is exchanged with the corresponding region of the heavy-chain Fd region, and the DuetMab technology (MedImmune), in which the native disulfide bonds in one Fab arm are replaced with engineered disulfide bonds. However, these methods require significant alterations to the native IgG form, which may result in compounds that are not fully analogous to natural antibodies.

[0010] Another strategy is to utilize amino acid substitutions in the constant and / or variable regions of the heavy and light chains in the form of IgG to reduce or eliminate heavy chain-light chain mismatches. To the inventors' knowledge, it has not been previously demonstrated that modification of only the CH1 domain addresses the chain association or mismatch issues frequently observed during the expression of multispecific antibodies. Instead, multispecific antibodies engineered to include CH1 domain variants further require modifications outside of the CH1 domain in order to address chain association issues such as the CL domain, and in some cases the VH, CH2, CH3, and / or VL domains. Examples thereof include Lewis et al., Nature Biotechnology 32(2):191-198 (2014) which generated mutant CH1 and CL domains, CRD1 (heavy chain substituted with D148K, F170T, V185F, and light chain substituted with K129D, L135F; EU numbering) and CRD2 (heavy chain substituted with H168A and F170T, and light chain substituted with L135Y, S176W) in an attempt to drive preferential pairing of the altered heavy and light chains and disfavor pairing of the heavy and light chain domains with wild-type constant domains. However, they reported that any pairing specificity obtained with mutant CH1 and CL domains in the absence of variable domains did not translate into the full-length IgG form without additional engineering within the VH-VL interface, i.e., substitutions within the VH-VL interface as well as CL and CH1 domain substitutions were required to achieve preferential heavy chain-light chain pairing. Engineering the CH1 and CL domains to contain charged amino acid residues has also been proposed to promote preferential heavy chain-light chain pairing (see, e.g., U.S. 10,047,163). Bispecific antibodies having at least two Fab fragments with different CH1 and CL domains are also known, where one Fab fragment has substitutions within the CH1 domain and the Cκ domain to drive preferential pairing (see US20180022829 and U.S. 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). Additional examples of specific CH1 domain substitutions that are alleged to promote preferential heavy chain-light chain pairing when the light chain or in some cases the CH2, CH3, and / or VH are also appropriately substituted to promote preferential pairing include: A141C / L, K147D, G166D, G166K or substitution with cysteine at positions 128, 129, 162, or 171 (WO2019183406 (Invenra Inc.)));Substitution of cysteine at position 126 or 220 with valine or alanine or substitution of non-cysteine at positions 128, 141 or 168 with cysteine, L145F, K147A, F170V, S183F or V185W / F (U.S. 9,527,927 (MedImmune, Inc.)); 172A and 174G (WO2020060924 (Dualogics, Inc. (Dualogics))); A172R and 174G or substitution of residue 190 with M or I (U.S. 10,047,167 (University of North Carolina Chapel Hill and Eli Lilly and Company)); L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and V185A / L (US20180177873 (Genentech, Inc.)); 131C / S, 133R / K, 137E / G, 138S / G178S / Y, 192N / S and / or 193F / L (U.S. 10,487,156 (Argenx BVBA)); 145D / E / R / H / K (IMGT position 26) (WO2018141894 (Merck & Co., Inc.)); 124K / E / R / D (U.S. 10,392,438 (Pfizer Inc.)); 133V, 150A, 150D, 152D, 173D or 188W (US20190023810 (Massachusetts Institute of Technology (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 (US20180179296 and U.S. 9,914,785 (Zymeworks Inc.)); 143A / E / R / K / D and 145T / L (U.S.10,077,298 (Zymeworks Inc.); 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, 139W / G / C, 179E or 186R (US20170204199 (Zymeworks Inc.)); substituted with cysteine at positions 126, 127, 128, 134, 141, 171 or 173 (Zenyaku Kogyo Co., Ltd.); L145Q, H168A, F170G, S183V and T187E (WO2020127354 (Alligator Bioscience)); 143D / E, 145T, 190E / D and 124R (WO2017 / 059551 (Zymeworks Inc.)). Additionally, it has been reported that in U.S. 9,150,639, Kyowa Hakko Kirin Co., Ltd. generated heavy chains including A140C, K147C or S183C for introducing cysteine to allow chemical modulation. Kirin Co., Ltd. indicated that antibody variants containing these heavy chain mutations could include wild-type light chains; however, there was no indication that this favored preferential heavy chain-light chain pairing.

[0011] Yet another strategy for minimizing heavy chain-light chain mismatching is to utilize different light chains, such as light chains with different constant domains. For example, Loew et al. generated multispecific antibodies with κ and λ light chains and observed minimal mismatching because certain naturally occurring κ light chains have high fidelity and do not pair with heavy chains from λ antibodies and vice versa (WO2018057955). Unfortunately, the applicability of this method is limited to those light chains with high fidelity. Others have generated multispecific antibodies using κ and λ light chains, where amino acid substitutions were used in both the heavy and light chains to electrostatically or sterically drive preferential pairing (see, for example, WO2017059551 (Zymeworks Inc.), US20140154254 (Amgen) and U.S. 10,047,163 (AbbVie Stemcentrx)). However, introducing numerous amino acid substitutions into both the heavy and light chains presents additional technical hurdles and may additionally have detrimental effects on antibody function and / or immunogenicity. Summary of the Invention

[0012] The object of the present invention is to provide engineered bispecific antibodies with proper heavy chain-light chain pairing. In one aspect, provided herein are CH1 domain variant polypeptides (also referred to herein as CH1 domain variants) that promote preferential pairing of a heavy chain with a specific light chain, and polypeptides including the same, such as antibodies. The CH1 domain variant contains at least one amino acid substitution (relative to the parent, e.g., wild-type sequence).

[0013] 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). The substitution promotes preferential pairing of the heavy chain containing the CH1 domain variant with a specific light chain. For example, CH1 domain variant 141 preferentially pairs with the λCL domain, as compared to the κCL domain, while CH1 domain variants 147F and / or 183R, 183K or 183Y preferentially pair with the κCL domain, as compared to the λCL domain.

[0014] In some embodiments, the CH1 domain variant contains at least one amino acid substitution at a CH1 domain position that forms an interface between the CH1 domain and VH, such as CH1 position 151 (EU numbering).

[0015] This preferential pairing of the constant domains is expected to drive the pairing of the full-length light and heavy chains, including the variable domains, thereby generating a solution to the chain pairing problem for bispecifics. Specifically, the CH1 domain variant polypeptide includes an amino acid substitution at one or more of the following 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. Optionally, such CH1 domain variant polypeptides preferentially pair with: (i) the κ light chain constant region (“CL”) domain, as compared to the λCL domain, and / or a κ light chain polypeptide, as compared to a λ light chain polypeptide; (ii) the λCL domain, as compared to the κCL domain, and / or a λ light chain polypeptide, as compared to a κ light chain polypeptide.

[0016] Optionally, in some embodiments, certain CH1 domain variants may be excluded and the CH1 domain variants according to the present invention may satisfy the following:

[0017] (a) 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, then the CL domain that the CH1 domain variant preferentially pairs with does not include amino acid substitutions;

[0018] (b) If position 126 or 220 on CH1 is substituted with valine or alanine, a non-cysteine at position 128, 141 or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F or V185W / F, then the CL domain that the CH1 domain variant preferentially pairs with does not include amino acid substitutions;

[0019] (c) If residue 172 on CH1 is substituted with 172R, residue 174 mutates to 174G, or residue 190 is substituted with 190M or 190I, then these are not the only substitutions included in CH1;

[0020] (d) If the CH1 substitutions consist of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, the CL domain that the CH1 domain variant preferentially pairs with is not modified;

[0021] (e) If the CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S and / or 193F / L, then these are not the only CH1 substitutions and / or in the bispecific antibody, the CH1 domain has the same human immunoglobulin subtype or allotype;

[0022] (f) If the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), there is no corresponding LC substitution, 129D / E / R / H / K (IMGT position 18);

[0023] (g) If the CH1 substitution consists of 124K / E / R / D, there is no corresponding substitution at position 176 of the LC, and the CH1 domain variant preferentially pairs with it;

[0024] (h) If the CH1 substitutions consist of 133V, 150A, 150D, 152D, 173D and / or 188W, there is no corresponding substitution in the LC that the CH1 domain variant preferentially pairs with;

[0025] (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 for which the CH1 domain variant would preferentially pair;

[0026] (j) If the CH1 substitution consists of 143A / E / R / K / D and 145T / L, there is no corresponding substitution in the LC for which the CH1 domain variant would preferentially pair;

[0027] (k) If the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, there is no corresponding substitution in the LC for which the CH1 domain variant would preferentially pair;

[0028] (l) If the CH1 substitution consists of a cysteine substitution at position 126, 127, 128, 134, 141, 171 or 173, the corresponding LC position is not modified to form a disulfide bond;

[0029] (m) If the CH1 substitution consists of L145Q, H168A, F170G, S183V and / or T187E, there is no corresponding substitution in the κ or λ LC for which the CH1 domain variant would preferentially pair;

[0030] (n) If the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there is no corresponding substitution in the LC for which the CH1 domain variant would preferentially pair; or

[0031] (o) If the CH1 substitution consists of A140C, K147C and / or S183C, there is a corresponding substitution in the LC for which the CH1 domain variant would preferentially pair.

[0032] In some embodiments, the CH1 domain variant polypeptide comprises an amino acid substitution at one or more of the following positions according to EU numbering: 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. Optionally, the CH1 domain variant polypeptide is preferentially paired with: (i) a κCL domain (or a polypeptide containing κCL), as compared to a λCL domain (or a polypeptide containing λCL); and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide.

[0033] In certain embodiments, such CH1 domain variants comprise an amino acid substitution at position 147, position 183, or both position 147 and 183.

[0034] In certain embodiments, such CH1 domain variants comprise 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 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; position 151 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 190 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.

[0035] In certain embodiments, the κ-preferring CH1 domain variant polypeptide can include: (i) an amino acid residue F, I, L, R, T, S, M, V, N, E, H, Y, or Q at position 147; and / or (ii) an amino acid residue I, W, F, E, Y, L, K, Q, N, or R at position 183.

[0036] In some preferred embodiments of the κ-preferring CH1 domain variant, the CH1 domain variant polypeptide can include: (i) an amino acid residue R, K, or Y at position 183; and / or (ii) an amino acid residue F at position 147.

[0037] In additional embodiments, the CH1 domain variant polypeptide includes: (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 residue Y at position 183. Optionally, such a CH1 domain variant can include the following amino acid sequences: (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.

[0038] In some embodiments, the CH1 domain variant polypeptide includes an amino acid substitution at a CH1 amino acid position within the interface between CH1 and VH. Optionally, the CH1 amino acid position within such an interface is position 151. Further optionally, such a CH1 domain variant can include an amino acid residue A or L at position 151.

[0039] In some embodiments, the CH1 domain variant polypeptide further includes one or more amino acid substitutions that increase the pairing of the CH1 domain with: (i) a κCL domain, as compared to a λCL domain; and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide.

[0040] In some embodiments, a CH1 domain variant polypeptide of any one of claims 2 to 10, which increases the pairing with the following 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%: (i) a κCL domain, as compared to a λCL domain; and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide. The increase in κ pairing can optionally be measured by liquid chromatography - mass spectrometry (LCMS).

[0041] In some embodiments, a CH1 domain variant polypeptide of any one of claims 2 to 10, which increases the pairing with the following by at least 1.2 - fold, at least 1.5 - fold, at least 2 - fold, 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: (i) a κCL domain, as compared to a λCL domain; and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide. The increase in κ pairing can optionally be quantified by flow cytometry, for example by comparing the mean fluorescence intensity (MFI) ratio of κCL staining to λCL staining.

[0042] In some embodiments, a CH1 domain variant polypeptide according to the present invention comprises an amino acid substitution at one or more of the following positions according to EU numbering: 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. Optionally, the CH1 domain variant preferably pairs with the following: (i) a λCL domain, as compared to a κCL domain; and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide.

[0043] In certain embodiments, a λ - preference 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.

[0044] In certain embodiments, the λ-preferred CH1 domain variant polypeptides comprise one or more of the following amino acid substitutions: substitution of R for the amino acid at position 119; substitution of V for the amino acid at position 124; substitution of V for the amino acid at position 126; substitution of G for the amino acid at position 127; substitution of H or S for the amino acid at position 130; substitution of Q, T, N, R, V or D for the amino acid at position 131; substitution of D, T, L, E, S or P for the amino acid at position 133; substitution of A, H, I, P, V, N or L for the amino acid at position 134; substitution of R for the amino acid at position 138; substitution of A for the amino acid at position 139; substitution of I, V, D, Y, K, S, W, R, L or P for the amino acid at position 140; substitution of D, K, E, T, R, Q, V or M for the amino acid at position 141; substitution of M for the amino acid at position 142; substitution of G for the amino acid at position 152; substitution of M for the amino acid at position 163; substitution of F, I or V for the amino acid at position 168; substitution of N, G, E, S or T for the amino acid at position 170; substitution of N, E, G, S, A or D for the amino acid at position 171; substitution of D or M for the amino acid at position 175; substitution of R or M for the amino acid at position 176; substitution of V, L, A, K or T for the amino acid at position 181; substitution of L or V for the amino acid at position 183; substitution of R for the amino acid at position 184; substitution of M, L, S, R or T for the amino acid at position 185; substitution of R, D, E, Y or S for the amino acid at position 187; substitution of S for the amino acid at position 197; substitution of D for the amino acid at position 203; substitution of I for the amino acid at position 208; substitution of T for the amino acid at position 210; substitution of A for the amino acid at position 211; substitution of N for the amino acid at position 212; substitution of E for the amino acid at position 213; substitution of R for the amino acid at position 214; substitution of G for the amino acid at position 216; and substitution of L, E, D, P, A, H, S, Q, N, T, I, M, G, C, K or W for the amino acid at position 218.

[0045] In still certain other embodiments, the λ-preferred CH1 domain variant polypeptides comprise any one or more of (i)-(xvii): (i) the amino acid residue V at position 126; (ii) the amino acid residue G at position 127; (iii) the amino acid residue V at position 131; (iv) the amino acid residue S at position 133; (v) the amino acid residue R at position 138; (vi) the amino acid residue I or V at position 140; (vii) the amino acid residue D, K, E or T at position 141; (viii) the amino acid residue M at position 142; (ix) the amino acid residue I at position 168; (x) the amino acid residue E, G or S at position 170; (xi) the amino acid residue E, D, G, S or A at position 171; (xii) the amino acid residue M at position 175; (xiii) the amino acid residue R at position 176; (xiv) the amino acid residue K, V, A or L at position 181; (xv) the amino acid residue R at position 184; (xvi) the amino acid residue R at position 185; (xvii) the 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.

[0046] In certain preferred embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise one or more of the following substitutions or consist of the following substitutions: 141D, 141E, 171E, 170E, 185R, and 187R.

[0047] In certain preferred embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise two or more of the following substitutions or consist of the following substitutions: 141D, 141E, 171E, 170E, 185R, and 187R.

[0048] In certain preferred embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise three or more of the following substitutions or consist of the following substitutions: 141D, 141E, 171E, 170E, 185R, and 187R.

[0049] In certain preferred embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise the following substitutions 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.

[0050] In still other embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise one or more substitutions at position 141 with D, K or E, said substitution optionally paired with a substitution at position 181 with K, and further optionally paired with a substitution at position 218 with L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

[0051] In still other embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention comprise a substitution at position 141 with D, K or E, said substitution paired with a substitution at position 181 with K and / or paired with a substitution at position 218 with L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

[0052] In additional embodiments, the λ-preferred CH1 domain variant polypeptides according to the present invention include any one or more of (i)-(xvii): (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) an 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; (ix) an amino acid residue P, A or E at position 218.

[0053] In additional embodiments, the λ-preferred CH1 domain variant polypeptides according to the invention include: (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) an 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) The amino acid residue D at position 141, the amino acid residue R at position 185, and the amino acid residue R at position 187; (xxv) The amino acid residue D at position 141, the amino acid residue E at position 170, and the amino acid residue R at position 185; (xxvi) The amino acid residue D at position 141, the amino acid residue E at position 170, and the amino acid residue R at position 187; (xxvii) The amino acid residue E at position 141, the amino acid residue E at position 171, and the amino acid residue R at position 187; (xxiii) The amino acid residue D at position 141, the amino acid residue E at position 171, and the amino acid residue R at position 185; or (xxix) The amino acid residue D at position 141, the amino acid residue E at position 171, and the amino acid residue R at position 187.;

[0054] Optionally, this CH1 domain variant comprises the following amino acid sequences: (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) SEQ ID NO:189.

[0055] In some preferred embodiments, the λ-preferred CH1 domain variants include: (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; or (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.

[0056] In additional preferred embodiments, the λ-preferred CH1 domain variants include amino acid substitutions consisting of: (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; or (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.

[0057] In certain preferred embodiments, the λ-preferred CH1 domain variants include amino acid substitutions consisting of: (i) SEQ ID NO:188; or (ii) SEQ ID NO:186.

[0058] In some embodiments, the λ-preferred CH1 domain variant polypeptide may further include one or more amino acid substitutions that increase the pairing of the CH1 domain with: (i) the λCL domain, as compared to the κCL domain; and / or (ii) the λ light chain polypeptide, as compared to the κ light chain polypeptide.

[0059] In some embodiments, the CH1 domain variant polypeptide may increase the pairing with: (i) the λCL domain, as compared to the κCL domain; and / or (ii) the λ light chain polypeptide, as compared to the κ light chain polypeptide, 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%. The increase in λ pairing can optionally be measured by liquid chromatography - mass spectrometry (LCMS).

[0060] In some embodiments, the CH1 domain variant polypeptide can pair with: (i) a λCL domain, as compared to a κCL domain; and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide, with 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. The increase in λ pairing can optionally be measured by flow cytometry, optionally by comparing the MFI value ratio of λCL staining to κCL staining.

[0061] On the other hand, the present disclosure further provides 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 one of those described above.

[0062] In some embodiments, the CH1 domain variant of such antibody heavy chain polypeptides comprises amino acid substitutions consisting of: (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) (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.

[0063] On the other hand, the present invention further provides an antibody or antibody fragment, which comprises 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 homologous pair; and (b) the first heavy chain polypeptide comprises a first CH1 domain variant, the first CH1 domain variant comprising an amino acid substitution at one or more of the following 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, 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, and the first CL domain is a wild-type CL domain. Further optionally, certain CH1 domain variants can be excluded as described above and the CH1 domain variants according to the present invention can meet one or more of the items (a)-(o) described above. The present invention also provides such an antibody or antibody fragment, which further comprises 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 homologous pair; and (b) the second heavy chain polypeptide comprises a second CH1 domain variant, the second CH1 domain variant comprising an amino acid substitution at one or more of the following 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, such that the second CH1 domain variant preferentially binds to the second light chain polypeptide comprising a second CL domain. Similarly, optionally, certain CH1 domain variants can be excluded as described above and the CH1 domain variants according to the present invention can meet one or more of the items (a)-(o) described above.Further optionally, such an antibody or antibody fragment comprises one or more of features (i)-(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 κ CL domain; (iv) the first CL domain is a λ CL domain; (v) the second CL domain is a κ CL domain; (vi) the second CL domain is a λ 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.

[0064] The present disclosure further provides an antibody or antibody fragment that comprises 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 homologous pair; (b) the first heavy chain polypeptide comprises a first CH1 domain variant according to any one of the κ -preferred CH1 domain variants described above; and (c) the first light chain polypeptide comprises a κ CL domain and is optionally a κ light chain polypeptide. Optionally, (i) the κ 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 of the CH1 domain, the one or more amino acid substitutions further promoting the preferential pairing of the heavy chain with: (i) a κ CL domain, as compared to a λ CL domain, and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide. The one or more amino acid substitutions outside of the CH1 domain can be, for example, in the VH.

[0065] The present disclosure also provides an antibody or antibody fragment that includes 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 homologous pair; (b) the second heavy chain polypeptide includes a second CH1 domain variant according to any one of the λ-preferred CH1 domain variants described above; and (c) the second light chain polypeptide includes a λCL domain and is optionally a λ light chain polypeptide. Optionally, (i) the λCL domain is a wild-type CL domain; and / or (ii) the second light chain polypeptide is a wild-type light chain polypeptide. In certain embodiments, the second heavy chain polypeptide optionally includes one or more amino acid substitutions outside of the CH1 domain, the one or more amino acid substitutions further promoting the preferential pairing of the heavy chain with: (i) a λCL domain, as compared to a κCL domain, and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide.

[0066] The present disclosure also provides an antibody or antibody fragment that includes 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 homologous pair; (b) the first heavy chain polypeptide includes a first CH1 domain that includes a CH1 domain variant according to any one of the κ-preferred CH1 domain variants described above; (c) the first light chain polypeptide includes a κCL domain and is optionally a κ light chain polypeptide; (d) the second heavy chain polypeptide and the second light chain polypeptide form a second homologous pair; (e) the second heavy chain polypeptide includes a second CH1 domain that includes a CH1 domain variant according to any one of the λ-preferred CH1 domain variants described above; and (f) the second light chain polypeptide includes a λCL domain and is optionally a λ light chain polypeptide. In certain embodiments, the first heavy chain polypeptide optionally includes one or more amino acid substitutions outside of the CH1 domain, the one or more amino acid substitutions further promoting the preferential pairing of the heavy chain with: (i) a κCL domain, as compared to a λCL domain, and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide. The one or more amino acid substitutions outside of the CH1 domain can be, for example, in the VH. In certain embodiments, the second heavy chain polypeptide optionally includes one or more amino acid substitutions outside of the CH1 domain, the one or more amino acid substitutions further promoting the preferential pairing of the heavy chain with: (i) a λCL domain, as compared to a κCL domain, and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide.

[0067] Any one of the antibodies or antibody fragments can be multispecific, optionally bispecific. Optionally, the structure of such an antibody or antibody fragment is depicted in any one of FIGS. 24 to 29.

[0068] In some embodiments, in the multispecific antibodies or antibody fragments as described above, the first CH1 domain variant and the second CH1 domain variant reduce the formation of non-homologous heavy-chain / light-chain pairs 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 antibodies or antibody fragments as described above, the first CH1 domain variant and the second CH1 domain variant increase the formation of homologous heavy-chain / light-chain pairs 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%.

[0069] In some embodiments, the reduction of non-homologous heavy-light pairing and / or the increase of homologous heavy-light pairing can be achieved by transfecting cells with HC (or VH plus CH1) including the CH1 of interest, κLC and λLC at a predetermined ratio such as HC:κLC:λLC = 2:1:1 and measuring the light-chain species by LCMS as in Example 7 and Figure 23 , 30 or 31 for quantification. In certain embodiments using such or similar quantification methods, an exemplary WT CH1 can generate HC-LC pairs, 60% of which are homologous pairs and 40% of which are non-homologous pairs, and by the CH1 variants according to the present disclosure, the percentage of homologous pairs can 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 percentage of non-homologous pairs can be reduced 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 percentage of homologous pairs can be increased to at least 85%, at least 90%, at least 95% or 100%, while the percentage of non-homologous pairs can be reduced to at least 15%, at least 10%, at least 5% or 0%.

[0070] In some embodiments, in the multispecific antibodies or antibody fragments as described above, the first CH1 domain variant and the second CH1 domain variant reduce the formation of non-homologous heavy chain-light chain pairs 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 antibodies or antibody fragments as described above, the first CH1 domain variant and the second CH1 domain variant increase the formation of homologous heavy chain-light chain pairs 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.

[0071] In some embodiments, the reduction of non-homologous heavy-light pairs and / or the increase of homologous heavy-light pairs can be quantified as follows: express HC (or VH+CH1) including the CH1 of interest, κLC, and λLC at a predetermined ratio simultaneously to allow the presentation of heavy-light pairs on cells (e.g., yeast cells), stain the cells with anti-κ and anti-λ antibodies, and quantify the presence of κ and λ by FACS, e.g., by comparing the MFI values as shown in Figures 2-5, 8-13, and 19-22. To compare the κ preference of a certain CH1, calculate the ratio of the MFI of cells stained with anti-κ: to the MFI of cells stained with anti-λ and divide by such ratio of WT CH1 to obtain the fold over parental (FOP) value. To compare the λ preference of a certain CH1, calculate the ratio of the MFI of cells stained with anti-λ: to the MFI of cells stained with anti-κ and divide by such ratio of WT CH1.

[0072] In certain embodiments using such or similar quantitative methods, when using the kappa-preferred CH1 variants according to the present disclosure, the FOP value (calculated for kappa preference, i.e., the MFI of kappa:lambda) can be increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, 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 quantitative methods, when using the lambda-preferred CH1 variants according to the present disclosure, the FOP value (calculated for lambda preference, i.e., the MFI of lambda:kappa) can be increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, 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.

[0073] In some embodiments, the second CH1 domain variant includes a substitution at position 141 and reduces the formation of non-cognate heavy-chain / light-chain pairs by at least 50%. In some embodiments, the second CH1 domain variant includes a substitution at position 141, and the first CH1 domain variant includes substitutions at positions 183 and optionally position 147, or vice versa, and reduces the formation of non-cognate heavy-chain / light-chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant includes 141D or 141E, and the second CH1 domain variant includes 183R, 183K, or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy-chain / light-chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant includes one or more of 141D or 141E, 170E, 171E, 181K, 185R, 187R, and 218P, and the first CH1 domain variant includes 183R, 183K, or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy-chain / light-chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant includes 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 includes 183R, 183K, or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy-chain / light-chain pairs by at least 50% to at least 75%.

[0074] In some embodiments, the first CH1 domain variant and the second CH1 domain variant provide 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 homologous pairs. In some embodiments, the first CH1 domain variant and the second CH1 domain variant provide for the formation of from about 85% to about 95% of the desired first and second homologous pairs. 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 position 147, and provides for the formation of from about 85% to at least about 95% of the desired first and second homologous 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 the formation of from about 85% to at least about 95% of the desired first and second homologous pairs. In some embodiments, the first CH1 domain variant and the second CH1 domain variant reduce the formation of non-homologous heavy chain-light chain 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 second CH1 domain variant comprises substitutions at positions 141, 170, 171, 181, 185, 187 and / or 218, and the first CH1 domain variant comprises substitutions at positions 183 and optionally position 147, or vice versa, and reduces the formation of non-homologous heavy chain-light chain pairs by 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 reduces the formation of non-homologous heavy chain-light chain pairs by less than about 15%, less than about 10% or less than about 5%.

[0075] In yet another aspect, the present disclosure further provides pharmaceutical and diagnostic compositions comprising: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above.

[0076] On the other hand, the present disclosure further provides therapeutic and diagnostic uses of antibodies and pharmaceutical compositions, said antibodies and pharmaceutical compositions comprising: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above.

[0077] In still another aspect, the present disclosure further provides nucleic acids encoding: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above.

[0078] In yet another aspect, the present disclosure further provides a vector comprising a nucleic acid encoding the following or a cell transfected with a nucleic acid encoding the following: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above and uses thereof for producing the above.

[0079] On the other hand, the present disclosure provides a method for generating a library of CH1 variant domains, said method comprising steps (a)-(c): (a) providing (i) one or more sets consisting of a polypeptide comprising a CH1 domain paired with a polypeptide comprising a κCL domain (“Cκ set”); (ii) one or more sets consisting of a polypeptide comprising a CH1 domain paired with a polypeptide comprising a λCL domain (“Cλ set”); and / or (iii) in VH in the C κ set and / or C λ set; (b) selecting one or more amino acid positions in the CH1 domain that contact one or more amino acid positions in the κCL domain in the Cκ set and / or the λCL 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 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 further optionally, the polypeptide comprising a κ or λCL domain further comprises a light chain variable region (VL).

[0080] Optionally: (I) In step (a), the CH1 domain, the κCL domain, and the λCL domain are wild-type and / or human; (II) In step (a), (i) the polypeptide comprising the CH1 domain paired with the polypeptide comprising the κCL domain and (ii) the polypeptide comprising the CH1 domain paired with the polypeptide comprising the λCL domain are both full antibodies or antigen-binding fragments (“Fab”); (III) In step (b), one or more amino acid positions of the CH1 domain are selected such that the amino acid residue at the one or more amino acid positions of the CH1 domain has a side chain atom within the distance of: (i) the side chain atom of the amino acid residue at the one or more amino acid positions in the κCL domain; (ii) the side chain atom of the amino acid residue at the one or more amino acid positions in the λCL domain; and / or (iii) the side chain atom of the amino acid residue at the one or more amino acid positions in the VH; and / or (IV) The generation in step (c) is carried out by degenerate codons, optionally degenerate RMW codons representing six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues.

[0081] In some embodiments, one or more CH1 amino acid positions selected in step (b): (i) are located at the interface with the κCL domain in at least 10% of the representative set of the Cκ set and have a fractional solvent-accessible surface area greater than 10% in at least 90% of the representative set of the Cκ set; (ii) are located at the interface with the λCL domain in at least 10% of the representative set of the Cλ group and have a fractional solvent-accessible surface area greater than 10% in at least 90% of the representative set of the Cλ group and / or (iii) are located at the interface with the VH in at least 10% of the representative set of the C κ and / or C λ and have a fractional solvent-accessible surface area greater than 10% in at least 90% of the representative set of the C κ and / or C λ set.

[0082] In some embodiments, the amino acid positions selected in step (b) include one or more positions among 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 can be excluded as described above and the CH1 domain variants according to the present invention can meet criteria (a) - (o) as described above.

[0083] In some embodiments, the synthetic polypeptide encoding the CH1 variant domain or the CH1 domain variant library in step (c) is expressed in a yeast strain. In some embodiments, the yeast strain is Saccharomyces cerevisiae. In some embodiments, a cell system such as a yeast strain co - expresses (i) one or more polypeptides including a κCL domain, such as a κ light chain, and (ii) one or more polypeptides including a λCL domain, such as a λ light chain. Optionally, the κ and / or λCL domain is wild - type. Further optionally, the κ and / or λCL domain is human.

[0084] In some embodiments, the method of the present disclosure further includes verifying that one or more substituted CH1 amino acid residues drive preferential pairing of the κ light chain or the λ light chain. In some embodiments, fluorescence - activated cell sorting is used to verify that one or more substituted CH1 amino acid residues drive preferential pairing of the κ light chain or the λ light chain.

[0085] In some embodiments, one or more κ constant (Cκ) domains, one or more λ constant (Cλ) domains, and one or more CH1 domains are wild - type. In some embodiments, one or more κ constant (Cκ) domains, one or more λ constant (Cλ) domains, and one or more CH1 domains are human.

[0086] In some embodiments, a method for generating a CH1 domain library comprises steps (a)-(c): (a) selecting one or more CH1 amino acid positions from the following CH1 amino acid positions numbered according to EU: 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); 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 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 position 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218 or any combination thereof. In certain embodiments, the generating in step (c) is performed by degenerate codons, optionally degenerate RMW codons representing six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues. In certain embodiments, in step (c), the amino acid positions selected in step (a) may be substituted with a predetermined amino acid, and the amino acid positions selected in (b) are substituted by degenerate codons. Optionally, the substitutions with a predetermined amino acid may include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P or any combination thereof.

[0087] In yet another aspect, the present disclosure provides methods for identifying one or more CH1 domain variant polypeptides that preferentially pair with: (A) a polypeptide comprising a κCL domain, as compared to a polypeptide comprising a λCL domain; or (B) a polypeptide comprising a λCL domain, as compared to a polypeptide comprising a κCL domain. Such methods comprise steps (a)-(c): (a) co-expressing one or more candidate CH1 domain variant polypeptides with (i) one or more polypeptides comprising a κCL domain and (ii) one or more polypeptides comprising a λCL domain; (b) comparing the amount of candidate CH1 domain variant polypeptides that pair with (i) polypeptides comprising a κCL domain to the amount of candidate CH1 domain variant polypeptides that pair with (ii) polypeptides comprising a λCL domain; (c) based on the comparison in step (b), selecting one or more CH1 domain variants that provide preferential pairing with: (A) a polypeptide comprising a κCL domain, as compared to a polypeptide comprising a λCL domain; or (B) a polypeptide comprising a λCL domain, as compared to a polypeptide comprising a κCL domain. In step (a), generally the total amount of the candidate CH1 domain variant polypeptides expressed and the total amount of the polypeptides comprising the expressed (κ and λ)CL domains may be substantially the same. Optionally, in step (a), the candidate CH1 domain variant polypeptides, the polypeptides comprising a κCL domain, and the polypeptides comprising a λCL domain are expressed at a ratio of approximately 2:1:1.

[0088] In some embodiments, in step (a), the (i) one or more polypeptides comprising a κCL domain and (ii) one or more polypeptides comprising a λCL domain are wild-type and / or human.

[0089] In some embodiments, in step (b), the amount is determined by fluorescence-activated cell sorting or by liquid chromatography-mass spectrometry.

[0090] In some embodiments, the method further comprises step (d): (d) co-expressing one or more control CH1 domain variants with (i) one or more polypeptides comprising a κCL domain and (ii) one or more polypeptides comprising a λCL domain, optionally wherein one or more of the one or more control CH1 domain variants are CH1 domain variants according to any of those described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1A -C is a schematic diagram of the binding of a CH1 domain variant to a Cλ domain or a Cκ domain. Figure 1A Heterodimerization of the wild-type CH1 domain with Cλ and Cκ is shown (the wild-type or unmodified CH1 domain is referred to as CH1 WT )Figure 1B Shows a CH1 domain variant preferentially paired with Cκ (such CH1 domain variants preferentially paired with Cκ are referred to as CH1κ). Figure 1C Shows a CH1 domain variant preferentially paired with Cλ (such CH1 domain variants preferentially paired with Cλ are referred to as CH1λ).

[0092] Figure 2A and 2B Shows exemplary FACS plots of multiple rounds of selection to identify CH1 domain variants with a λCL domain preference ( Figure 2A ) or a κCL domain preference ( Figure 2B ). R1 = first round of selection, R2 = second round of selection, R3 = third round of selection. The x-axis shows the λ light chain labeled with PE, and the y-axis shows the κ light chain labeled with FITC.

[0093] Figure 3 Shows individual unique clones expressing CH1 domain variants with a λCL domain preference or a κCL domain preference. The ratio of the median fluorescence intensity (MFI) of anti-κ to anti-λ of the clones (κ:λ ratio) was scored. The κ:λ ratio of any individual clone was compared to a matched strain with a wild-type CH1 sequence ("parent"). FOP means fold over parent.

[0094] Figure 4 Shows individual unique clones expressing CH1 domain variants with amino acid substitutions at positions 141, 147, or 183 (EU numbering). The ratio of the anti-κ MFI to the anti-λ MFI of the clones was scored and compared to the parent to determine the FOP. CH1 positions 147 and 183 were identified as the two positions providing a κCL domain preference. CH1 position 141 was identified as the position providing a λCL domain preference.

[0095] Figure 5Shows specific amino acid substitutions at positions 141, 147, and / or 183 (EU numbering S183K) in the CH1 domain with λCL domain preference (A141T, Q, D, or R) or κCL domain preference (K147V, A, F, Y, or M), as measured by the ratio of anti-κ MFI to anti-λ MFI. Amino acid substitutions shown as white dots (V134; T141, V147; A151, and K183) were identified after initial selection from a library with diversity at multiple positions, and amino acid substitutions shown as black dots were identified after additional rounds of selection from a diversified library targeting positions 141, 147, and 183. Parental κ:λ ratio (wild-type signal): GAL1 Cκ; GAL10 Cλ: 3.58 and GAL1 Cλ; GAL10 Cκ: 0.3. The parental ratio is the average of experimental replicates. For CH1 variants with substitutions at 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 183 (e.g., Y x F means a CH1 variant with substitutions K147Y and S183F).

[0096] Figure 6A -E shows representative binding data indicating that compared to the wild-type CH1 domain (BsAb1 and BsAb15), the CH1 domain variants do not alter the target binding of the bispecific antibodies (BsAb2 - BsAb14). Figure 6A Shows IL12B and EGFR binding data for BsAbs1 - 3. Figure 6B Shows IL12B and EGFR binding data for BsAbs 5, 7, and 4. Figure 6C Shows IL12B and EGFR binding data for BsAbs 9, 10, and 6. Figure 6D Shows IL12B and EGFR binding data for BsAbs 11, 12, and 8. Figure 6E Shows IL12B and EGFR binding data for BsAbs13 - 15. Pani = Panitumumab; Uste = Ustekinumab.

[0097] Figure 7 Shows an increase in correct heavy-chain - light-chain pairing (HC1 - LC1 or HC2 - LC2) and a concomitant decrease in heavy-chain - light-chain mismatches (HC1 - LC2 and HC2 - LC1) in bispecific antibodies (BsAb2 - BsAb14) containing CH1 domain variants compared to bispecific antibodies (BsAb1) containing the wild-type CH1 domain.

[0098] Figure 8Shows the λ - preference FOP values of WT clones, A141D clones, and individual clones with different amino acid substitutions at positions 141, 181, and 218 of the CH1 domain obtained from the output of the 141x181x218 library selection in Example 5. The 13 data points marked in the rectangle correspond to the clones with the highest FOP values, and the amino acid residues at CH1 positions 141, 181, and 218 and the FOP values of each clone are provided in Table 8.

[0099] Figure 9 Shows the λ - preference FOP values of WT clones, A141D clones, and individual clones with D at position 141, K at position 181, and different amino acid substitutions at position 218 of the CH1 domain in the 141x181x218 library selection output in Example 5. The hollow data points represent the FOP of individual clones with the same CH1 sequence, and the solid data points represent the average FOP value.

[0100] Figure 10 Shows the λ - preference FOP values measured with recloned clones as well as WT and A141D clones, which confirms the maintenance of λ - preference.

[0101] Figure 11 Shows an exemplary scatter plot of HEK293 - produced IgG with CH1 having one of the nine 141x181x218 leaders selected in Example 5 and stained for κCL and λCL for WT and A14D. The scatter plot of individual clones overlaps with the WT plot. The x - axis shows the λ light chain labeled with PE, and the y - axis shows the κ light chain labeled with FITC.

[0102] Figure 12 Shows the λ - preference FOP values of the nine leaders from Example 5 as well as WT and A141D. Three CH1 variants with the highest FOP values (D_K_WT, D_K_P, and D_K_A) were selected for subsequent double - strand (κ or λ) transfection in HEK293.

[0103] Figure 13 Compares the λ - preference FOP values among 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.

[0104] Figure 14 Shows the light - chain species (comparing κ and λ) % of the nine leader - full - length IgG produced in HEK293, as measured by liquid chromatography - mass spectrometry (“LCMS”). 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.

[0105] Figure 15 Shows the exemplary process yields of three leader sequences (D_K_WT, D_K_P, and D_K_A) and the yield of A141D relative to WT, shown as the fold over parental ("FOP") value.

[0106] Figure 16 Shows the Tm (°C) of Fabs with κ pairing and Fabs with λ pairing having one of three leader CH1 variants (D_K_WT, D_K_P, and D_K_A) or A141D or WT.

[0107] Figure 17 Shows the relative λ Tm (°C), as defined as: [Change in Tm of variant Fab with λ pairing relative to WT Fab with λ pairing ("ΔλTm")] - [Change in Tm of variant Fab with κ pairing relative to WT Fab with κ pairing ("ΔκTm")].

[0108] Figure 18 Provides the sequencing results from the recloning output in Example 6, visualizing the frequent amino acid substitutions observed in the output clones.

[0109] Figure 19 Shows the leader sequences from the recloning output in Example 6 and the λ - preference FOP values (λMFI:κMFI) of some of the 141x181x218 leader sequences (DKP, DKA, KKE, KKP, and EKK) from Example 5, which were expressed as IgG in yeast. At least seven leader sequences marked with arrows have FOP values equal to or higher than those of the tested 141x181x218 leader sequences.

[0110] Figure 20 Shows the λ - preference FOP values of 14 leader sequences from Example 7 and DKP, A141D, and wild - type identified in Example 5. Two of the leader sequences marked with arrows, "A414D_P171E_V185R" and "A141D_F170E_T187R", show higher FOP values than DKP. The FOP values of all 14 leader sequences are higher than that of the wild - type.

[0111] Figure 21 Shows exemplary λ - preference FACS plots comparing 14 CH1 domain variants in Example 7 and three controls (DKP, A141D, and wild - type identified in Example 5). The x - axis shows the λ light chain labeled with PE, and the y - axis shows the κ light chain labeled with FITC. The numbers in each plot are the sort # 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.

[0112] Figure 22 shows Figure 21 Exemplary FACS plot overlays of a single plot (labeled "a"), wild-type plot (labeled "b"), and DKP plot (labeled "c").

[0113] Figure 23 Shows the light chain species (comparing κ and λ) % of 14 leaders and three control full-length IgGs produced in HEK293, as measured by LCMS in Example 7. The three controls are shown by hollow arrows. "A414D_P171E_V185R" and "A141D_F170E_T187R" (solid arrows) show higher λ% and lower κ chain % compared to the positive control "DKP".

[0114] Figures 24 - 29 provide exemplary and non - limiting examples of various multispecific antibody structures that can be used with the CH1 domain variants disclosed herein. In Figures 24 - 29, unless otherwise noted, the following apply: (1) Each domain is presented as a rectangle, and the text therein shows the domain name (e.g., CH1, VH1, etc.); (2) Solid rectangles and dotted rectangles are CH1 domain variants with κ or λ preference, which can be the CH1 domain variants disclosed herein; (3) "CH1κ" is a CH1 domain variant with κCL preference, "CH1λ" is a CH1 domain variant with λ preference, and "CH1" without the indication of "κ" or "λ", with or without light - chain allotype preference, is any CH1 domain, wild - type or variant; (4) "Cκ" is the κCL domain, "Cλ" is the λCL domain, and "CL" without the indication of "κ" or "λ", when shown paired with a solid or dotted CH1 domain, represents the CL domain of the allotype (κ or λ) preferred by the paired solid or dotted CH1 domain; (5) When more than one solid and / or dotted CH1 domain is present in a multispecific structure, at least one is a CH1 domain variant disclosed herein, and the rest may or may not be CH1 domain variants disclosed herein; (6) When both solid and dotted CH1 domains are present in a multispecific structure, the solid and dotted represent CH1 domains with different light - chain allotype preferences (i.e., when the solid represents a CH1 domain with κ preference, the dotted represents a CH1 domain with λ preference, and vice versa); (7) VH1 and VL1 form the antigen - binding site for the first epitope, VH2 and VL2 form the antigen - binding site for the second epitope, VH3 and VL3 form the antigen - binding site for the third epitope, VH4 and VL4 form the antigen - binding site for the fourth epitope, VH5 and VL5 form the antigen - binding site for the fifth epitope, and VH6 and VL6 form the antigen - binding site for the sixth epitope; (8) All of the first through sixth epitopes can be different from each other, or not all of the first through sixth epitopes can be different from each other, as long as the specific combination as a whole makes the presented structure multispecific; (9) A group of multiple domains connected to each other represents a polypeptide (e.g., a heavy - chain polypeptide, a light - chain polypeptide, etc.); (10) The domain orientation within a polypeptide follows the text direction of the domain names shown from the N - terminus to the C - terminus; (11) Linkers or hinges can be used between domains as needed, and disulfide bonds can be present between polypeptides (and / or within domains), perhaps to enable the proper formation of antigen - binding sites, even if the figure does not explicitly show the linker, hinge, or disulfide bond; (12) The CH2 and / or CH3 domains shown in the figure can be omitted as much as possible and, when appropriate, replaced with a hinge; (13) The CH1, CH2, and CH3 domains can be individually wild - type or variant and can individually have any (heavy - chain) allotype;And (14) when there is more than one CH1 domain in the structure, the CH1 domains can be or can not be the same isotype, when there is more than one CH2 domain in the structure, the CH2 domains can be or can not be the same isotype, and when there is more than one CH3 domain in the structure, the CH3 domains can be or can not be the same isotype.;

[0115] Figures 24A - 24C Some exemplary and non-limiting embodiments of various multispecific antibody structures in which the CH1 domain variants disclosed herein can be used are provided. In Figure 24A , a κ-preferred CH1 domain (“CH1κ”) is used for one polypeptide. Another CH1 domain can or can not prefer a λCL domain and can be or can not be a CH1 domain variant disclosed herein. In Figure 24B , a λ-preferred CH1 domain (“CH1λ”) is used for one polypeptide. Another CH1 domain can or can not prefer a κCL domain and can be or can not be a CH1 domain variant disclosed herein. In Figure 24C , CH1κ is used for one polypeptide and CH1λ is used for one polypeptide. This general structure allows the production of bispecific compounds with minimal or less effort for removing mismatched compounds, with or without. At least one of the CH1κ and CH1λ domains is a CH1 domain variant disclosed herein. As described above in (10), the domain orientation within a polypeptide is according to the text orientation of the domain names shown from the N-terminus to the C-terminus. Thus, in the case of the upper left compound of Figure 24A , in the direction from the N-terminus to the C-terminus, 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. Figures 24A - 24C (and all other applicable figures) represent mechanisms that promote heterodimerization of two non-identical polypeptides, such as “stapler” engineering. Figures 24A - 24C (and all other applicable figures) show a hinge structure that connects a polypeptide containing CH1κ and a polypeptide containing CH1λ. Although two bonds (e.g., disulfide bonds) are explicitly shown connecting the two polypeptides, the number of bonds and the exact location / position of the bonds can vary and be appropriately selected. In Figure 24C , in the lower right of, “+” indicates a mixture of two different Fab fragments.

[0116] Figures 25A - 25B Additional exemplary and non-limiting embodiments of various multispecific antibody structures in which the CH1 domain variants disclosed herein can be used are provided. The structure is similar to that in Figures 24A - 24C , but the domain order is different. InFigure 25A In, CH1κ and VL are in the same polypeptide, and CH1λ and VL are in the same polypeptide. In Figure 25B , Cλ and CH2 are in the same polypeptide (upper three and lower left), and Cλ is in a heavy-chain-like polypeptide (polypeptide containing a hinge) (lower right).

[0117] Figures 26A - 26C Additional exemplary and non-limiting embodiments of various multispecific antibody structures are provided, the multispecific antibody structures comprising two sets of two antigen-binding sites in tandem and thus being tetravalent. The structure 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.

[0118] Figures 27A - 27C Additional exemplary and non-limiting embodiments of various multispecific antibody structures are provided, the multispecific antibody structures being similar to Figures 26A - 26C those in, but with a different domain order. As described above in (10), the domain orientation within a polypeptide is according to the text direction of the domain names shown from the N-terminus to the C-terminus. Thus, in the case of the upper left structure of Figure 27A , in the direction from the N-terminus to the C-terminus, the first polypeptide comprises VH3-VH1-CH1 (solid)-CH2-CH3, the second polypeptide comprises VL1-VL3-CL, the third polypeptide comprises VH4-VH2-CH1 (dotted)-CH2-CH3, and the fourth polypeptide comprises VL2-VL4-CL. In Figures 27A - 27C any structure, appropriate linkers can be used between domains to effect proper formation of the antigen-binding sites.

[0119] Figures 28A - 28D Additional exemplary and non-limiting embodiments of various multispecific antibody structures containing at least one scFv are provided. Any of the structures provided in Figures 24-29 can additionally include or be modified to include one or more scFv-containing moieties, for example conjugated to any one of a heavy-chain constant domain, a light-chain constant domain and / or an antigen-binding domain. By way of example, Figures 28A - 28C structures are provided in which Figure 24A the upper left structure of 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 some cases, more than two scFvs can be conjugated. By way of example,Figures 28A - 28C provides a structure in which Figure 24A the upper left structure is conjugated with four scFvs, allowing specificity for up to six epitopes.

[0120] Figures 29A - 29D Also provided are additional exemplary and non-limiting embodiments of various multispecific antibody structures containing two additional Fab fragments. Although the two Fab fragments are conjugated to the CH3 domain, it should be noted that the Fab fragments can be conjugated to any other part of the structure, and it should also be noted that one (or three or more than three) rather than two Fab fragments can be conjugated. In Figure 29A , the two CH1 domains are in the same polypeptide as the CH2 and CH3 domains. In the intermediate structure, a κ-preferred CH1 domain and a λ-preferred CH1 domain are present within the same polypeptide (for two of the two CH1-containing polypeptides). When the two CH1-containing polypeptides are the same, the structure facilitates the production of tetravalent bispecific compounds without the need for a mechanism (such as "knobs-into-holes" engineering) to promote heterodimerization of two non-identical polypeptides, for example by simply using the triple transfection system used in the examples. In Figure 29B , the two polypeptides do not contain any CH1 domains. In the intermediate structure, when the two CH1-free polypeptides are the same, the structure facilitates the production of tetravalent bispecific compounds without the need for a mechanism (such as "knobs-into-holes" engineering) to promote heterodimerization of two non-identical polypeptides, for example by simply using the triple transfection system used in the examples. In Figure 29C and 29D , each polypeptide contains a CH1 domain. In Figure 29C and 29D , in the intermediate structure, if the first and third epitopes are the same epitope and the second and fourth epitopes are the same epitope but different from the first and third epitopes, the structure is bispecific. In such structures, if the two CH2 / CH3-containing polypeptides are the same, the structure facilitates the production of tetravalent bispecific compounds without the need for a mechanism (such as "knobs-into-holes" engineering) to promote heterodimerization of two non-identical polypeptides, for example by simply using the triple transfection system used in the examples.

[0121] Figure 30Exemplary process yields normalized to the process yield of wild-type (WT) are shown for full IgG containing one of the top two λ-preferred CH1 variants identified in Example 7 (“A141D P171E V185R” or “A141D F170E T187R”) or the κ-preferred CH1 variant identified in Example 4 (“K147F S183R”) or WT CH1. Striped bars (paired with κ) and solid bars (paired with λ) represent process yields normalized to the WT corresponding yields. Open diamonds (paired with κ) and solid triangles (paired with λ) represent the raw process yields (mg / L).

[0122] Figure 31 Exemplary process yields normalized to the process yield of wild-type (WT) are shown for Fab containing one of the top two λ-preferred CH1 variants identified in Example 7 (“A141D P171E V185R” or “A141D F170E T187R”) or the λ-preferred CH1 variants identified in Example 4 or 5 (“A141D” or “A141D S181K K218P”) or the κ-preferred CH1 variant identified in Example 4 (“K147F S183R”) or WT CH1. Yields are normalized to the WT corresponding yields. Striped bars represent Fab containing κ LC and solid bars represent Fab containing λ LC.

[0123] Figure 32 The wild-type CH1-Cλ interface in its electron density is shown. Representative electron density in the region of interest of the Fab crystal structure of the panitumumab variable fragment (Fv) paired with the wild-type λ constant domain (Cλ) and wild-type IgG1-CH1. Heavy chain (HC) carbon atoms are colored light gray, λ light chain (λLC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown as a stick representation. The 2Fo-Fc electron density map is shown as a gray mesh contoured at 1σ with carve. The data for this crystal structure extends to atomic resolution.

[0124] Figure 33 The A141D CH1-Cλ interface in its electron density is shown. Representative electron density in the region of interest of the Fab crystal structure of the panitumumab variable fragment (Fv) paired with the wild-type λ constant domain (Cλ) and A141D-substituted IgG1-CH1. Heavy chain (HC) carbon atoms are colored light gray, λ light chain (λLC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown as a stick representation. The 2Fo-Fc electron density map is shown as a gray mesh contoured at 1σ with carve. The data for this crystal structure extends to Atomic resolution.

[0125] Figure 34 Shows the wild-type CH1-Cκ interface in its electron density. Representative electron density in the region of interest of the Fab crystal structure of the panitumumab variable fragment (Fv) paired with the wild-type κ constant domain (Cκ) and wild-type IgG1-CH1. Heavy chain (HC) carbon atoms are colored light gray, κ light chain (κLC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown as a stick representation. The 2Fo-Fc electron density map is shown as a gray mesh contoured at 0.9σ with carve. The data for this crystal structure extends to near atomic resolution.

[0126] Figure 35 Shows the K147F-S183R CH1-Cκ interface in its electron density. Representative electron density in the region of interest of the crystal structure of the panitumumab variable fragment (Fv) paired with the wild-type κ constant domain (Cκ) and K147F-S183R-substituted IgG1-CH1. Heavy chain (HC) carbon atoms are colored light gray, κ light chain (κLC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown as a stick representation. The 2Fo-Fc electron density map is shown as a gray mesh contoured at 0.9σ with carve. The data for this crystal structure extends to near atomic resolution.

[0127] Figure 36 A-36D shows that the HC-A141D substitution allows hydrogen bonding with λLC while destabilizing κ pairing through steric hindrance with κLC. Figure 36 A-36D provides views of the pairing interfaces around the HC-Ala141 position between WT CH1 and λLC ( Figure 36 A), between WT CH1 and κLC ( Figure 36 B), between A141D CH1 and λLC ( Figure 36 C), or between A141D CH1 and κLC ( Figure 36 D). The κ light chain constant domain (κLC) interface contains the three hydrophobic residues Phe116, Phe118, and Leu135 illustrated in Figure 36 B. The presence of Thr116 in λLC at the structurally equivalent κLC-Phe116 position allows hydrogen bonding with the carboxyl hydrogen of HC-Asp141, as shown by the black dashed line ( Figure 36 C). In Figure 36In panel D, the HC alignment of A141DCH1-constant λ (Cλ) and WT CH1-Cκ shows steric hindrance between the HC-Asp141 side chain and the κLC-Phe116 side chain. 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 shown as stick representations with a transparent molecular surface, and backbone atoms are shown as wireframe representations.

[0128] Figure 37 Panels A and 37B show that the wild-type CH1 sequence sequesters HC-Gln175 within an intrachain hydrogen bond network, which may be disrupted by the K147F substitution, allowing HC-Gln175 to freely interact with κLC-Gln160. Figure 37 Panels A and 37B provide a view of the ternary hydrogen bond network in the HC, involving Lys147, Asp148, and Gln175 in the panitumumab wild-type CH1-constant κ (Cκ) structure ( Figure 37 panel A) and the panitumumab K147F-S183R-CH1-Cκ structure ( Figure 37 panel B). Heavy chain (HC) carbon atoms are colored light gray, κ light chain (κLC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are shown as stick representations, and backbone atoms are shown as wireframe representations. Hydrogen bonds are shown as dashed lines.

[0129] Figure 38 Panel A-38D shows that hydrogen bonding between HC-Arg183 and κLC-Thr178 can drive κ pairing, while steric hindrance between HC-Arg183 and λLC-Tyr178 reduces the preference for λ pairing. Figure 38 Panel A-38D provides a view of the region around the S183R substitution in IgG1-CH1 and of the hydrogen bonds between HC-Ser183 and λLC-Thr178 in the panitumumab wild-type CH1-constant λ (Cλ) structure ( Figure 38 panel B) and between HC-Arg183 and κLC-Thr178 in the panitumumab K147F-S183R-CH1-constant κ (Cκ) structure ( Figure 38 panel C). Figure 38 Panel A shows that HC-Ser183 and κLC-Thr178 are too far apart to hydrogen bond. 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 shown as stick representations. The side chain of λLC-Tyr178 is also shown as a transparent molecular surface. Hydrogen bonds are shown as black dashed lines. Figure 38D provided a model in which the HC of the panitumumab K147F-S183R-CH1-Cκ structure was superimposed on the HC of the panitumumab wild-type CH1-Cλ structure. The resulting model showed a significant steric hindrance between HC-Arg183 and λLC-Tyr178. Detailed Description

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" encompasses 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0131] It should be understood that the aspects and embodiments of the disclosure described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments.

[0132] The present disclosure provides an engineered CH1 domain comprising at least one amino acid substitution that prevents heavy chain-light chain mismatching by promoting preferential pairing of the heavy chain comprising the CH1 domain with a κCL domain (or κ light chain) or a λCL domain (or λ light chain). The term “preferential pairing” refers to the pairing of a heavy chain (or CH1 domain) with a light chain (or CL domain) of 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 may pair equally well with both L1 and L2, resulting in a mixture of approximately 50:50 H1:L1 to H1:L2. For example, if the amount of H1:L1 heterodimer formed is greater than the amount of H1:L2 heterodimer formed when H1 is co-expressed with L1 and L2, then “preferential pairing” will occur between H1 and L1. In this example, H1 pairs with L1 relative to L2. If H1 is inherently biased to pair with L1 as compared to L2 (such that the ratio of H1:L1 to H1:L2 is not 50:50, but is instead, for example, 60:40 or 70:30, in which case the formation of H1:L2 is still undesirable), then preferential pairing between the desired pairings, i.e., H1:L1, will occur when the number of pairings between H1:L1 is improved (increased) as compared to H1:L2. As used herein, the term “preferential pairing” encompasses the pairing of a heavy chain and a light chain (as described above) as well as the pairing of a CH1 domain and a CL domain. By way of example, if the amount of CH1:Cκ formed is greater than the amount of CH1:Cλ formed when CH1 is co-expressed with Cκ and Cλ, then “preferential pairing” will occur between the CH1 domain and the κCL domain. Similarly, if the amount of CH1:Cλ formed is greater than the amount of CH1:Cκ formed when CH1 is co-expressed with Cλ and Cκ, then “preferential pairing” will occur between the CH1 domain and the λCL domain.

[0133] Certain positions within the CH1 domain that are found to be part of the CH1-CL interface (for both Cκ and Cλ) affect the binding of the heavy chain to the light chain. Additionally, positions within the CH1 domain at the CH1:VH interface are also shown to 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 where the chain pairing or meeting or contact is termed an "interface". Further, within the heavy chain, the CH1 domain also contacts a portion of the VH, and this space where the CH1 domain and VH are very close is also covered by the term "interface". The 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, which 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 includes the CH1 domain and the VH domain of the heavy chain. The "interface" preferably derives from an IgG antibody or its Fab.

[0134] The CH1 variant domains described herein contain amino acid substitutions at one or more CH1:CL interface (CH1:κCL or CH1:λCL) positions, such as positions 141, 147, 170, 171, 175, 181, 183, 184, 185, 187, and / or 218 or one or more CH1:VH interface positions, such as position 151, as compared to the parent. The term "parent" refers to the polypeptide (and the amino acid sequence encoding said polypeptide) that is subsequently modified to generate the variant. The parent polypeptide can be a wild-type or naturally occurring polypeptide or a variant or engineered form thereof. Thus, a "parent CH1 domain" refers to the 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 can be a wild-type or naturally occurring CH1 domain or a variant or engineered form thereof, such as a wild-type CH1 domain modified to conjugate a toxin or a small molecule drug. Such a parent CH1 domain can be isolated or be part of a larger construct, such as a Fab, F(ab′)2, or IgG, which can optionally contain additional modifications, such as CH3 modifications that promote heterodimerization, alter Fc receptor binding, extend the half-life, and / or CH2 and / or CH3 modifications that link additional binding domains.

[0135] The resulting CH1 variant domain preferentially pairs with a κCL (Cκ) domain or a λCL (Cλ) domain, which Cκ and Cλ domains can be part of a light chain. Amino acid variation at one or both of positions 147 and 183 (EU numbering) in the CH1 domain promotes binding to Cκ (and concomitantly blocks pairing with Cλ), while amino acid variation at position 141 in the CH1 domain promotes binding to Cλ (and concomitantly blocks pairing with Cκ). The κ and λ CL domains can be present in any number of forms, including but not limited to wild-type or chimeric Fab or IgG, such as a Fab or IgG containing Vκ and Cκ, Vκ and Cλ, Vλ and Cκ, or Vλ and Cλ. By improving the fidelity of heavy-chain to light-chain pairing while maintaining the native IgG structure of the bispecific antibody, such CH1 variant domains can be used to engineer multispecific antibodies, which is advantageous due to its well-recognized properties as a therapeutic molecule, including a long in vivo half-life and the ability to elicit effector functions.

[0136] 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 from positions 118 - 215 (EU numbering), and the hinge region is the amino acid sequence from positions 216 - 230 (EU numbering). As used herein, the term "CH1 domain variant" refers to an amino acid sequence that includes the entire CH1 domain (positions 118 - 215 according to EU numbering) or a fragment thereof that includes at least 7 of residues 118 - 215 of CH1 (according to EU numbering), wherein such fragment includes 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 contains variation in the hinge region, such as positions 216 and 218.

[0137] The CH1 domain pairs with the CL domain of the light chain. In some embodiments, the light chain is a κ chain. In some embodiments, the light chain is a λ chain. The terms "κ constant domain", "κCL domain", or "Cκ" refer to the constant domain of the κ light chain. The terms "λ constant domain", "λCL domain", or "Cλ" refer to the constant domain of the λ light chain. A single disulfide bond covalently links the CH1 having 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.

[0138] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including (but not limited to): monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and / or antibody fragments (preferably those fragments that exhibit the desired antigen-binding activity, which are also referred to as "antigen-binding antibody fragments").

[0139] "Monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations and / or substitutions or arising during the production of the monoclonal antibody preparation), such variants generally being present in minor amounts. Each monoclonal antibody in a monoclonal antibody preparation binds to a single determinant (epitope) on the antigen, as compared to polyclonal antibody preparations which typically contain different antibodies directed against different determinants (epitopes).

[0140] "Multispecific antibody" may also be referred to herein as "multispecific compound" and 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, the multispecific antibody comprises (1) a first heavy chain and a first light chain that form a homologous pair and bind to a first antigen, and (2) a second heavy chain and a second light chain that form a homologous pair and bind to a second antigen.

[0141] As used herein, a “bispecific antibody,” also referred to as a “bispecific compound,” is a type of multispecific antibody and refers to an antibody that includes 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. Bispecific antibodies can target, for example, two different surface receptors on the same or different (e.g., immune cells and cancer cells) cells, two different cytokines / chemokines, a receptor and a ligand. Combinations of antigens that can be targeted by bispecific antibodies can include, but are not limited to: 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 LTβR; EGFR and IGFR; VEGFR2 and VEGFR3; VEGFR2 and PDGFRα; PDGFRα and PDGFRβ; 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-1α and IL-1β; IL-12 and IL-18; VEGF and osteopontin; VEGF and Ang-2; VEGF and PDGFRβ; VEGF and Her2; VEGF and DLL4; FAP and DR5; FcgRII and IgE; CEA and DTPA; CEA and IMP288; and LukS-PV and LukF-PV.

[0142] “Different antigens” can refer to different and / or distinct pluralities of proteins, polypeptides, or molecules; and different and / or distinct pluralities of epitopes that can be contained within one protein, polypeptide, or other molecule. Thus, a bispecific antibody can bind to two epitopes on the same polypeptide.

[0143] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule, called a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the interaction affinity. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope can comprise determinants that are chemical reactive surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge characteristics.

[0144] In some instances, an antibody comprises four polypeptide chains: two heavy (H) chains and two light (L) chains that are 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 a full antibody, CH comprises the domains CH1, CH2, and CH3. In the case of an antibody fragment, CH can comprise the CH1, CH2, and / or CH3 domains, and in some preferred embodiments, CH comprises at least the CH1 domain. The CH1 domain variants disclosed herein can be used in combination with wild-type CH2 and / or CH3 domains or CH2 and / or CH3 domains that comprise one or more amino acid substitutions, such as amino acid substitutions that alter or improve antibody stability and / or effector function. 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 hypervariable regions called complementarity determining regions (CDRs) that are interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or its antigen-binding fragment) can be identical to human germline sequences or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on a side-by-side analysis of two or more CDRs. Thus, the CDRs in the heavy chain are named "CDRH1", "CDRH2", and "CDRH3" respectively, and the CDRs in the light chain are named "CDRL1", "CDRL2", and "CDRL3". In other cases, an antibody can comprise its multimer (e.g., IgM) or its antigen-binding fragment.

[0145] In some cases, VH and CL can be present in a polypeptide. In some cases, VL and CH1, CH2, and / or CH3 domains can be present in a polypeptide. For example, in some antibodies or antibody fragments, although the first polypeptide comprises VH1 and CH1, and the second polypeptide comprises VL1 and CL (VH1 and VL form the antigen-binding site for a first epitope), the third polypeptide comprises VH2 and CL, and the fourth polypeptide comprises VL2 and CH1 (VH2 and VL2 form the antigen-binding site for a second epitope). In another antibody or antibody fragment, although the first polypeptide comprises VH1 and CH1, and the second polypeptide comprises VL1 and CL (VH1 and VL form the antigen-binding site for a first epitope), the third polypeptide comprises VL2, CL, and one or more of the CH2 and / or CH3 domains, and the fourth polypeptide comprises VH and CH1. The present invention encompasses any antibody or antibody fragment comprising any CH1 variant of the CH1 variants disclosed herein, which provides preferential pairing with κCL or preferential pairing with λCL, regardless of whether the CH1 domain is in the heavy chain or the light chain.

[0146] As used herein, the terms "cognate pair" or "cognate pairing" refer to a pair or pairing of two antibody chains (e.g., a heavy chain and a light chain), each containing variable regions (e.g., VH and VL), wherein the combination of the variable regions provides the desired binding specificity to an epitope or antigen. As used herein, the terms "non-cognate pair" or "non-cognate pairing" refer to a pair or pairing of two antibody chains (e.g., a heavy chain and a light chain), each containing variable regions (e.g., VH and VL), wherein the combination of the variable regions does not provide the desired binding specificity to an epitope or antigen.

[0147] There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0148] Unless otherwise expressly stated, the term "antibody" as used herein encompasses molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (sometimes referred to as "full-length antibody" or "intact antibody" or "whole antibody" etc., in all cases referring to an antibody having a structure substantially similar to a native antibody) and antigen-binding antibody fragments thereof. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of a full-length antibody or a combination of portions derived from one or more full-length antibodies, and binds an antigen to which a full-length antibody or full-length antibodies bind.

[0149] An antigen-binding fragment of an antibody comprises any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Exemplary antibody fragments include, but are not limited to: Fv; fragment antigen-binding ("Fab") fragments; Fab' fragments; Fab'('Fab'-SH') containing a free sulfhydryl group; F(ab')2 fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., single-chain variable fragments ("scFv"), nanobodies or VHHs, or only VH or VL domains); and monospecific or multispecific compounds formed from one or more of the foregoing antibody fragments. In some embodiments, the antigen-binding fragment of a bispecific antibody described herein is an scFv. In preferred embodiments, the antigen-binding fragment includes a CH1 domain that preferentially pairs with κCL or λCL.

[0150] Like the intact antibody molecule, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). A multispecific antigen-binding fragment of an antibody can include at least two different variable domains, wherein each variable domain is capable of specifically binding a separate antigen or a different epitope of the same antigen.

[0151] The present disclosure provides CH1 domain variants that preferentially pair (or bind to) with the CL domain of the κ light chain or the CL domain of the λ light chain. In one embodiment, the CH1 domain variant does not exhibit a reduced binding to the κ or λ light chain and at the same time exhibits exclusivity or increased preference for binding to the other class of light chain (in this instance, λ or κ respectively). These CH1 domain variants can be used to fully or partially address heavy and light chain mismatching when generating multispecific, such as bispecific antibodies, by promoting proper heavy and light chain pairing. In one embodiment, the CH1 domain variant can optionally be used in combination with other variants outside of the CH1 domain to further promote preferential pairing with the CL domain of the κ light chain or the CL domain of the λ light chain (e.g., VH:VL substitutions such as Q​39E / K:Q​38K / E (Dillon et al., MAbs 2017 9(2):213-230); or Q​39K+R62E:Q​38D+D1R or Q​39Y+Q105R:Q​38R+K42D (Brinkmann et al., MAbs 2017 9(2):182-212). More specifically, bispecific antibodies comprising these CH1 variant domains will form fewer unwanted product-related contaminants, i.e., molecules containing mismatched domains, and eliminating such contaminants during downstream processing can be challenging. For example, by engineering the heavy chain CH1 domain of antibody A into a κ-preferring CH1 domain variant (e.g., 147Phe and / or 183Arg, Lys, Tyr) and engineering the heavy chain CH1 domain of antibody B into a λ-preferring CH1 domain variant (e.g., 141Asp), a bispecific antibody comprising (i) the heavy and light chains of antibody A (where the light chain is a κ light chain) and (ii) the heavy and light chains of antibody B (where the light chain is a λ light chain) may be produced more efficiently, i.e., with fewer unwanted product-related contaminants. Thus, the heavy chain of antibody A will favor binding to the light chain of antibody A (and disfavor binding to the light chain of antibody B), while the heavy chain of antibody B will favor binding to the light chain of antibody B (and disfavor binding to the light chain of antibody A). See FIGS. 1 and 7 and Table 6.

[0152] In some embodiments, the CH1 domain variant reduces the formation of mismatches, i.e., non-homologous HC1-LC2 and / or HC2-LC1 pairs, 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, a 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 the formation of mismatches, i.e., non-homologous HC1-LC2 and / or HC2-LC1 pairs, by at least 25% to at least 80%. 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, reduces the formation of mismatches, i.e., non-homologous HC1-LC2 and / or HC2-LC1 pairs, by at least 50%. 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, reduces the formation of mismatches, i.e., non-homologous HC1-LC2 and / or HC2-LC1 pairs, by at least 75%.

[0153] In some embodiments, the CH1 domain variant preferentially pairs (binds) with the cognate CL domain (Cκ or Cλ) or the cognate light chain containing the corresponding CL domain (Cκ or Cλ), resulting in 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, namely HC1-LC1 and / or HC2-LC2. In some embodiments, the CH1 domain variant preferentially pairs (binds) with the cognate CL domain (Cκ or Cλ) or the cognate light chain containing the corresponding CL domain (Cκ or Cλ), resulting in the formation of from about 80% to about 99% or more specifically, from at least about 85% to at least about 95% of the desired first and second cognate pairs, namely HC1-LC1 and / or HC2-LC2. 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, provides the formation of from about 85% to at least about 95% of the desired first and second cognate pairs, namely HC1-LC1 and / or HC2-LC2.

[0154] In some embodiments, the CH1 domain variant reduces the formation of mispaired heavy chain-light chain heterodimers, namely HC1-LC2 and / or HC2-LC1 pairs, to 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, 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 the formation of mispaired heavy chain-light chain heterodimers to less than about 15%, less than about 10% or less than about 5%.

[0155] Several CH1 domain positions have been identified as affecting light chain binding preference, i.e., preferentially pairing with either the κCL domain or the λCL domain, 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). Substituting wild-type amino acid residues at any one or more of these positions in the CH1 domain with variant (non-wild-type) amino acid residues generates heavy chains that have a preferential pairing for light chains containing either the κCL domain or the λCL domain. For example, each of positions 147 and 183 has been identified as having a pairing preference for the κCL domain, and positions 141, 170, 171, 175, 181, 184, 185, 187, and 218 have been identified as having a pairing preference for the λCL domain.

[0156] Substituting the wild-type amino acid residue (Ala) at position 141 in the CH1 domain with Thr, Asp, Lys, Glu, Arg, Met, Val, or Gln is shown to increase the heavy chain preference for binding to a light chain containing the λCL domain. Substituting the wild-type amino acid residue (Phe) at position 170 in the CH1 domain with Glu, Gly, Ser, Asn, or Thr; substituting the wild-type amino acid residue (Pro) at position 171 in the CH1 domain with Glu, Gly, Ser, Asn, Asp, or Ala; substituting the wild-type amino acid residue (Met) at position 175 in the CH1 domain with Asp or Met; substituting the wild-type amino acid residue (Ser) at position 181 in the CH1 domain with Val, Leu, Ala, Lys, or Thr; substituting the wild-type amino acid residue (Ser) at position 184 in the CH1 domain with Arg; substituting the wild-type amino acid residue (Val) at position 185 in the CH1 domain with Met, Leu, Ser, Arg, Thr; substituting the wild-type amino acid residue (Thr) at position 187 in the CH1 domain with Arg, Asp, Glu, Tyr, or Ser; and / or substituting the wild-type amino acid residue (Lys) at position 218 in the CH1 domain with Leu, Glu, Asp, Pro, Ala, His, Ser, Gln, Asn, Thr, Ile, Met, Gly, Cys, Lys, or Trp also helps to increase the pairing of the heavy chain with a light chain containing the λCL domain.

[0157] 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 shows increased heavy chain preference for binding to light chains containing the κCL domain. 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 shows increased heavy chain preference for binding to light chains containing the κCL domain (see Figure 5 ). The effect of a given variant amino acid residue at a specific position can vary, but all variants show improved preferential pairing with Cκ or Cλ based on the amino acid position including the variant residue. Additionally, given the high similarity in the CH1 regions of IgG1, IgG2, IgG3, and IgG4, it is expected that the CH1 domain variants described herein will display similar preferential pairing properties in each isotype.

[0158] Compared to the wild-type CH1 domain sequence (Ala at position 141), the first round of selection identified Thr at position 141 to promote preferential pairing with Cλ, but compared to Thr, additional rounds of selection identified Asp, Arg, and Gln to provide increased preferential pairing (see Figure 5 ). Additional screening strategies identified Lys and Glu also to provide increased λ preference (see Example 5, Figures 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 λ preference (see Examples 5-7). In addition, the applicant showed that specific substitution combinations that increase λ preference include, but are not limited to: 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 141, 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. In additional screening efforts, "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 λ-preferred CH1 domain substitution combinations (see Figure 20 , 23 , 30 and 31).

[0159] Similarly, compared to the wild-type CH1 domain sequence, the first round of selection identified Val or Ala at position 147 and Lys at position 183 as promoting preferential pairing with Cκ, but compared to 147Val or Ala or 183Lys respectively, additional 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 providing increased preferential pairing. These CH1 domain variants, either alone or in combination with other amino acid substitutions, can improve the preferential pairing of heavy chains containing such CH1 domain variants with light chains containing Cκ or Cλ.

[0160] The present disclosure provides variant CH1 domains that include amino acid substitutions at one or more of the following positions according to EU numbering, and thus, the CH1 domain variants exhibit preferential pairing to Cκ or Cλ (or light chains including such domains): 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. As shown herein, different amino acid residue substitutions at one or more of these positions can result in CH1 domains that preferentially pair with Cκ or Cλ (see Tables 3 and 4). In some embodiments, the amino acid substitution at position 147 (EU numbering) is not cysteine. In some embodiments, the amino acid substitution at position 183 (EU numbering) is not cysteine or threonine. In some embodiments, the amino acid substitution at position 147 (EU numbering) is not cysteine and the amino acid substitution at position 183 (EU numbering) is not cysteine or threonine.

[0161] In some embodiments, the CH1 domain variant includes an amino acid substitution at one or more of the following positions to drive preferential pairing of the CH1 domain variant (or the heavy chain comprising such a domain) with Cκ (or the light chain comprising such a 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 substitution is 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; 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 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, or R; position 185 is substituted with W; position 190 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 some embodiments, the CH1 domain variant includes amino acid substitutions at positions 147 and 183 to drive preferential pairing (binding therewith) with the κ light chain. 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 a particular embodiment, the CH1 domain variant includes R or K or Y alone at position 183 or in combination with F at position 147. Non-limiting examples of κ-preferred CH1 domain variants can include the amino acid sequences of SEQ ID NO: 137, 138, 139, 60, 41, or 136.

[0162] In some embodiments, the CH1 domain variant comprises an amino acid substitution at one or more of the following positions to drive preferential pairing of the CH1 domain variant (or the heavy chain comprising such a domain) with Cλ (or the 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 substitution is 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 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, 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 G; position 171 is substituted with N, E, G, S, A, D, preferably D, E, G, or S; position 175 is substituted with D or M, preferably M; position 181 is substituted with V, L, A, K, or T, preferably 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 R; 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 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 with the λ light chain. 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 a particular embodiment, the CH1 domain variant comprises Asp or Glu at position 141. In some embodiments, the amino acid substitution at position 141 can be combined with one or more substitutions within CH1, such as Lys at position 181 or Lys at position 181 and Ala or Pro at position 218.An Asp or Glu at position 141 can 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 λ-preferred CH1 domain variants can include the amino acid sequences 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.

[0163] 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 additional embodiments, the CH1 domain variant comprises the amino acid sequence of SEQ ID NO:188, 186, or 143.

[0164] The present disclosure also contemplates polypeptides comprising a CH1 domain variant, such as an antibody. Such polypeptides can be bispecific antibodies that comprise a first heavy chain containing a first CH1 domain variant and a second heavy chain containing 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 comprising a first CH1 domain. In some embodiments, the antibody further comprises a second heavy chain comprising a second CH1 domain that comprises an amino acid sequence different from the CH1 domain of the first heavy chain.

[0165] In some embodiments, the first CH1 domain variant can preferentially pair with (or bind to) Cκ, and the second CH1 domain variant can preferentially bind to Cλ. In such cases, 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 κ light chain (Cκ and Vκ) or a chimeric light chain (Cκ and Vλ), and the second light chain is a λ light chain (Cλ and Vλ) or a chimeric light chain (Cλ and Vκ).

[0166] 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. In such cases, 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 λ light chain (Cλ and Vλ) or a chimeric light chain (Cλ and Vκ), and the second light chain is a κ light chain (Cκ and Vκ) or a chimeric light chain (Cκ and Vλ).

[0167] The first and second light chains can include (or can not include) amino acid substitutions that drive preferential pairing with the CH1 domain. In some embodiments, the CL domain of the light chain is not modified to alter binding to the heavy chain, such as the CH1 domain. In some embodiments, the first light chain contains a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain. In some embodiments, the second light chain contains a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain. The wild-type κ light chain or Cκ domain can be encoded by IGKC. The wild-type λ light chain or Cλ domain can be encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0168] 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 form containing a CH1 domain, such as but not limited to the structures depicted in FIGS. 24-29. See also, e.g., Brinkmann and Kontermann at Table 2, MAbs 9(2):182-212 (2017), which is incorporated herein by reference in its entirety.

[0169] The multispecific antibody can include one or more of the CH1 domain variants having the amino acid sequences listed in Tables 3, 4, 7, 9, 12, or 13. In some embodiments, the antibody includes a first heavy chain and a first light chain containing a first CH1 domain variant, and the first heavy chain and the first light chain form a first homologous pair. The first CH1 domain variant can include amino acid substitutions at one or more of the following 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, 218. Such a first CH1 domain variant preferentially binds to the first light chain. The CL domain of the first light chain can or can not be modified to alter binding to the first heavy chain.

[0170] In some embodiments, the antibody further comprises a second heavy chain and a second light chain containing a variant of the second CH1 domain, and the second heavy chain and the second light chain form a second homologous pair. The variant of the second CH1 domain may comprise an amino acid substitution at one or more of the following 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, 218. Such a variant of the second CH1 domain preferentially binds to the second light chain. The CL domain of the second light chain may or may not be modified to alter the binding to the second heavy chain.

[0171] In certain embodiments of a multispecific antibody or antibody fragment, the antibody or antibody fragment may comprise a κ-preferred CH1 domain variant and a λ-preferred CH1 domain variant. In some cases, the κ-preferred CH1 domain variant may be a κ-preferred CH1 domain variant as disclosed herein, and the λ-preferred CH1 domain may or may not be the λ-preferred CH1 domain described herein. In some cases, the λ-preferred CH1 domain variant may be a λ-preferred CH1 domain variant as disclosed herein, and the κ-preferred CH1 domain may or may not be the κ-preferred CH1 domain described herein. In certain cases, both the κ-preferred CH1 domain variant and the λ-preferred CH1 domain variant are variants as disclosed herein.

[0172] Any of the CH1 domain variants disclosed herein can be used to provide a pairing preference for a κCL domain or a λCL domain, and the CL domain can be wild-type or non-wild-type. Further, any of the CH1 domain variants disclosed herein can be used to provide a κ / λ pairing preference in an antibody or antibody fragment structure, with or without the introduction of further amino acid changes to the rest of the antibody structure, such as the CH2, CH3, VH, VL, or CL domains. For example, the CH1 domain variants disclosed herein can be used in combination with VH substitutions that can further enhance the light chain pairing preference (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).

[0173] Without wishing to limit the scope of the invention, it is emphasized that the CH1 domain variants provided herein provide κ / λ pairing preference in the context of a wild-type light chain (or a polypeptide comprising a wild-type CL domain) without the need for another modification in the CH2, CH3, or variable domains, although such non-CH1 modifications can optionally be used in combination with the novel CH1 domain variants discovered by the inventors herein. This is particularly unexpected given the many reported failures in generating antibodies, particularly multispecific antibodies, where modifying only the CH1 domain provided a meaningful κ or λ preference.

[0174] In some embodiments, the antibody is part of a pharmaceutical composition. Such compositions can contain multiple polypeptides, such as antibodies, comprising the CH1 domain variants described herein.

[0175] The present disclosure also contemplates methods for obtaining such CH1 domain variants. The variant CH1 domains described herein can be identified by rational design (computer) or can be identified randomly, for example, using ePCR or other mutagenesis techniques known in the art. In one embodiment, a rational design method is employed to design the variant CH1 domain. For such methods, a set of structures, such as experimentally derived protein structures, such as Fab crystal structures, can be assembled and analyzed to identify solvent-exposed positions involved in contacts spanning the CH1-CL domain interface (also referred to as CH1-CL domain interface positions). The set can be curated by selecting structures having certain properties, such as a high percentage identity to a reference (wild-type) CH1, Cκ, and Cλ. In some embodiments, a position is described or defined as contacting another residue (or being "in contact") if a pair of side-chain atoms is within the cutoff distance of. A "CH1 interface residue" can be defined as a residue in the CH1 domain that contacts a residue in the Cκ domain or the Cλ domain. In this context, the terms "residue" and "position" can be used interchangeably. The inventors also unexpectedly found that amino acid substitutions at CH1 positions in the CH1-VH interface (e.g., CH1 position 151) alter the light chain allotype preference. Thus, in some embodiments, CH1 positions that contact VH residues (e.g., a pair of side-chain atoms is within the cutoff distance of) can also be selected for rational CH1 domain variant identification.

[0176] The selection of amino acid positions, singly or in combination (e.g., singlets, doublets, triplets, etc.) to be altered may depend on a variety of different parameters, e.g., the consistent role of the position in forming the interface between CH1 and CL or between CH1 and VH in different structures, the accessibility of the position in the overall structure, the relationship of the position to positions that affect antigen binding, or the likelihood that the residue allosterically affects the formation of the CH1:CL or CH1:VH interface without directly participating in intermolecular contacts across the interface. In some embodiments, amino acid residues in the CH1 domain are selected for mutagenesis if: 1) the residue is at the interface with the light chain constant domain in at least 10% of the structures that converge on Cκ and has a fractional solvent accessible surface area (SASA) greater than 10% in at least 90% of the structures that converge on Cκ (see Example 1) or 2) the residue is at the interface with the light chain constant domain in at least 10% of the structures that converge on Cλ and has a fractional SASA greater than 10% in at least 90% of the structures that converge on Cλ or 3) the residue is at the interface with VH in at least 10% of a representative set of the C κ and / or C λ set and has a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the C κ and / or C λ set.

[0177] In addition, for each of the specific amino acid substitutions in the CH1 domain provided herein to confer κ or λ preference, the amino acid included as a result of the substitution can be further substituted by a conservative amino acid substitution to obtain another CH1 domain variant that provides equivalent κ or λ preference. Alternatively, for each CH1 domain variant, one or more amino acid positions in the CH1 domain variant that are not affected relative to the wild-type sequence can be altered by conservative substitution to obtain another CH1 domain variant that provides equivalent κ or λ preference.

[0178] "Conservative amino acid substitutions" are known in the art and involve amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituting for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting 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 substituting for another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain substituting for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0179] Next, a library in which the CH1 domain residues are varied can be generated. One or more CH1 domain residues can be varied in the library. In some embodiments, about one to six CH1 domain residues are varied in the library. Amino acid diversity at each residue position can be generated by degenerate codons such as NNK to allow representation of at least all 20 naturally occurring amino acids at a given CH1 domain position. The selected CH1 domain positions can be varied individually to generate point substitutions (also called singlets), or subsets of combinations of positions can be varied in combination, such as to generate double substitutions and triple substitutions (also called doublets and triplets). In some embodiments, variant combinations are generated that contain CH1 domain positions that are adjacent in 3D space, such as positions 147x[124, 126, 145, 148, 175, and 181].

[0180] In some embodiments, a method for preparing a library of CH1 domain variants includes: a) providing a set of structures that contain 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 substitute positions contacting one or more Cκ domain positions and / or one or more Cλ domain positions; c) substituting the one or more CH1 domain positions identified in step b) with any amino acid other than the parental amino acid; and d) synthesizing a polypeptide encoding the CH1 variant domain of step c) to assemble a library of CH1 variant domains.

[0181] 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 in step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In a particular embodiment, the Cλ amino acid sequence in step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions that span the CH1-CL interface or the CH1-VH interface.

[0182] In some embodiments, one or more CH1 amino acid residues selected for substitution (i) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cκ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of the representative set of CH1:Cκ structures; (ii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of the representative set of CH1:Cλ structures; or (iii) are located at the interface with the VH in at least 10% of a representative set of C κ and / or C λ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of the representative set of C κ and / or C λ structures.

[0183] In some embodiments, a library is generated by altering one or more CH1 positions disclosed herein as altering light chain allotype preference (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 certain embodiments, a library can be generated by combining a predetermined substitution at one or more CH1 positions disclosed herein as altering light chain allotype preference (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. In a particular instance, the predetermined substitution can include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P or any combination thereof.

[0184] In some embodiments, a library is screened to identify CH1 domain variants that exhibit preferential binding to kappa or lambda light chains. Such screening can begin by expressing the library in a suitable host cell, such as a eukaryotic cell, such as a yeast cell, such as Saccharomyces cerevisiae. After expressing the CH1 variant domains contained in the library in the host cell, the variant library can be screened, for example, by FACS or MACS to identify those variants with the desired binding properties.

[0185] In some embodiments, a method for identifying CH1 domain variants with preferential Cκ or Cλ domain binding comprises: a) providing a set of constructs comprising 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 substitute positions contacting one or more Cκ domain positions and / or one or more Cλ domain positions; c) substituting the one or more CH1 domain positions identified in step b) with any amino acid other than the parental amino acid; d) synthesizing polypeptides encoding the CH1 variant domains of step c) to assemble a CH1 variant domain library; and e) screening the library of d) to identify CH1 domain variants with preferential Cκ or Cλ domain binding.

[0186] 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 in step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC1,IGLC2,IGLC3,IGLC6, or IGLC7. In certain embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions spanning the CH1-CL interface or the CH1-VH interface.

[0187] In some embodiments, one or more CH1 amino acid residues selected for substitution (i) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cκ structures and have a fractional solvent-accessible surface area greater than 10% in at least 90% of the representative set of CH1:Cκ structures; (ii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a fractional solvent-accessible surface area greater than 10% in at least 90% of the representative set of CH1:Cλ structures; or (iii) are located at the interface with CH1:C κ and / or CH1:Cλ at the interface of VH in at least 10% of a representative set of structures, and in CH1:C κ and / or CH1:C λ the fractional solvent accessible surface area is greater than 10% in at least 90% of a representative set of structures.

[0188] The methods described herein can further include verifying that one or more substituted CH1 amino acid residues drive preferential pairing of the heavy chain to the κCL domain (or light chain comprising the κCL domain) relative to the λCL domain (or light chain comprising the λCL domain), and vice versa. A variety of methods can be used to evaluate preferential light chain pairing, including but not limited to fluorescence activated cell sorting (FACS), LC-MS, αLISA, and SDS-PAGE. In some embodiments, one or more CH1 domain positions selected for substitution in step c) occur at the interface of light chains 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, one or more CH1 domain positions selected for substitution in step c) have a fractional solvent accessible surface area greater than about 10% in at least about 90% or more of the structures in any given Cκ or Cλ set. In some embodiments, one or more CH1 domain positions selected for substitution in step c) occur at the interface of VH regions with a predetermined frequency, e.g., in any given set of wild-type antibody structures, the selected CH1 domain positions contact VH in at least 10% of the structures.

[0189] By employing the methods described herein for identifying CH1 domain variants, the following CH1 domain positions are 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). Substituting any one or combination of these CH1 domain positions can result in the CH1 domain having preferential pairing to a particular CL domain. Thus, a heavy chain comprising such a CH1 domain variant and a light chain comprising a particular CL domain are more likely to form a homologous pair, i.e., there is preferential pairing between the heavy chain and the light chain, and the heavy chain and the light chain form a homologous pair driven at least in part by substitution of one or more CH1 domains.

[0190] In one embodiment, the CH1 domain variant preferentially pairs with Cκ, thus driving preferential pairing of the light chain containing the Cκ domain and the heavy chain containing the CH1 domain variant. In another embodiment, the CH1 domain variant preferentially pairs with the Cλ domain, thus driving preferential pairing of the light chain containing the Cλ domain and the heavy chain containing the CH1 domain variant. Certain exemplary CH1 domain substitutions have been identified to promote preferential heavy chain pairing with κ light chains, such as 147F and / or 183R, 183K or 183Y, while other CH1 domain substitutions have been identified to promote preferential heavy chain pairing with λ light chains, such as 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 generate improved fidelity in heavy chain-light chain pairing. In some embodiments, the bispecific antibody comprises a first heavy chain comprising CH1λ (such as 141D, 141E or 141K and 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 (such as 147F and / or 183R, 183K or 183Y), each of the heavy chains preferentially pairing with its cognate light chain. In some embodiments, the bispecific antibody comprises a first heavy chain comprising CH1κ (such as 147F and / or 183R, 183K or 183Y) and a second heavy chain comprising CH1λ (such as 141D, 141E or 141K and 170E, 170G, 171E, 171D, 171G, 171S or 175M and / or 181K, 181B, 184R, 185R, 187R, 218A and / or 218P), such as "141D, 171E and 185R" or "141D, 170E and 187R", each of the heavy chains preferably pairing with its cognate light chain.

[0191] Polypeptides encoding CH1 variant domains obtained by the methods described herein can be recombinantly expressed in host cells, such as eukaryotic cells. 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 κ light chains and one or more wild-type λ light chains.

[0192] The examples provided below are intended to illustrate the invention. These examples are not intended to limit the invention to any specific application or theory of operation.

[0193] Examples

[0194] Example 1: Computer Selection of Diverse CH1 Domain Positions in a Library

[0195] A set of Fab crystal structures was assembled from the Protein Data Bank (PDB) and used in a structure-guided method to identify CH1-CL interface residues for diversification.

[0196] The set of initial 2,367 Fab crystal structures was narrowed by selecting structures with a high percentage identity to reference (wild-type) CH1, Cκ, and Cλ sequences (shown below). The reference sequence for the CH1 alignment spanned the appropriate CH1 (EU residues 118 - 215) plus a portion of the IgG1 hinge (EU residues 216 - 229). The Cκ and Cλ reference sequences spanned EU residue numbers 108 - 214 and 107A - 215, respectively.

[0197] CH1 (plus upper to middle hinge) reference:

[0198] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (SEQ ID NO:1).

[0199] Cκ reference:

[0200] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).

[0201] Cλ reference:

[0202] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:3).

[0203] If a pair of side-chain atoms is within the cutoff distance, the residues are defined as being in "contact". CH1 interface residues are defined as those that contact one or more Cκ or Cλ residues in a single structure.

[0204] The solvent-accessible surface area (SASA) of individual heavy and light chain residues was calculated in the "free state", i.e., without pairing to the light and heavy chains, respectively. The fractional SASA was defined as the ratio of the residue SASA to that of a Gly-X-Gly tripeptide model isolated with the same amino acid as the residue (i.e., X). Solvent-exposed residues were defined as those with a fractional SASA greater than 10%.

[0205] Narrowing an initial set of crystal structures by high percent identity led to the identification of a set of 183 CH1:Cκ structures ("Cκ set") and 43 CH1:Cλ structures ("Cλ set"). After considering gaps in the alignment due to missing amino acids in the structures, all entries in the Cκ set had 100% identity to the reference CH1 and Cκ sequences, while entries in the Cλ set had >99% identity to the reference sequences.

[0206] A structure-based sequence alignment between Cκ and Cλ is shown below. CH1 forms a stable interface with Cκ and Cλ, despite low sequence identity between the latter domains. According to the BLOSUM62 scoring, conserved and semi-conserved substitutions are depicted using "|" and ":", respectively. The sequence identity between the domains is 38.3% (41 identities out of 107 Cκ residues).

[0207] Cκ: -RTVAAP S V F I FP P SDEQ LK S G T A S V V C L L NN FYPREAKVQWKVDNALQSGNS Q E S V T AAPSV|FPPS:E|L:::A::VCL|::FYP:V WK:D:::::|::

[0208] Cλ: GQPKAAPSV T L FP P SSEE LQ A N K A T L V C L I SD FYPGAVTVAWKADSSPVKAGV E T TT PCκ: EQ D SKDSTY S L S ST L T LSKADYEKHKVYACEVTHQGLSSPVT K SFNRG E C-(SEQ ID NO:2)

[0209] :::S::Y:SS L:L::|::H|Y:C|VTH|G S|V K:EC

[0210] Cλ: S K QS N-NKY A A S S Y L S LTPEQWKSHRSYSCQVTHEG—-STVE K TVAPT E CS(SEQ ID NO:3)

[0211] The underlined amino acids represent the Cκ and Cλ residues that contact the CH1 domain. This determination is based on a consensus of Fab structures focused on Cκ and Cλ. There are 25 Cκ interface residues and 26 Cλ interface residues. A 2×2 matrix was constructed that focused on positions at the interface of Cκ or Cλ (N = 28) and depended on (1) whether the residue at a given position contacts the CH1, and (2) whether the amino acid at that position is the same between Cκ and Cλ (see Table 1).

[0212] Table 1. Cκ and Cλ Amino Acid Positions at the CH1:CL Interface

[0213]

[0214] Table 1 highlights a structurally conserved set of 14 Cκ and Cλ positions, i.e., the same EU residue number but with different amino acid identities, that contact the CH1 domain. Table 2 lists the 14 amino acid positions (EU numbers) and shows the amino acids present in the κ and λ light chains. Such differences in Cκ and Cλ interface residue identities can be exploited to generate mutant CH1 domains that bind specifically to either Cκ or Cλ but not both.

[0215] Table 2. Structurally Conserved CH1 Contact Positions with Non-Identical Amino Acid Residues for Cκ and Cλ

[0216]

[0217]

[0218] As an initial threshold for selecting library variants, individual CH1 domain positions need to meet the following criteria: 1) the position is at the interface of the light chain constant domain in at least 10% of the structures of the Cκ set, and the fractional SASA of the residue at the position is greater than 10% in at least 90% of the structures of the Cκ set; or 2) the position is at the interface of the light chain constant domain in at least 10% of the structures of the Cλ set, and the fractional SASA of the residue at the position is greater than 10% in at least 90% of the structures of the Cλ set; or 3) the position is at the interface of the VH region in at least 10% of the structures of the CH1:Cκ set (Cκ set) or CH1:Cλ set (Cλ set), and the fractional SASA of the residue at the position is greater than 10% in at least 90% of the structures of the Cκ and / or Cλ sets. The interface definition takes into account contacts of CH1 residues with any CL domain residues, i.e., includes but is not limited to a set of fourteen CL domain residues listed in Table 2 or contacts of CH1 residues with any VH residues.

[0219] Based on this threshold criterion, a set of thirty CH1 amino acid positions were identified for potential inclusion (after excluding Cys220). From this larger set, a set of 25 CH1 positions were selected for variation in the library. Amino acid diversity at each position was generated using degenerate NNK codons representing all 20 natural amino acids (Stemmer et al., Proceedings of the National Academy of Sciences of the United States of America, October 25, 1994; 91(22):10747 - 51). Amino acid substitutions were made individually at each of the 25 CH1 positions, and subsets of single substitutions were selectively combined, for example, to generate double mutants and triple mutants. The final library design consisted of 89 CH1 oligonucleotides representing 25 singlets (NNK codon diversification at a single CH1 position), 48 doublet mutants (NNK codon diversification at two CH1 positions), and 16 triplet mutants (NNK codon diversification at three CH1 positions).

[0220] Example 2: Library of CH1 domain variants in yeast co-expressing Cκ and Cλ light chains

[0221] 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 different expression levels to allow subsequent selection of Cλ-preferred CH1 substitutions and Cκ-preferred CH1 substitutions).

[0222] Two-way expression plasmids (pAD7064 and pAD4800) were constructed. Each of the plasmids contains a Saccharomyces cerevisiae Gal1 / Gal10 promoter region flanked by wild-type human IgG light chain κ and λ constant domains, as well as the Saccharomyces cerevisiae URA3 gene (selectable marker). Plasmids pAD7064 and pAD4800 differ in the orientation of the κ and λ constant domains relative to the Gal1 / 10 promoter region. Unique restriction enzyme sites (PME-I and SFI-I) were placed upstream of the κ and λ constant domains in each plasmid. pAD7064 and pAD4800 were digested with PME-I and SFI-I, respectively, and then co-transformed into an engineered yeast strain together with PCR-amplified DNA inserts (ADI-26140 light chain region; Gal1 / 10 promoter region; and differentially encoded ("degenerate") ADI-26140 light chain variable region (IDT gblock) with 5' and 3' ends that is directed to assemble into the plasmid by homologous recombination). The transformed yeast was inoculated onto solid agar plates lacking URA3+, grown at 30 °C for 48 hours, and then clones were picked and DNA was extracted and purified. After sequencing, two double light chain DNA constructs were identified: (1) Gal10::ADI-26140-VL-CκGal1::ADI-26140-VL-Cλ (human Cλ under the control of a dominant promoter, allowing subsequent selection of Cκ-preferred CH1 replacement); and (2) Gal10::ADI-26140-VL-CλGal1::ADI-26140-VL-Cκ (human Cκ under the control of a dominant promoter, allowing subsequent selection of Cλ-preferred CH1 replacement). ADI-26140 is an anti-hen egg lysozyme (HEL) IgG.

[0223] For heavy chain expression, a DNA vector (pAD4466) was constructed, which contains a Gal1 promoter, an SFI-I restriction site, the CH2-CH3 domains of human IgG heavy chain (IgG1 (N297A)), and TRP1 (selectable marker).

[0224] In parallel, two independent pools of CH1 domain variant DNA fragments were generated for insertion into pAD4466. The first pool was generated using the computer-aided design method described in Example 1. The second pool was generated by error-prone PCR (ePCR). Briefly, mutagenic nucleotide analogs dPTP (0.01 mM) and 8-oxo-DGTP (0.01 mM) were included in the PCR reaction at dilutions of (a) 1:100 and 1:100 or (b) 1:100 and 1:10, respectively.

[0225] pAD4466 was digested with Sfi-I and introduced into a yeast strain expressing Cκ and Cλ, along with PCR-amplified DNA encoding the variable regions of ADI-26140HC and CH1 domain variant DNA from rational design work or ePCR. Each DNA fragment had appropriate DNA sequences at the 5' and 3' ends to direct assembly (by homologous recombination) with the digested plasmid or PCR fragment (ADI-26140 heavy chain variable region or CH1 protein domain).

[0226] Assembly of individual libraries was carried out by the native Saccharomyces cerevisiae homologous recombination method. Dilutions of the transformed cells of each library were plated on media lacking uracil and tryptophan to quantify the number of members of each library. The number of members of each library was greater than 10 7 members. The remainder of the transformed cells was cultured in liquid media lacking uracil and tryptophan to select for the presence of each (HC and dual LC) plasmid.

[0227] Example 3: Identification of the position of the CH1 domain affecting light chain binding

[0228] Libraries were generated as previously described (see, e.g., WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng Des Sel. October 2013; 26(10):663 - 70). Briefly, after induction and presentation of IgG, yeast cells (about 10^7 - 10^8) were stained at 4°C with 1:100 diluted goat anti-human F(ab')2κ-FITC (Southern Biotech, Birmingham, Alabama, catalog number 2062 - 02) and 1:100 diluted goat anti-human F(ab')2λ-PE (Southern Biotech, Birmingham, Alabama, catalog number 2072 - 09) in PBSF for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL PBSF and transferred to a filter-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined so as to (1) increase the λ light chain while correspondingly losing the κ light chain ( Figure 2A ) or (2) increase the κ light chain while correspondingly losing the λ light chain ( Figure 2B ). After three rounds of selection, the yeast was plated on media lacking uracil and tryptophan to generate single isolates for sequence identification.

[0229] Single clones representing unique sequences were cultured in 96-well plates. After induction and presentation of IgG, approximately 2 × 10 6 yeast cells were stained for 15 minutes at 4 °C with goat anti-human F(ab')2κ-FITC (SouthernBiotech, Birmingham, AL, catalog number 2062-02) diluted 1:100 in PBSF and goat anti-human F(ab')2λ-PE (SouthernBiotech, Birmingham, AL, catalog number 2072-09) diluted 1:100. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.1 mL wash buffer and evaluated on a BD FACS Canto instrument equipped with a 96-well plate handler. The ratio of the median fluorescence intensity (MFI) of anti-κ to anti-λ (κ:λ ratio) ( Figure 3 ) for individual unique clones was scored and then compared to a matched strain with the wild-type CH1 sequence (“parent”) to calculate the FOP.

[0230] The following CH1 domain positions (EU numbering) were identified as affecting light chain binding preference, i.e., preferential binding to the κCL domain (or light chain containing the κCL domain) or the λCL domain (or light chain containing the λCL domain): 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 of preferential κ light chains. Bold amino acid residues in the sequence column indicate substitution positions, i.e., amino acid substitutions different from the parent (SEQ ID NO:1). Table 4 provides a list of CH1 sequences identified from the selection of preferential λ light chains. Bold amino acid residues in the sequence column indicate substitution positions.

[0231] Table 3. CH1 domain sequences preferentially binding to Cκ

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] Table 4. CH1 domain sequences of the preferred binding to Cλ

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Δ indicates amino acid deletion.

[0247] Surprisingly, it was found that some CH1 amino acid substitutions located at the VH:CH1 interface rather than the CH1:light chain interface produced κ-binding preference in the three-chain system. Specifically, the mutant groups K147V+P151A and P151L+N201S (SEQ ID NO: 36 and 70, Table 3) returned κFOP values of 18.1 and 10.4, respectively. Although position CH1:147 is located at the CH1:LC interface, CH1:201 is not (it is fully solvent-exposed and not part of any domain interface); thus, the occurrence of the P151 substitution in these high FOP clones suggests the potential role of this position in determining κ over λ preference. Without wishing to be bound by theory, for the reasons discussed below, such distal mutations are thought to affect HC:LC pairing, and thus mutations at the VH:CH1 interface may be exploited for preferred κ over λ pairing.

[0248] First, P151 is part of the so-called "ball and socket joint" between the VH and CH1 domains (Lesk A.M. et al., Nature 1988 Sep 8; 335(6186):188-90; Landolfi N.F. et al., Journal of Immunology Feb 1, 2001; 166(3):1748-54). This joint has been hypothesized to regulate flexibility within the domains by its effect on the "elbow angle" between the antibody variable and constant domains (Stanfield R.L. et al., Journal of Molecular Biology (J Mol Biol.) Apr 14, 2006; 357(5):1566-74). Substitutions in the ball and socket joint can have functional consequences, as in the case of an anti-IFN-γ monoclonal antibody with reduced neutralizing activity due to a single amino acid substitution in this region (Landolfi N.F. et al., Journal of Immunology Feb 1, 2001; 166(3):1748-54). This effect has been attributed to altered flexibility and allosteric mechanisms rather than direct changes at the antigen-binding interface. Second, it is also known that Fabs with λ constant domains have a greater range of elbow angles relative to Fabs with κ domains (Stanfield R.L. et al., Journal of Molecular Biology Apr 14, 2006; 357(5):1566-74. doi:10.1016 / J.jmb.2006.01.023. Epub Jan 25, 2006). This hyperflexibility has been attributed to a single residue insertion in the so-called switch region between the VL and CL domains. Third, further analysis of Fab crystal structures (Adimab unpublished data) has revealed differences in atomic packing in the ball and socket joint region between κ and λ Fabs. Thus, the modulation of Fab flexibility by the ball and socket joint, together with the inherent differences between Fabs with κ and λ light chains, suggests a new mechanism for deriving differential κ and λ preferences through mutations at the VH:CH1 interface.

[0249] Example 4: Identification and characterization of CH1 domain variants with κ- or λ-preferred light chain pairing

[0250] Based on the MFI ratio between κ and λ, clones obtained from selections with increased Cκ and Cλ preferences were chosen for further characterization (see Figure 4)。Isolate and amplify DNA pools mutated to each of the 20 amino acids (NNK) at each position of interest (141, 147, or 183). These single-position targeted libraries were constructed in the manner previously described using appropriate light chain alkaline strains. Four libraries were constructed in the presence of variations at positions 141, 147, 183, or 147 + 183 in the CH1 domain. κ or λ preference selection was performed as described above. The output was sequenced as previously described, and κ or λ preference relative to the appropriate parent was quantified based on FACS to determine the amino acid substitutions that provide preferential pairing of the light chain κ or λ.

[0251] A number of CH1 domain variants with amino acid residue substitutions at each of positions 141, 147, and 183 were identified as having a pairing preference for the κCL domain (or a light chain containing the κCL domain) or the λCL domain (or a light chain containing the λCL domain). At position 141 in the CH1 domain, substitution with D, R, or Q (compared to wild-type A) increased the preferential pairing with the λCL domain (or a light chain containing the λCL domain) (i.e., a reduced κ:λ MFI ratio) (see Figure 5 ). At position 147 in the CH1 domain, substitution with F, I, T, Y, L, R, N, E, H, M, or Q (compared to wild-type K) increased the preferential pairing with the κCL domain (or a light chain including the κCL domain) (i.e., an increased κ:λ MFI ratio) (see Figure 5 ). Substitution at position 183 in the CH1 domain with R, K, Y, W, E, F, or Q (compared to wild-type S) increased the preferential pairing with the κCL domain (or a light chain containing the κCL domain) (i.e., an increased κ:λ MFI ratio) (see Figure 5 ). Table 5 shows the number of observed CH1 domain variants with specific amino acid substitutions that drive pairing preference.

[0252] Table 5. Amino acid substitutions observed in CH1 domain variants with light chain preference

[0253] Amino Acid Substitutions Observed Counts Light Chain Preference A141D 35 λ A141R 7 λ A141Q 5 λ K147F 24 κ K147I 5 κ K147T 3 κ K147Y 3 κ K147L 2 κ K147R 2 κ K147N 2 κ K147E 1 κ K147H 1 κ K147M 1 κ K147Q 1 κ S183R 19 κ S183K 11 κ S183Y 5 κ S183W 3 κ S183E 2 κ S183F 1 κ S183Q 1 κ

[0254] Next, the effects of the identified CH1 domain variants were evaluated on a control standard bispecific antibody (2 heavy chains × 2 light chains) in an IgG-like format (2 Fab regions connected to a dimeric Fc molecule at the N-terminus). VH-CH1 sequences derived from two approved clinical therapeutic antibodies: ustekinumab and panitumumab were used. 'Knob' (S354C; T366W) and 'hole' (Y349C; T366S; L368A; Y407V) mutations were introduced to promote the desired heterodimer pairing of the heavy chains. DNA plasmids were confirmed by Sanger sequencing before transfection into HEK293 cells using standard protocols.

[0255] Transfected HEK cells were cultured in CD optiCHO medium (Invitrogen) and on day 6 post-transfection, supernatants were collected and subjected to protein A-based affinity purification. Purified IgG was treated with ELISA (Genevis AB) to enzymatically cleave the Fab region from the Fc portion.

[0256] The purified Fab was subjected to LCMS to confirm the sequence of each IgG component (2 heavy chains x 2 light chains) and to determine the relative percentage 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 a polyclonal antibody (Applied Biosystems) maintained at 65°C. The HPLC was performed on an Agilent 1100 series HPLC with an R2 10 μm column (2.1×30 mm, 0.1 mL). After injection, the sample was eluted from the column using a 0.21 minute gradient of 2-95% acetonitrile at a flow rate of 2 mL / min (mobile phase A: H2O containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid). A diverter valve was used to load a total flow of 150 μL / min into a Bruker maXis 4G mass spectrometer. The mass spectrometer was operated in positive ion mode with an m / z range of 700 to 2500. The remaining source parameters were set as follows: the capillary was set to 5500 V, the nebulizer was set to 4.0 bar, the drying gas was set to 4.0 L / min, and the drying temperature was set to 200°C. The acquired MS spectra were analyzed using Bruker Compass data analysis version 4.1. The detection of intact Fab species was confirmed based on mass measurements compared to the theoretical sequence. The relative quantification of each species was calculated based on the intensity of each species' peak compared to the sum of all peak intensities.

[0257] When both heavy chains are wild-type, incorrect pairing occurs about 30% of the time; however, when the heavy chains include CH1 variant domains as described herein, there is a significant improvement in the correct pairing of heavy and light chains (see Figure 7 and Table 6). The Pani light chain is wild-type. The Uste light chain is a λ fusion. HC1 is pani; LC1 is paniκ; HC2 is uste; LC2 is usteλ. For example, when the first heavy chain (HC1) contains K147F and S183R / K / Y and the second heavy chain contains A141D (BsAbs 10, 12, and 14, respectively), the mismatch is reduced by at least half, i.e., it occurs only 6.8, 10.5, or 11% of the time. In fact, a single substitution at position 141 (141D) results in a 50% reduction in mismatch, i.e., 6.1% versus 3.1% for HC1-LC2 and 22.8% versus 9.9% for HC2-LC1 (BsAb2). Based on this, the applicant provides exemplary CH1 domain sequences with κ or λ light chain / CL domain preferences in Table 7.

[0258] Table 6. Percentage of heavy chain-light chain product formation

[0259]

[0260]

[0261] Table 7. CH1 domains with κ or λ chain preferences

[0262]

[0263] The expression and quality of the purified antibody were evaluated by size exclusion chromatography (SEC). Briefly, column chromatography (TSKgel Super SW3000 column) was monitored using an Agilent 1100 HPLC. The column was pre-treated with highly glycosylated and aggregated IgG to minimize the possibility of antibody-column interaction and equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride pH 6.8) before use. 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 monitored at a wavelength of 280 nm. The total assay time was approximately 11 minutes. The data were analyzed using ChemStation software. The SEC profiles confirmed that the CH1 domain substitutions had no effect on the variant profiles compared to the wild-type (data not shown).

[0264] The binding affinity and kinetics of the purified bispecific antibodies that bind to human IL-12B (Uste) and human EGFR (Pani) were measured to confirm that the CH1 variant domains do not affect target binding (see Figures 6A - 6E ). Using The QKe instrument (ForteBio) captured the bispecific IgG sample on the anti-hIgG Fc sensor tip and measured the binding kinetics to IL12B or EGFR (association rate: 180 seconds and dissociation rate: 180 seconds). BLI analysis was performed at 29 °C using 1× kinetics buffer (ForteBio) as the assay buffer. The anti-human IgG Fc capture (AHC) biosensor (ForteBio) was first pre-soaked in the assay buffer for more than five minutes. The bispecific IgG sample (5 μg / mL) was captured on the sensor for 300 seconds. Then the sensor was soaked in the assay buffer for 120 seconds to establish a baseline before measuring the binding to IL12B or EGFR protein (100 nM concentration). Dissociation of IL12B or EGFR was measured by moving the sensor into the assay buffer for 180 seconds. Stirring for all steps was 1000 rpm. Using reference subtraction, dissociation-based inter-step correction, 1-to-1 binding model, and global fitting (Rmax not linked by the sensor), kinetic parameters were generated by Data analysis software version 8.2.0.7. Association rate constant (ka), dissociation rate constant (kd), and equilibrium constant (K D ) values were assigned individually for each measurement result.

[0265] Example 5: 141×181×218 Library Construction and Selection

[0266] Additional CH1 amino acid substitutions that provide preferential pairing with the λCL domain were also identified. Based on previous selection data and structural analysis, a set of three CH1 positions (141, 181, and 218) were chosen for additional mutagenesis. Amino acid diversity at position 141 was generated by a degenerate codon RMW representing six naturally occurring amino acids (D, T, A, E, K, and N). Amino acid diversity at positions 181 and 218 was generated by a degenerate codon NNK representing all 20 naturally occurring amino acids. The library design included all possible combinations of amino acids at these three positions with a diversity of 2,400. Using the light chain strain and the λ light chain under the GAL10 promoter (GAL1::ADI-26140VL-Ck x GAL10::ADI-26140VL–Cl), this library was constructed in the manner described previously. λ-preference selection was performed by staining with anti-human κ-FITC and anti-human λ-PE antibodies, followed by multiple rounds of cell sorting, as described previously. The output (96 clones) was sequenced as described previously, and quantification of FACS-based λ-preference relative to the parental strain was performed. Wild type (“WT”) and the previously identified lead clone A141D were included in the analysis. Based on these data, the amino acid combinations and A141D that provided the greatest improvement in light chain λ preference relative to the parent were identified.

[0267] Figure 8 As determined by the FOP values, most of the output clones had a higher preference for pairing with the λ chain. Table 8 provides the FOP values of the λ:κ MFI ratios for the CH1 domain substitutions and Figure 8 the top 13 clones labeled in.

[0268] Table 8. The top 13 FOP values from the output clones

[0269] Amino Acid Residues at Positions 141, 181, and 218 FOP EIL 7.34 KKE 6.84 EKP 6.44 KLD 5.76 KKP 5.54 KKA 5.49 KKE 5.25 KKP 5.03 KKH 4.99 EKD 4.98 KKP 4.96

[0270] Analysis showed that substitutions with D, K, or E at position 141 paired with substitutions with K at position 181 and substitutions with L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W at position 218 occurred frequently in the output clones and increased λ light chain preference (increased λ:κ MFI ratio) relative to A141D. Figure 9 The individual and average FOP values measured in clones with D at position 141, K at position 181, and various amino acids at position 218 of CH1 are shown. The lead CH1 sequence was cloned back into the LC staining (this process was subsequently adopted in all assays) and the clones and λ preference were confirmed by calculating the FOP values in triplicate ( Figure 10 ).

[0271] Additional analysis generated nine unique candidate CH1 sequences for mammalian IgG production (see Table 9).

[0272] Table 9. CH1 domains with κ or λ chain preference

[0273]

[0274]

[0275] The nine candidate CH1 sequences, along with WT (i.e., "ASK") and A141D (i.e., "DSK"), were cloned into a mammalian expression vector by standard methods. To determine λ preference, plasmids representing the desired heavy chain, λ light chain, and κ light chain were transfected into HEK293 cells at a 2:1:1 plasmid ratio. The transfected HEK cells were cultured, and IgG was purified using the previously described protocol. Without wishing to be bound by theory, expressing approximately equal amounts of total heavy and light chain polypeptides (HC:κLC:λLC = "2:1:1" here resulting in total HC:total LC = 1:1) (i.e., no excess HC and no excess LC) appears to allow the inventors to avoid various biases, leading to visualization of the true κ or λ preference of the CH1 domain variants.

[0276] Quantification of λ preference of mammalian-produced IgG was performed by FACS. Figure 11 FACS plots were provided and Figure 12 and Table 10 provides the FOP values (λ:κ MFI) for the nine CH1 variants as well as WT and A141D (i.e., "DSK"). Figure 13 It is shown that when CH1 has a D at position 141, additional substitutions at position 181 or positions 181 and 218 further improve λ preference (based on the λ:κ MFI ratio).

[0277] Table 10. FOP values for nine CH1 variants

[0278] CH1 Substitutions (at 141, 181, and 218) FOP D_K_P 4.33 D_K_A 3.83 D_K_WT 3.57 K_WT_WT 2.24 E_WT_WT 2.04 K_K_WT 1.82 E_K_WT 1.70 D_WT_WT 1.61 K_K_P 1.24 K_K_E 1.18 WT_WT_WT 1.00

[0279] In addition, LCMS data of reduced full-length IgG was used to determine the relative amounts of λ and κ light chains in the purified IgG samples. Figure 14 The species % paired with κ light chain (LC) and the species % paired with λ light chain were compared.

[0280] Analysis of these data yielded three CH1 sequences (SEQ ID NO: 143, 142, and 141, having DKP, DKA, and DKK substitutions, respectively), with improved λ preference relative to the parental and previously identified lead sequence A141D.

[0281] To determine whether these CH1 sequences pair with the κ light chain, candidate CH1 heavy chain plasmids were transfected into HE293 cells with 1.) κ light chain or 2.) λ light chain. K147F S183R, WT, A141D, which have κ preference for CH1, were also included as controls. The transfected HEK cells were cultured and purified by standard methods. Linked heavy chain Fab and light chain Fab were generated from the purified IgG using previously described methods. Process yields were determined using standard methods and normalized relative to the WT process yield to calculate the "FOP" process yield. Based on the process yield FOP, when only κLC (but not λLC) was present, A141D, A141D S181K, A141D S181K K218A, and A141D S181K K218P all remained bound to κLC, but more binding occurred with λLC compared to κLC( Figure 15 ). Using BioRad CFX96 RT PCR, the FabTm of κ- and λ-Fab was measured by differential scanning fluorimetry( Figure 16 ). For each CH1 variant, the relative gain in Tm of the λ-paired Fab ("relative λTm gain" or "net λTm gain") was calculated as defined: [Change in Tm of the λ-paired variant Fab relative to the λ-paired WT Fab ("ΔλTm")] - [Change in Tm of the κ-paired variant Fab relative to the κ-paired WT Fab ("ΔκTm")]( Figure 17 ). As shown in Figure 17 , the relative λTm gain increased with additional substitutions at S181 or S181 and K218. Without wishing to be bound by theory, based on Figure 16 and 17 , the destabilization of κLC pairing appears to contribute to the relative λTm gain and increased pairing with λCL.

[0282] Example 6: 141xALL Library Construction and Selection

[0283] When paired with substitutions at position 141, additional libraries were constructed to sample additional residues in CH1 to drive λ-preferred binding. Six new libraries (LAD11522 - LAD11527) were designed to have up to three substitutions across three regions (DOR1, DOR2, and DOR3) of CH1 (Table 11). The six libraries together represent every possible set of substitutions that includes two substitutions within the three domains of interest paired with position 141. In all libraries, amino acid diversity at position 141 was generated by the degenerate codon RMW, and amino acid diversity at the other two variant positions was generated by the degenerate codon NNK. The libraries were constructed using previously described methods. λ-preferred selection was performed as previously described.

[0284] Table 11. Library Design and Construction

[0285]

[0286] Starting from after the second round of FACS selection, the selected output was separated to recover the CH1 diversity and recloned into the appropriate double-stranded light chain strain to restore the reduced κ light chain expression in the library. PCR amplification with appropriate primers and standard DNA purification was used to isolate the CH1 diversity. This pool of DNA fragments was then electroporated with the ADI-26140 heavy chain variable region and the plasmid was digested into the appropriate double-stranded light chain strain.

[0287] As previously described ( Figure 18 ), the output was sequenced and the quantification of FACS-based λ preference relative to the parental strain was determined. The previously identified lead clone A141DS181K K218P was included in the analysis. Based on these data, the amino acid combinations with the greatest improvement in light chain λ preferential pairing relative to the parental were determined.

[0288] The first 46 clones containing 28 unique CH1 sequences (Table 12) were expressed as IgG in yeast. The new CH1 sequences were compared with the WT, A141D (or "DSK") and some of the lead sequences from the 141×181×218 series (DKP, DKA, KKE, KKP and EKK) from Example 5 for the FOP values determined by flow cytometry (λMFI:κMFI)( Figure 19 ). Corresponding to the data points marked with arrows in Figure 19 , at least seven with CH1 sequences SEQ ID NO:155, 157, 159, 162, 163, 164 or 165 showed FOP values equal to or higher than those of the tested 141×181×218 lead sequences.

[0289] Table 12. 28 Unique CH1 Sequences with λ Preference from 141×ALL Sequences

[0290]

[0291]

[0292]

[0293] Example 7: Construction and Screening of 141×(170 / 171)×(185 / 187) Series

[0294] Analysis of the results in Example 6 yielded four new positions / residues of interest, including F170, P171, V185, and T187. Based on the amino acids frequently observed at positions 170, 171, 185, and 187 and at position 141 that produced high FOP values in previous studies (e.g., frequent E and D at position 141; frequent E at position 170 or 171 in the 141×ALL output; and frequent R at position 185 and / or 187 when position 141 was substituted and independently when substituted by position 171), 14 unique CH1 domain variants with up to three amino acid substitutions per CH1 domain (Table 13) were rationally designed as candidates for the lead λ preference substitution group. The 14 leader sequences 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.

[0295] Table 13. New CH1 sequences from the 141×(170 / 171)×(185×187) series

[0296]

[0297]

[0298] As described above, the heavy chain containing one of the 14 CH1 domain variant sequences was cloned into mammalian (HEK) cells that co-expressed κ and λ light chains (ratio of heavy chain (HC): λ light chain (LC): κ LC = 2:1:1, i.e., the HC:LC ratio was always 1:1). The wild type (ADI-26140 heavy chain), "A141D" variant, and "A141D_S181K_K218P" variant were also included as controls. The λ preference was determined using the same assay as described above.

[0299] The λ MFI to κ MFI ratio was evaluated by flow cytometry. The FOP values and individual FACS plots for the 14 leader sequences are provided in Table 14 and Figures 20 - 22 . The numbers in each plot are the sort # shown in Table 14. Among the 14 leader sequences, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed even higher FOP values than the leader sequence "A141D_S181K_K218P" identified in Example 5. Many of the other variants among the 14 leader sequences also showed higher FOP values compared to "A141D", and all 14 leader sequences showed higher FOP values compared to the wild type.

[0300] Table 14. FOP values of 14 CH1 variant leader sequences and controls (sorted based on FOP values)

[0301]

[0302]

[0303] The amounts of κ and λ LC in each sample were quantified using LCMS (Table 15 and Figure 23 ). Similar to the results of the FACS-based λ preference assessment, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed even higher λ chain % and even lower κ chain % compared to the leader sequence "A141D_S181K_K218P" identified in Example 6. Many other variants among the 14 leader sequences also showed higher λ % and lower κ % compared to "A141D", and all 14 leader sequences showed higher λ % and lower κ % compared to wild type.

[0304] Table 15. λLC% and κLC% measured by LCMS (with FOP values in Table 14)

[0305] Substitutions in CH1 %κLC %λLC Score A141D_P171E_V185R 4％ 96％ 4.71 A141D_F170E_T187R 6％ 94％ 3.29 A141D_S181K_K218P 9％ 91％ 2.90 A141E_V185R_T187R 10％ 90％ 2.30 A141E_P171E_V185R 10％ 90％ 2.29 A141D_F170E_V185R 15％ 85％ 2.18 A141D_V185R_T187R 15％ 85％ 2.11 A141E_F170E_T187R 12％ 88％ 2.00 A141D_V185R 18％ 82％ 1.76 A141E_V185R 20％ 80％ 1.70 A141D_P171E_T187R 19％ 81％ 1.68 A141E_P171E_T187R 20％ 80％ 1.60 A141D 28％ 72％ 1.47 A141D_T187R 31％ 69％ 1.37 A141E_F170E_V185R 24％ 76％ 1.31 A141E_T187R 27％ 73％ 1.18 WT 40％ 60％ 1.00

[0306] To determine whether the top two λ-preferred CH1 variants ("A141D_P171E_V185R" and "A141D_F170E_T187R") pair with κ light chains, the CH1 variant heavy chain plasmids were transfected into HEK293 cells with 1.) κ light chain or 2.) λ light chain (heavy chain:light chain ratio = 1:1). K147F S183R, WT, which is κ-preferred for CH1, was also included as a control. The transfected HEK cells were cultured and the IgG was purified using a protein A column by standard methods. The process yield (mg / L) was determined using standard methods and normalized relative to the WT process yield. Based on the normalized process yield, "A141D_P171E_V185R" and "A141D_F170E_T187R" still bound to κLC when only κLC (but not λLC) was present, but more binding occurred to λLC compared to κLC ( Figure 30 ).

[0307] The process yields of the Fab format were also evaluated. IgG with CH1 variant heavy chains was generated and purified using the same method. K147F S183R, WT, A141D, and A141D S181K K218P with κ preference in CH1 were also included as controls. Using standard methods, linked heavy chain Fab and light chain Fab were generated from the purified IgG via papain enzymatic digestion and CH1 column purification. The normalized Fab digest was calculated as the % recovery of Fab recovered from the IgG digest normalized to the % parental recovery per light chain (% amount of recovered Fab / amount of IgG in the digest). The process yields were determined using standard methods and normalized relative to the WT process yield. Consistent with Figure 15 the data, the process yields of "A141D" and "A141D S181K K218P" with λ LC were higher than those with κ LC, and "K147F S183R" showed extremely high κ preference ( Figure 31 ). When only κ CH1 (but not λ CH1) was present, "A141D_P171E_V185R" and "A141D_F170E_T187R" still bound to κ CH1, but the yields obtained with λ LC were significantly higher than those obtained with κ LC ( Figure 31 ). Adding "P171E_V185R" or "F170E_T187R" to the "A141D" mutation further enhanced the λ preference of "A141D".

[0308] Example 8: Structural analysis of "A141D" and "K147F S183R" variants

[0309] Method

[0310] Crystallization and structure determination of panitumumab wild-type CH1-Cλ

[0311] Panitumumab wild-type CH1-constant lambda (Cλ) Fab protein at 6.5 mg / ml was centrifuged at 14,000×g for 5 minutes at 4 °C. 305 nL of the protein was mixed with 150 nL of the reservoir droplet and 50 nL of the seed solution and equilibrated at 20 °C in a MRC 3-well plate with 40 μl of the reservoir solution. Seeds identified from BCS screening (molecular size) were used for 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.) crystallization experiments to obtain crystals grown in 0.1 M phosphate / citrate pH 5.5 and 36% (v / v) PEG Smear Low and transferred to 0.1 M phosphate / citrate pH 5.5, 38% PEG Smear Low and 4% glycerol and then flash frozen in liquid nitrogen. Collected at 100 K at Diamond Light Source beamline I03 in Didcot, UK, on an Eiger2 XE 16M detector (DECTRIS). Diffraction data. The data sets were integrated in autoPROC (Vonrhein, C. et al. (2011) "Data processing and analysis with the autoPROC toolbox" Acta Crystallographica D67, 293 - 302.) using the following: XDS (Kabsch W. (2010) "XDS" Acta Crystallographica Section D - Biological Crystallography 66, 125 - 132.), and scaled using Aimless from the CCP4 software suite (Evans P.R. and Murshudov, G.N. (2013) "How good are my data and what is the resolution" Acta Crystallographica Section D - Biological Crystallography 69, 1204 - 1214.) (Winn M.D. et al. (2011) "Overview of the CCP4 suite and current developments" Acta Crystallographica Section D - Biological Crystallography 67, 235 - 242.235 - 242.). The crystal consists of 2 molecules per asymmetric unit (ASU) in the P1211 space group.Structure solution using the automated molecular replacement system MoRDA (Vagin A. and Lebedev A. (2015) “MoRDa, an automatic molecular replacement pipeline” Acta Crystallogr. A. 71, s19.) (incorporating MOLREP (Vagin A., Teplyakov A. (1997) “MOLREP: an automated program for molecular replacement” J. Appl. Cryst. 30, 1022–1025.) and Refmac5 (Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F. and Vagin, A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355–367.)), the system selected Protein Data Bank (Berman H.M. et al. (2000) “The Protein Data Bank” Nucleic Acids Res., 28.) entries 5N7W and 5SX4 as initial search models. Automated model building was completed using the BUCCANEER software (Cowtan K. (2006) “The Buccaneer software for automated model building. 1. Tracing protein chains” Acta Crystallogr. D62, 1002–1011.).The model was refined to the final R and R by manual refinement in Coot (Emsley P., Lohkamp, B., Scott, W.G. and Cowtan K. (2010) “Features and development of Coot” Acta Crystallographica Section D - Biological Crystallography 66, 486 - 501.) and refinement improvements in Refmac5 (Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F. and Vagin, A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallographica Section D - Biological Crystallography 67, 355 - 367.) and Buster (Bricogne G, Blanc E, Brandl M, Flensburg C, Keller P, Paciorek W, Roversi P, Sharff A, Smart O, Vonrhein C, Womack T. (2011). BUSTER version 2.11.7. Global Phasing Ltd, Cambridge, United Kingdom.). 游离 14.5% and 16.9% respectively ( Figure 32 ).

[0312] Crystallization and structure determination of panitumumab A141D CH1 - Cλ, wild - type CH1 - Cκ and K147F - S183R CH1 - Cκ

[0313] Panitumumab A141D CH1-Cλ, panitumumab wild-type CH1-constant κ (Cκ), and panitumumab K147F-S183R CH1-Cκ Fab were centrifuged at 14,000×g for 5 minutes 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 drops, and 50 nL of seed solution was equilibrated with 40 ul of reservoir solution. Seed crystals identified from BCS screening were used in the MMS experiment to find the optimal crystallization conditions. 0.1 M phosphate / citrate buffer pH 5.5 and 36% (v / v) PEG Smear Low were used for panitumumab A141D CH1-Cλ and 0.1 M sodium acetate pH 4.5 with 30% v / v PEG Smear Low for panitumumab K147F-S183R CH1-Cκ. 150 nL of 19.2 mg / ml wild-type CH1-Cκ was mixed with 150 nL of reservoir drops and added to 40 ul of reservoir solution and screened using a PACT kit (molecular size). The final crystallization conditions consisted of 0.1 M MES pH 6.0 and 0.2 M calcium chloride dihydrate with 20% w / v PEG 6000. Crystals were transferred to cryo-solutions consisting of: 0.1 M phosphate / citrate buffer pH 5.5, 38% PEG Smear Low, 4% glycerol; 0.07 M MES, pH 6.0, 21% PEG6000, 0.2 M CaCl2, 23.5% glycerol; and 0.1 M 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 collected at 100 K at Diamond Light Source beamline I03 in Didcot, UK, equipped with an Eiger2 XE 16M detector (Dectris). Crystallographic data for the resolution. The data were indexed and integrated in iMOSFLM (Battye, T.G.G., Kontogiannis, L., Johnson, O., Powell, H.R., and Leslie, A.G. (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, P.R. and Murshudov, G.N. (2013) "How good is my data and what is the resolution?" Acta Crystallographica Section D - Biological Crystallography 69, 1204 - 1214.) as follows: CCP4 suite (Winn, M.D. et al. (2011) "Overview of the CCP4 suite and current developments" Acta Crystallographica Section D - Biological Crystallography 67, 235 - 242. 235 - 242.).

[0314] 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 restraint refinement were carried out in Refmac5 (Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallographica Section D: Biological Crystallography, 67, 355 - 367.), where the TLS factors were applied in the last few rounds of refinement. The site occupancies of A141D CH1 - Cλ were assigned based on those of wild - type CH1 - Cλ and were manually adjusted in Coot during iterative refinement (Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010) "Features and development of Coot" Acta Crystallographica Section D: Biological Crystallography, 66, 486 - 501.). The R and R 游离 values of the final structure solved in P1211 with 2 molecules per ASU were 15.2% and 17.0% ( Figure 33 ).

[0315] The structures of panitumumab wild-type CH1-Cκ and K147F-S183R-CH1-Cκ were solved by molecular replacement carried out with Phaser (McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C. and Read, R.J. (2007). Phaser crystallographic software. Journal of Applied Crystallography, 40(4), 658-674.), using the coordinates of panitumumab Fab fragments complexed with EGFR (PDB code 5SX4) and the solved wild-type CH1-Cκ structure, respectively, followed by iterative manual model building with Coot (Emsley P., Lohkamp, B., Scott, W.G. and Cowtan K. (2010) "Features and development of Coot" Acta Crystallographica Section D - Biological Crystallography 66, 486-501.) and automatic refinement in Refmac5 (Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F. and Vagin, A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallographica Section D - Biological Crystallography 67, 355-367.). Translation non-crystallographic symmetry was observed for the wild-type CH1-Cκ structure, and thus the structure was solved in the lower space group (P1211) with 6 Fab molecules in the ASU. The structure was refined to final R and R 游离 values of 19.8% and 23.2% respectively ( Figure 34 ). The K147F-S183R CH1-Cκ structure was solved to final R and R 游离 values of 19.8% and 23.3% respectively ( Figure 35 ).

[0316] Structural analysis and interpretation

[0317] λLC preference mediated by HC-A141D

[0318] Without wishing to be bound by theory, the enhanced λ preference of panitumumab A141D CH1-Cλ may be mediated by an intermolecular hydrogen bond formed between the side-chain carboxyl of HC-Asp141 and the side-chain hydroxyl of λLC-Thr116 ( Figure 36 C), which cannot form with HC-Ala141 in panitumumab wild-type CH1-Cλ ( Figure 36A). The κLC region around HC-Ala141 consists of the hydrophobic residues Phe116, Phe118, and Leu135, while κLC-Phe116 is replaced by the polar residue Thr116 in λLC ( Figure 36 B). Thus, introducing a charge via the A141D mutation can reduce κ preference by disrupting the hydrophobicity of the CH1-κLC interface while stabilizing the CH1-λLC pairing through hydrogen bonding with λLC-Thr116. Additionally, without wishing to be bound by theory, κ preference can be further reduced by steric hindrance between HC-Asp141 and κLC-Phe116, as shown by the alignment of panitumumab A141D CH1-Cλ and wild-type CH1-κLC ( Figure 36 D).

[0319] In the hydrogen bond between HC-Asp141 and λLC-Thr116, the 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. Thus, another amino acid with a hydrogen acceptor atom in its side chain can also form a hydrogen bond with Thr116 of λLC, providing λ preference. Based on the fact that the side chain of glutamic acid also has a hydrogen acceptor atom (O) and glutamic acid is similar to aspartic acid in size and shape, glutamic acid may form a hydrogen bond with Thr116 of λLC while causing steric hindrance with κLC as shown in Figure 36 D, overall providing λ preference. Indeed, the A141E substitution provides strong λ preference as demonstrated in the examples above, confirming the applicant's structural analysis.

[0320] κLC preference mediated by HC-K147F-S183R

[0321] The observed κ preference of panitumumab K147F-S183R CH1-Cκ can 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 sequesters HC-Gln175, contributing to the baseline κ pairing preference ( Figure 37 A). One explanation is that replacing CH1 HC-Lys147 with phenylalanine at this position disrupts this network and releases the HC-Gln175 side chain, which interacts with κLC by hydrogen bonding with the formamide oxygen of κLC-Gln160, thus increasing κ preference ( Figure 37 B). Additionally, without wishing 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 37 B, Figure 38C). In contrast, without wishing to be bound by theory, the hydrogen bonding observed between HC-Ser183 and λLC-Tyr178 at the HC 183 position in wild-type panitumumab CH1-Cλ was eliminated by severe steric hindrance between HC-Arg183 and the side chain of λLC-Tyr178 in the modeled pairing of K147F-S183R CH1 and λLC, destabilizing the λ pairing in favor of κLC( Figure 38 B and 38D).

[0322] In the hydrogen bond between HC-Arg183 and κLC-Thr178, the 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. Thus, another amino acid with a hydrogen donor atom in its side chain can also form a hydrogen bond with Thr178 of κLC, providing κ preference. Larger side chains such as that of Arg can help generate steric hindrance with Tyr178 of λLC, thus providing additional κ preference. For example, the side chains of both lysine and tryptophan have large side chains containing a hydrogen donor atom (H). Thus, lyase and tryptophan may form a hydrogen bond with Thr178 of κLC and may experience steric hindrance with λLC as shown in Figure 38 D, overall providing κ preference. The side chain of threonine can also act as a hydrogen donor through the H atom of -OH. Thus, the applicant further conceives that an amino acid with a relatively large side chain that can act as a hydrogen acceptor can also form a hydrogen bond with Thr178 of κLC to provide κ preference. For example, glutamate, glutamine, histidine, or tyrosine with a relatively large side chain (with a hydrogen acceptor atom) can also provide κ preference when placed at residue 183 of HC. In fact, most of these newly proposed amino acid substitutions at residue 183 were actually identified as κ preference in Example 3 (see Table 3).

[0323] As described above, substituting Lys147 with Phe disrupts the hydrogen bond between Lys147 and Gln175, thus releasing Gln175 for hydrogen bonding with Gln160 of κLC and thus contributing to κ preference. Thus, substituting Lys147 with another amino acid whose side chain does not contain a hydrogen donor or acceptor atom, such as alanine, glycine, isoleucine, leucine, or valine, may also contribute to κ preference. In fact, most of these newly proposed amino acid substitutions at residue 147 were actually identified as κ preference 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 the following 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, Optionally, the CH1 domain variant polypeptide is caused to preferentially pair with: (i) a kappa light chain constant region ("CL") domain, as compared to a lambda CL domain, and / or a kappa light chain polypeptide, as compared to a lambda light chain polypeptide; or (ii) a lambda CL domain, as compared to a kappa CL domain, and / or a lambda light chain polypeptide, As compared with kappa light chain polypeptides; With the proviso that one or more of the following substitution combinations are optionally excluded: (a) if residue 141 of CH1 is substituted with C or L, residue 166 is substituted with D or K, residues 128, 129, 162, or 171 of CH1 are substituted with C, and / or residue 147 is substituted with D, then the CL does not comprise an amino acid substitution; (b) if position 126 or 220 on CH1 is substituted with valine or alanine, The non-cysteine residue at position 128, 141, or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F, or V185W / F, then the CL does not include an amino acid substitution; (c) if residue 172 on CH1 is substituted to 172R, residue 174 is mutated to 174G, or residue 190 is substituted to 190M or 190I, these are not the only CH1 substitutions; (d) if 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, then CL is unmodified; (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 containing the CH1 domains are of the same human immunoglobulin subtype or allotype; (f) if the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), there is no corresponding LC substitution at 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) if the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, then there is no corresponding LC substitution; (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 there is no corresponding LC substitution; (j) if the CH1 substitution consists of 143A / E / R / K / D and 145T / L, then there is no corresponding LC substitution; (k) if the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, then there are no corresponding LC substitutions; (1) if the CH1 substitution consists of substitution by cysteine at position 126, 127, 128, 134, 141, 171 or 173, the corresponding LC position is not modified to form a disulfide bond; (m) if the CH1 substitution consists of L145Q, H168A, F170G, S183V and / or T187E, then there are no corresponding κ or λ LC substitutions; (n) if the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there are no corresponding CL substitutions; or (o) CH1 substitutions consist of A140C, K147C and / or S183C, with corresponding CL substitutions present.

2. The CH1 domain variant polypeptide of claim 1 , comprising an amino acid substitution at one or more of the following positions according to EU numbering: 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, Optionally, the CH1 domain variant polypeptide is caused to preferentially pair with: (i) a kappa CL domain, as compared to a lambda CL domain; and / or (ii) kappa light chain polypeptides, as compared to lambda light chain polypeptides. 3 . The CH1 domain variant polypeptide according to claim 2 , comprising an amino acid substitution at position 147, position 183, or positions 147 and 183.

4. The CH1 domain variant polypeptide according to 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 by H; f. Position 129 is substituted by P; g. Position 131 is substituted by A; h. Position 132 is substituted by P; i. Position 134 is substituted with G; j. Position 136 is substituted by E; k. Position 139 is substituted by I; l. Position 143 is substituted by 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 replaced by L or S; r. Position 151 is substituted with A or L; s. Position 153 is replaced by S; t. Position 154 is substituted with M or G; u. Position 170 is substituted with G or L; v. Position 172 is replaced by V; w. Position 175 is substituted by G, L, E, A; x. Position 176 is substituted by 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 by W; bb. Position 190 is substituted by P; cc. Position 191 is substituted by I; dd. Position 197 is substituted by A; ee. Position 201 is replaced by S; ff. Position 203 is replaced by S; gg. Position 204 is substituted by Y; hh. Position 205 is substituted by Q; ii. Position 206 is substituted by S; jj. Position 210 is replaced by R; kk. Position 212 is replaced by G; ll. Position 213 is substituted by E or R; mm. Position 214 is substituted with R; and nn. Position 218 is substituted by Q.

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

6. The CH1 domain variant polypeptide according to any one of claims 2 to 5, comprising: (i) amino acid residue R, K or Y at position 183; and / or (ii) amino acid residue F at position 147.

7. The CH1 domain variant polypeptide according to any one of claims 2 to 6, comprising: (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, The CH1 domain variant polypeptide optionally comprises the following amino acid sequence: (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.

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

9. A CH1 domain variant polypeptide, which comprises: (i) the amino acid residue F at position 147 and the amino acid residue R at position 183; (ii) the amino acid residue F at position 147 and the amino acid residue K at position 183; (iii) the amino acid residue F at position 147 and the amino acid residue Y at position 183; (iv) the amino acid residue R at position 183; (v) the amino acid residue K at position 183; or (vi) the amino acid residue Y at position 183.

10. The CH1 domain variant polypeptide according to claim 9, which comprises the amino acid substitutions consisting of: (i) the amino acid residue F at position 147 and the amino acid residue R at position 183; (ii) the amino acid residue F at position 147 and the amino acid residue K at position 183; (iii) the amino acid residue F at position 147 and the amino acid residue Y at position 183; (iv) the amino acid residue R at position 183; (v) the amino acid residue K at position 183; or (vi) the amino acid residue Y at position 183.

11. The CH1 domain variant polypeptide according to claim 10, which comprises the following amino acid sequences: (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.

12. The CH1 domain variant polypeptide according to any one of claims 2 to 11, which further comprises one or more amino acid substitutions that increase the pairing of the CH1 domain with: (i) the κCL domain, as compared to the λCL domain; and / or (ii) the κ light chain polypeptide, as compared to the λ light chain polypeptide.

13. The CH1 domain variant polypeptide according to any one of claims 2 to 11, which pairs with: (i) the κCL domain, as compared to the λCL domain; and / or (ii) the κ light chain polypeptide, as compared to the λ light chain polypeptide, increasing 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%, optionally as measured by liquid chromatography - mass spectrometry (LCMS), or 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 as measured by flow cytometry, optionally by comparing the ratio of mean fluorescence intensity (MFI) of kappa CL staining to lambda CL staining.

14. The CH1 domain variant polypeptide of claim 1 , comprising an amino acid substitution at one or more of the following positions according to EU numbering: 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, Optionally, the CH1 domain variant is preferentially paired with: (i) a lambda CL domain, as compared to a kappa CL domain; and / or (ii) a lambda light chain polypeptide, as compared to a kappa light chain polypeptide. 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. The CH1 domain variant polypeptide according to claim 14 or 15, comprising one or more of the following amino acid substitutions: a. Position 119 is replaced by R; b. Position 124 is replaced by V; c. Position 126 is substituted with V; d. Position 127 is substituted with G; e. Position 130 is substituted by 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 by 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 replaced by M; n. Position 152 is replaced by G; o. Position 163 is substituted by 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 by 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 by L or V; Position 184 is replaced by R; Position 185 is replaced by M, L, S, R or T; Position 187 is replaced by R, D, E, Y or S; Position 197 is replaced by S; Position 203 is replaced by D; Position 208 is replaced by I; Position 210 is replaced by T; Position 211 is replaced by A; Position 212 is replaced by N; Position 213 is replaced by E; Position 214 is replaced by R; Position 216 is replaced by G; and Position 218 is replaced by L, E, D, P, A, H, S, Q, N, T, I, M, G, C, K or W.

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

23. The CH1 domain variant polypeptide of any one of claims 14 to 17, comprising a substitution at position 141 to D, K or E 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. The CH1 domain variant polypeptide according to any one of claims 14 to 17, comprising any one or more of (i)-(ix): (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 / or (ix) amino acid residue P, A or E at position 218.

25. The CH1 domain variant polypeptide according to any one of claims 14 to 17, comprising: (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 K at position 141; (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 E 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, amino acid residue V at position 181, amino acid residue R at position 184, and amino acid residue R at position 187; (xviii) amino acid residue E at position 141 and amino acid residue R at position 185; (xix) amino acid residue E at position 141 and amino acid residue R at position 187; (xx) amino acid residue E at position 141, amino acid residue E at position 170, and amino acid residue R at position 185; (xxi) amino acid residue E at position 141, amino acid residue E at position 170, and amino acid residue R at position 187; (xxii) amino acid residue D at position 141 and amino acid residue R at position 185; (xxiii) 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; The CH1 domain variant polypeptide optionally comprises the following amino acid sequence: (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)SEQ ID NO:

189.

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

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

143.

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

29. The CH1 domain variant polypeptide of any one of claims 14 to 28, which pairs with: (i) a lambda CL domain, as compared to a kappa CL domain; and / or (ii) a lambda light chain polypeptide, as compared to a kappa light chain polypeptide, 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 Increased 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, optionally such as by flow cytometry, optionally measured by comparing the MFI value ratio of λCL staining to κCL staining.

30. 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 one of claims 1 to 29, and the antibody heavy chain polypeptide optionally further comprises one or more amino acid substitutions outside the CH1 domain, and the one or more amino acid substitutions further promote the preferential pairing of the heavy chain with the following: (I) (i) a κCL domain, as compared to a λCL domain, and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide; or (II) (i) a λCL domain, as compared to a κCL domain, and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide.

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

32. 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 homologous pair; and (b) the first heavy chain polypeptide comprises a first CH1 domain variant, the first CH1 domain variant comprising an amino acid substitution at one or more of the following 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, such that the first CH1 domain variant preferentially binds to the first light chain; optionally wherein the first light chain polypeptide comprises a first CL domain, and the first CL domain is a wild-type CL domain; provided that optionally one or more of the following substitution combinations in the first CH` domain are excluded: (a) 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, then the CL does not comprise an amino acid substitution; (b) If position 126 or 220 on CH1 is substituted with valine or alanine, a non-cysteine at position 128, 141 or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F or V185W / F, then the CL does not include amino acid substitution; (c) If residue 172 is substituted with 172R, residue 174 mutates to 174G, or residue 190 is substituted with 190M or 190I, then these are not the only CH1 substitutions; (d) If 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, then the CL is not modified; (e) If the CH1 substitution consists of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S and / or 193F / L, then these are not the only CH1 substitutions and / or in a bispecific antibody, the CH1 domain containing has the same human immunoglobulin subtype or allotype; (f) If the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), then there is no corresponding LC substitution at 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) If the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, then there is no corresponding LC substitution; (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 there is no corresponding LC substitution; (j) If the CH1 substitution consists of 143A / E / R / K / D and 145T / L, then there is no corresponding LC substitution; (k) If the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, then there is no corresponding LC substitution; (l) If the CH1 substitution consists of being substituted with cysteine at positions 126, 127, 128, 134, 141, 171 and / or 173, then the corresponding LC positions are not modified to form disulfide bonds; (m) If the CH1 substitution consists of L145Q, H168A, F170G, S183V and / or T187E, then there is no corresponding κ or λ LC substitution; (n) if the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there are no corresponding CL substitutions; or (o) CH1 substitutions consist of A140C, K147C and / or S183C, with corresponding CL substitutions present.

33. The antibody or antibody fragment of claim 32, 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 comprises a second CHI domain variant comprising an amino acid substitution at one or more of the following 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, such that the second CHI domain variant preferentially binds to the second light chain polypeptide comprising a second CL domain, with the proviso that one or more of the following substitution combinations in the second CH1 domain are optionally excluded: (a) if residue 141 of CH1 is substituted with C or L, residue 166 is substituted with D or K, residues 128, 129, 162, or 171 of CH1 are substituted with C, and / or residue 147 is substituted with D, then the CL does not comprise an amino acid substitution; (b) if position 126 or 220 on CH1 is substituted with valine or alanine, the non-cysteine at position 128, 141 or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F or V185W / F, then the CL does not include an amino acid substitution; (c) if residue 172 is substituted to 172R, residue 174 is mutated to 174G, or residue 190 is substituted to 190M or 190I, these are not the only CH1 substitutions; (d) if 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, then CL is unmodified; (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 containing the CH1 domains are of the same human immunoglobulin subtype or allotype; (f) if the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), there is no corresponding LC substitution at 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) if the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, then there is no corresponding LC substitution; (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 there is no corresponding LC substitution; (j) if the CH1 substitution consists of 143A / E / R / K / D and 145T / L, then there is no corresponding LC substitution; (k) if the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, then there are no corresponding LC substitutions; (1) if the CH1 substitution consists of substitution by cysteine at position 126, 127, 128, 134, 141, 171 or 173, the corresponding LC position is not modified to form a disulfide bond; (m) if the CH1 substitution consists of L145Q, H168A, F170G, S183V, and T187E, then there are no corresponding κ or λ LC substitutions; (n) if the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there are no corresponding CL substitutions; or (o) CH1 substitutions consist of A140C, K147C, and / or S183C, with corresponding CL substitutions present; Further optionally wherein the antibody or antibody fragment comprises one or more of features (i)-(ix): (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 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 substitution in the first CH1 domain variant is different from the amino acid substitution in the second CH1 domain variant.

34. 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 claims 2 to 13; and (c) The first light chain polypeptide comprises a κ CL domain and is optionally a κ light chain polypeptide, Optionally, wherein: (i) The κ 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, wherein the first heavy chain polypeptide comprises one or more amino acid substitutions outside the CH1 domain, and the one or more amino acid substitutions further promote the preferential pairing of the heavy chain with: (i) The κ CL domain, as compared to the λ CL domain, and / or (ii) The κ light chain polypeptide, as compared to the λ light chain polypeptide.

35. 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 homologous 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 λ CL domain and is optionally a λ light chain polypeptide; Optionally, wherein: (i) The λ 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, wherein the second heavy chain polypeptide comprises one or more amino acid substitutions outside the CH1 domain, and the one or more amino acid substitutions further promote the preferential pairing of the heavy chain with: (i) The λ CL domain, as compared to the κ CL domain, and / or (ii) The λ light chain polypeptide, as compared to the κ light chain polypeptide.

36. 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 homologous pair; (b) The first heavy chain polypeptide comprises a first CH1 domain, and the first CH1 domain comprises a CH1 domain variant according to any one of claims 2 to 13; (c) The first light chain polypeptide comprises a κ CL domain and is optionally a κ light chain polypeptide; (d) The second heavy chain polypeptide and the second light chain polypeptide form a second homologous pair; (e) The second heavy chain polypeptide comprises a second CH1 domain, and the second CH1 domain comprises a CH1 domain variant according to any one of claims 14 to 29; and (f) The second light chain polypeptide comprises a λ CL domain and is optionally a λ light chain polypeptide, Further optionally, wherein the first heavy chain polypeptide comprises one or more amino acid substitutions outside the CH1 domain, and the one or more amino acid substitutions further promote the preferential pairing of the heavy chain with: (i) The κ CL domain, as compared to the λ CL domain, and / or (ii) The κ light chain polypeptide, as compared to the λ light chain polypeptide, And further optionally wherein the second heavy chain polypeptide comprises one or more amino acid substitutions outside of the CH1 domain, said one or more amino acid substitutions further promoting preferential pairing of the heavy chain with: (i) a lambda CL domain, as compared to a kappa CL domain, and / or (ii) a lambda light chain polypeptide, as compared to a kappa light chain polypeptide.

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

38. The antibody or antibody fragment of claim 32 or 36, which is multispecific, wherein the first CH1 domain variant and the second CH1 domain variant: (i) reducing the formation of non-homologous heavy chain-light chain pairs 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-fold .5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 21 times, at least 22 times, at least 23 times, at least 24 times, or at least 25 times; (ii) provides 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 homologous pairs; (iii) providing for about 85% to about 95% formation of the desired first homologous pair and the second homologous pair; and / or (iv) reduces the formation of non-cognate heavy chain-light chain 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%, Optionally wherein 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 features (i)-(iv): (i) said first CH1 domain variant comprises a substitution at position 147 and / or 183 and reduces the formation of non-cognate heavy chain-light chain pairs 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 the formation of non-cognate heavy chain-light chain pairs 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 the formation of non-cognate heavy chain-light chain pairs by at least about 50% to at least about 75%; or (iv) 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 provides for about 85% to at least about 95% formation of the desired first and second homologous pairs.

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

41. The antibody or antibody fragment of claim 33, 36, 38 or 39, which is multispecific and wherein: (a) said first CH1 domain variant comprises amino acid substitutions consisting of: (i) amino acid residue F at position 147; and / or (ii) amino acid residue R, K or Y at position 183; and (b) said second CH1 domain variant comprises an amino acid substitution 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; (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187; or (iii) amino acid residue D at position 141, amino acid residue K at position 181, and amino acid residue P at position 218.

42. The antibody or antibody fragment of claim 33 or 36, wherein: (a) The first CH1 domain variant comprises the following amino acid sequence: (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; and (b) the second CH1 domain variant comprises the following amino acid sequence: (i) SEQ ID NO: 188; (ii) SEQ ID NO: 186; or (iii) SEQ ID NO:

143.

43. The antibody or antibody fragment of any one of claims 40 to 42, wherein the first CH1 variant and the second CH1 variant: (i) reducing the formation of non-cognate heavy chain-light chain pairs by at least 50% to at least 75%; and / or (ii) providing for about 85% to at least about 95% formation of the desired first homologous pair and the second homologous pair.

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

45. A method for generating a CH1 domain variant library, the method comprising: (a) Provide (i) one or more sets ("C κ sets") consisting of a polypeptide comprising a CH1 domain paired with a polypeptide comprising a κCL domain and / or (ii) one or more sets ("C λ sets"), optionally wherein the polypeptide comprising a CH1 domain further comprises a heavy chain variable region (VH), and further optionally wherein the polypeptide comprising a κ or λCL domain further comprises a light chain variable region (VL); (b) selecting the CH1 domain and (i) the C κ The κCL domain is concentrated, (ii) the C λ concentrated in the λCL domain and / or (iii) the C κ Set and / or the C λ One or more amino acid positions that contact one or more amino acid positions in the VH of the focus; 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 wherein: (I) in step (a), the CH1 domain, the κCL domain and the λCL domain are wild-type and / or human; (II) in step (a), both (i) the polypeptide comprising a CH1 domain paired with the polypeptide comprising a κCL domain and (ii) the polypeptide comprising a CH1 domain paired with the polypeptide comprising a λCL domain are intact antibodies or antigen-binding fragments ("Fab"); (III) In step (b), one or more amino acid positions of the CH1 domain are selected when the amino acid residues at the one or more amino acid positions of the CH1 domain have side chain atoms within the distance: (i) the side chain atoms of the amino acid residues at the one or more amino acid positions in the κCL domain; (ii) the side chain atoms of the amino acid residues at the one or more amino acid positions in the λCL domain; and / or (iii) the side chain atoms of the amino acid residues at the one or more amino acid positions in the VH; and / or (IV) The generation in step (c) is performed by degenerate codons, optionally degenerate RMW codons representing 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 method of claim 45, wherein the one or more CH1 amino acid positions selected in step (b) are: (i) Located in the same area as the C κ At least 10% of the representative sets are at the interface of the κCL domain and at the C κ The fractional solvent accessible surface area in at least 90% of the representative sets of the set is greater than 10%; (ii) located in the same area as the C λ At least 10% of the representative sets are at the interface of the λCL domain and at the C λ The fractional solvent accessible surface area in at least 90% of a representative set of sets is greater than 10%; and / or (iii) located at the interface of the VH in at least 10% of the representative set of the C κ and / or C λ set, and the fractional solvent-accessible surface area is greater than 10% in at least 90% of the representative set of the C κ and / or C λ set.

47. The method of claim 45 or 46, wherein 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; With the proviso that one or more of the following substitution combinations are optionally excluded: (a) if residue 141 of CH1 is substituted with C or L, residue 166 is substituted with D or K, residues 128, 129, 162, or 171 of CH1 are substituted with C, and / or residue 147 is substituted with D, then the CL does not comprise an amino acid substitution; (b) if position 126 or 220 on CH1 is substituted with valine or alanine, the non-cysteine at position 128, 141 or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F or V185W / F, then the CL does not include an amino acid substitution; (c) if residue 172 is substituted to 172R, residue 174 is mutated to 174G, or residue 190 is substituted to 190M or 190I, these are not the only CH1 substitutions; (d) if 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, then CL is unmodified; (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 containing the CH1 domains are of the same human immunoglobulin subtype or allotype; (f) if the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), there is no corresponding LC substitution at 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) if the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, then there is no corresponding LC substitution; (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 there is no corresponding LC substitution; (j) If the CH1 substitution consists of 143A / E / R / K / D and 145T / L, there is no corresponding LC substitution; (k) If the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, there is no corresponding LC substitution; (l) If the CH1 substitution consists of substitution by cysteine at positions 126, 127, 128, 134, 141, 171 or 173, the corresponding LC position is not modified to form a disulfide bond; (m) If the CH1 substitution consists of L145Q, H168A, F170G, S183V and T187E, there is no corresponding κ or λ LC substitution; (n) If the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there is no corresponding CL substitution; or (o) The CH1 substitution consists of A140C, K147C and / or S183C, and there is a corresponding CL substitution.

48. The method according to any one of claims 45 to 47, wherein the library of CH1 domain variants in step (c) is expressed in: (I) A yeast strain; (II) Saccharomyces cerevisiae; and / or (III) A cell system co-expressing (i) one or more polypeptides comprising a κ CL domain and (ii) one or more polypeptides comprising a λ CL domain, optionally wherein the κ and / or λ CL domain is wild-type, Further optionally, the κ and / or λ CL domain is human.

49. A method for generating a library of CH1 domain variants, the method comprising: (a) Selecting one or more CH1 amino acid positions from the following CH1 amino acid positions numbered according to EU: 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 different from the positions selected in step (a); 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 amino acid positions selected in steps (a) and (b) are substituted with any non-wild-type amino acid, Optionally wherein: (I) The amino acid positions selected in (a) include position 141, 147, 151, 170, 171, 181, 183, 185, 187 or 218 or any combination thereof; (II) said generating in step (c) is performed by 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 by a degenerate codon, optionally wherein 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. A method of identifying one or more CH1 domain variant polypeptides that preferentially pair with: (A) a polypeptide comprising a kappa CL domain, as compared to a polypeptide comprising a lambda CL domain; or (B) A polypeptide comprising a lambda CL domain, as compared 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 according to any one of claims 44 to 49 with (i) one or more polypeptides comprising a κ CL domain and (ii) one or more polypeptides comprising a λ CL domain; (b) comparing (i) the amount of the candidate CH1 domain variant polypeptide paired with a polypeptide comprising a κ CL domain to (ii) the amount of the candidate CH1 domain variant polypeptide paired with a polypeptide comprising a λ CL domain; (c) based on the comparison in step (b), selecting one or more CH1 domain variants that provide preferential pairing with: (A) a polypeptide comprising a kappa CL domain, as compared to a polypeptide comprising a lambda CL domain; or (B) A polypeptide comprising a lambda CL domain, as compared to a polypeptide comprising a kappa CL domain, wherein in step (a), optionally the total amount of said candidate CH1 domain variant polypeptide expressed and the total amount of said polypeptide comprising expressed (κ and λ)CL domains are approximately the same, Optionally wherein 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. 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. The method of claim 50 or 51, wherein in step (b) the amount is determined by fluorescence activated cell sorting or by liquid chromatography-mass spectrometry.

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

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