Variant CH1 and CL domains engineered for preferential chain pairing and multispecific antibodies containing same

Abstract

Variant CH1 domains, variant CL domains, and variant CH1-CL domain sets are provided that contain at least one amino acid substitution that promotes preferential CH1-CL pairing. Also provided are polypeptides, molecules, and multispecific antibodies that contain such variants, as well as compositions that include any of the foregoing. Also provided are methods of generating variant CH1 and / or variant CL domain libraries, and methods of using the libraries to identify one or more variant CH1 and / or variant CL domains and / or variant CH1-CL domain sets. Additionally provided are methods of screening for suitable combinations of CH1-CL sets for multispecific antibodies and / or antigen-binding antibody fragments.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 136,091, filed January 11, 2021, entitled "CH1 AND KAPPA CL DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL CHAIN ​​PAIRING AND MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME," the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to variant CH1 domain and variant CL domain polypeptides comprising at least one amino acid substitution that promotes preferential chain pairing between a heavy chain comprising the variant CH1 domain and a light chain comprising the variant CL domain, polypeptides, molecules, and multispecific antibodies or antigen-binding antibody fragments comprising such variants, and compositions comprising any of the foregoing. The present invention further relates to polynucleotides encoding such variant CH1 and / or CL domain polypeptides, molecules, multispecific antibodies, or antigen-binding antibody fragments comprising the variant CH1 and / or CL domain polypeptides, and compositions and libraries comprising any of the foregoing. The present invention further relates to methods of generating variant CH1 and / or CL domain libraries and methods of using the libraries to identify one or more variant CH1 and / or CL domains and libraries, and methods for identifying two polypeptides that preferentially pair with each other. [Background technology]

[0003] There are ongoing efforts to develop antibody therapeutics with more than one antigen-binding specificity, such as bispecific antibodies. Bispecific antibodies can be used to block multiple surface receptors associated with cancer, autoimmune diseases, inflammation, or other diseases and conditions. Bispecific antibodies can also be used to bring targets into close proximity, regulate protein complex formation, or drive cell-cell contact. The production of bispecific antibodies was first reported in the early 1960s (Nisonoff et al., Arch Biochem Biophys 1961 93(2):460-462), and the first monoclonal bispecific antibodies were generated using hybridoma technology in the 1980s (Milstein et al., Nature 1983 305(5934):537-540). Interest in bispecific antibodies has grown significantly over the past decade due to their therapeutic potential, and bispecific antibodies are now in clinical use; for example, blinatumomab and emicizumab have been approved for the treatment of certain 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 production methods and characteristics of bispecific antibodies approved for pharmaceutical use).

[0004] While bispecific antibodies have demonstrated significant advantages over monospecific antibodies, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, their lack of stability, and their lack of a long half-life in humans. Bispecific antibodies can be formed by coexpressing two different heavy chains and two different light chains. However, because heavy chains bind to light chains in a relatively promiscuous manner, coexpression of two heavy chains and two light chains can result in a mixture of 16 possible combinations, representing 10 different antibodies, only one of which corresponds to the desired bispecific antibody (if complete promiscuity exists, the maximum yield in the mixture is 12.5%). This mismatch (also known as the linkage association problem) poses a major challenge in producing bispecific antibodies, and various technologies have been developed to address this issue.

[0005] One strategy used to mitigate such chain mispairing is to design bispecific antibodies with a common light chain, i.e., two different heavy chains and two identical light chains (see, e.g., Merchant et al., Nat. Biotech. 16:677-681 (1998)). However, this strategy requires identifying two antibodies with different specificities but the same light chain, i.e., differing only in the heavy chain, which is difficult and tends to compromise the specificity of each binding arm, significantly reducing diversity (see, e.g., Wang et al., MABS 10(8):1226-1235 (2018)).

[0006] Another strategy is to modify the heavy chain constant region 1 ("CH1") domain or CH1 domain and the light chain constant region ("CL") domain to promote CH1 pairing with a light chain of a particular isotype (kappa ("κ") or lambda ("λ")). For example, a kappa CL domain ("CLκ")-preferring CH1 domain (which may be referred to as a "CH1κ") preferentially pairs with a CLκ domain (or a variant CLκ domain) rather than a CLλ domain (or a variant CLλ domain), and a lambda CL domain ("CLλ")-preferring CH1 domain (which may be referred to as a "CH1λ") preferentially pairs with a CLλ domain (or a variant CLλ domain) rather than a CLκ domain (or a variant CLκ domain). Much technical effort has been devoted to combining CH1 and CLκ domains to promote proper heavy chain pairing. Although several CH1 and / or CLκ domain modifications have been reported that purportedly increase the propensity for preferential pairing between a given CH1 domain and a CLκ domain over pairing of a variant CH1 domain with another CL domain and / or pairing of a variant CLκ domain with another CH1 domain, previous techniques appear to have drawbacks, such as not being universally applicable to multispecific antibodies combining different specificities, not achieving sufficiently preferential CH1-CLκ pairing, requiring the incorporation of multiple substitutions in the CH1 domain and / or the CLκ domain, and / or the need to incorporate additional substitutions in the variable regions to achieve highly preferential CH1-CLκ pairing. Thus, despite the foregoing, there remains a need for improvement, particularly in light of the recent clinical focus on generating multispecific, e.g., bispecific, antibodies or antigen-binding antibody fragments for use in human therapy. Summary of the Invention

[0007] It is an object of the present invention to provide engineered variant CH1 domain polypeptides, or heavy chains comprising such variant CH1 domain polypeptides, that can preferentially pair with a given CL domain or variant CL domain polypeptide, or with a light chain comprising such a CL domain or variant CL domain polypeptide. The variant CH1 domain polypeptides according to the present invention can be incorporated into polypeptides, molecules, or antibodies or antigen-binding antibody fragments, such as multispecific (e.g., bispecific) antibodies or antigen-binding antibody fragments.

[0008] In one aspect, provided herein are variant immunoglobulin heavy chain constant region 1 ("CH1") domain polypeptides (also referred to herein as variant CH1 domains), and also provided herein are heavy chain polypeptides comprising such variant CH1 domain polypeptides.

[0009] In some embodiments, such a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant CLκ or CLλ domain polypeptide rather than with another given CL domain polypeptide (a WT CLκ or CLλ domain polypeptide, or another variant CLκ or CLλ domain polypeptide), or a light chain comprising such a variant CL domain polypeptide.

[0010] In some embodiments, the variant CH1 domain polypeptide comprises at least one amino acid substitution (e.g., relative to a parent, e.g., wild-type sequence, such as SEQ ID NO: 1, or an allelic variant thereof, such as, but not limited to, SEQ ID NO: 3).

[0011] In some embodiments, the variant CH1 domain polypeptide may comprise an amino acid substitution, which may comprise or consist of an amino acid substitution at one or more of the following CH1 amino acid positions according to EU numbering: 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187. (Also, in each instance in this application where applicant refers to a specific position in an immunoglobulin polypeptide, the position is according to EU numbering unless otherwise specified.) Optionally, the variant CH1 domain polypeptide is a variant of the CH1 domain of human IgG, further optionally human IgG1, human IgG2, or human IgG4.

[0012] In some embodiments, variant CH1 domain polypeptides may include one or more additional amino acid substitutions at CH1 positions outside of positions 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and / or 187. In some cases, such additional positions may optionally be selected from the CH1 positions listed in Table 1.

[0013] In some embodiments, such a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant immunoglobulin kappa light chain constant region (CLκ) or lambda light chain constant region (CLλ) domain polypeptide, or a light chain polypeptide comprising a variant CLκ or CLλ domain, rather than with another given immunoglobulin light chain constant region (CL) domain or variant CL domain polypeptide (a wild-type (WT) CLκ or CLλ domain polypeptide, or another variant CLκ or CLλ domain polypeptide), or rather than with a light chain comprising a wild-type or another given variant CL domain polypeptide.

[0014] In some embodiments, the variant CLκ or CLλ domain polypeptide, or the light chain comprising such a variant CL domain polypeptide, to which such a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, preferentially pairs may comprise at least one amino acid substitution, which may comprise or consist of an amino acid substitution at one or more of the following CLκ or CLλ amino acid positions according to EU numbering: 114, 120, 124, 127, 129, 133, 135, 137, 138, 178, and / or 180.

[0015] In some embodiments, such variant CH1 domain polypeptides are variants of the CH1 domain of human IgG, and optionally human IgG1, human IgG2, or IgG4.

[0016] Such variant CH1 domain polypeptides may not be part of the existing CH1-CL set listed in Table 1.

[0017] In some embodiments, the amino acid substitutions in the variant CH1 domain polypeptide may comprise or consist of amino acid substitutions at: (I) positions 185 and / or 187; (II) positions 145, 147, and / or 148; (III) positions 147 or 148; (IV) positions 145; (V) positions 166 and / or 187; (VI) positions 145 and / or 147; or (VII) positions 124 and / or 147.

[0018] In further embodiments, such variant CH1 domain polypeptides, or heavy chain polypeptides comprising such variant CH1 domain polypeptides, may preferentially pair with variant CLκ or CLλ domain polypeptides, or light chain polypeptides comprising such variant CL domain polypeptides, and the variant CL domain polypeptides may comprise at least one amino acid substitution, and the amino acid substitution position in the variant CL (CLκ or CLλ) domain polypeptide may comprise or consist of an amino acid substitution at: (I) position 135, (II) position 124, (III) position 129, (IV) position 133, (V) positions 137 and / or 138, (VI) positions 178 and / or 180, or (VI) position 127.

[0019] In some embodiments, the combination of substitution positions in the CH1-CL set may follow the combination of substitution positions in any one of the CH1-CLκ sets in Table 2 or any one of the CH1-CLλ sets in Table 28.

[0020] In some embodiments, the amino acid substitutions in the variant CH1 domain polypeptide are at any combination of the following positions: (i) 145, 147, and 181; (ii) 128 and 147; (iii) 168, 185, and 187; (iv) 147 and 185; (v) 148; (vi) 139, 141, and 187; (vii) 166 and 187; (viii) 168 and 185. (ix) 124 and 147; (x) 147 and 148; (xi) 145; (xii) 145 and 181; (xiii) 124, 145, and 147; (xiv) 166 and 187; (xv) 147 and 175; (xvi) 147, 175, and 181; (xvii) 145 and 147; or (xviii) 147 and 185.

[0021] In further embodiments, such variant CH1 domain polypeptides, or heavy chain polypeptides comprising such variant CH1 domain polypeptides, may preferentially pair with variant CLκ domain polypeptides, or light chain polypeptides comprising such variant CLκ domain polypeptides, optionally with amino acid substitution positions in such variant CLκ domain polypeptides at: (i) positions 129, 178, and 180; (ii) positions 124, 133, and 178; (iii) position 135; (iv) positions 135 and 178; (v) positions 124 and 129; (vi) positions 114, 135, and 138; (vii) positions 137 and 138; (viii) positions 135; (ix) positions 127 and 129; (x) positions 127 and 129; (xi) positions 133 or 124 and 133; (xii) positions 133 or 120, 178, and 180; (xiii) positions 127, 129, and 178; (xiv) positions 114, 137, and 138; (xv) positions 129, 178, and 180; (xvi) positions 129 and 180; (xvii) positions 133 and 180; or (xviii) positions 129 and 180.

[0022] In further embodiments, such variant CH1 domain polypeptides, or heavy chain polypeptides comprising such variant CH1 domain polypeptides, may preferentially pair with variant CL λ domain polypeptides, or light chain polypeptides comprising such variant CL λ domain polypeptides, and optionally, amino acid substitution positions in such variant CL λ domain polypeptides are: (i) positions 129, 178, and 180; (ii) positions 133 and 178; (iii) position 135; (iv) positions 135 and 178; (v) positions 129, 178, and 180; (xiii) positions 127, 129, and 178; (xiv) positions 114, 137, and 138; (xv) positions 129, 178, and 180; (xvi) positions 129 and 180; (xvii) positions 133 and 180; or (xviii) positions 129.

[0023] In still further embodiments, such variant CH1 domain polypeptides may comprise one or more of the following amino acid substitutions: 124R, 128R, 139R, 141Q, 145Q, 145S, 147E, 147H, 147N, 147Q, 147R, 147T, 148E, 148R, 166K, 168R, 168S, 175D, 175E, 181E, 181Q, 185E, 185Q, 185S, 185Y, 187D, 187K, and / or 187Q.

[0024] In certain embodiments, the amino acid substitutions in such variant CH1 domain polypeptides are: (i) 145Q, 147E, and 181E; (ii) 128R and 147R; (iii) 168S, 185S, and 187D; (iv) 147T and 185Q; (v) 148R; (vi) 139R, 141Q, and 187Q; (vii) 166K and 187K; (viii) 168R and 185E; (ix) 124R and 147R; (x) 147H and 148E, (xi) 145S, (xii) 145S and 181Q, (xiii) 145S, (xiv) 145Q and 181E, (xv) 124R, 145S, and 147Q, (xvi) 166K and 187K, (xvii) 147R and 175D, (xviii) 147R, 175E, and 181Q, (xix) 145S and 147N, or (xx) 147N and 185Y.

[0025] In certain embodiments, a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant CLκ domain polypeptide, or a light chain polypeptide comprising such a variant CLκ domain polypeptide, optionally with amino acid substitutions in the variant CLκ domain polypeptide selected from the group consisting of: (i) 129R, 178R, and 180Q; (ii) 124E, 133Q, and 178E; (iii) 135R; (iv) 135S and 178R; (v) 124S and 129E; (vi) 114D; (xiv) 127T, 129D, and 178R; (xv) 114Q, 137T, and 138E; (xvi) 129D, 178R, and 180H; (xvii) 129D and 180Q; (xviii) 133Y and 180R; or (xix) 129R and 180S.

[0026] In certain embodiments, a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant CLλ domain polypeptide, or a light chain polypeptide comprising such a variant CLλ domain polypeptide, optionally with amino acid substitutions in the variant CLλ domain polypeptide, selected from the group consisting of: (i) 129R, 178R, and 180Q; (ii) 133Q and 178E; (iii) 135R; (iv) 135S and 178R; (v) 124S and 129 (xiv) 127T, 129D, and 178R, (xv) 114Q, 137T, and 138E, (xvi) 129D, 178R, and 180H, (xvii) 129D and 180Q, (xviii) 133Y and 180R, or (xix) 129R.

[0027] In certain embodiments, the amino acid substitutions in such variant CH1 domain polypeptides may comprise or consist of: (i) 145Q, 147E, and 181E; (ii) 128R and 147R; (iii) 168S, 185S, and 187D; or (iv) 147T and 185Q.

[0028] In certain embodiments, a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant CLκ domain polypeptide, or a light chain polypeptide comprising such a variant CLκ domain polypeptide, and optionally, the amino acid substitutions in the variant CLκ domain polypeptide may comprise or consist of: (i) 129R, 178R, and 180Q; (ii) 124E, 133Q, and 178E; (iii) 135R; or (iv) 135S and 178R.

[0029] In certain embodiments, a variant CH1 domain polypeptide, or a heavy chain polypeptide comprising such a variant CH1 domain polypeptide, may preferentially pair with a variant CLλ domain polypeptide, or a light chain polypeptide comprising such a variant CLλ domain polypeptide, and the amino acid substitutions in the variant CLλ domain polypeptide may include or consist of: (i) 129R, 178R, and 180Q; (ii) 133Q and 178E; (iii) 135R; or (iv) 135S and 178R.

[0030] In certain embodiments, the variant CH1 domain polypeptide may comprise an amino acid sequence according to any one of SEQ ID NOs: 31, 21, 11, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201.

[0031] In some preferred embodiments, the variant CH1 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 31, 21, 11, or 41.

[0032] In some embodiments, a heavy chain polypeptide according to the present disclosure may comprise any of the variant CH1 domain polypeptides described above.

[0033] Another object of the present invention is to provide engineered variant CL domain (e.g., variant CLκ or CLλ domain) polypeptides, or light chains comprising such variant CL domain polypeptides, that can preferentially pair with a given CH1 domain or variant CH1 domain polypeptide, or with a heavy chain comprising such a CH1 domain or variant CH1 domain polypeptide. The variant CLκ or CLλ domain polypeptides according to the present invention can be incorporated into polypeptides, molecules, or antibodies or antigen-binding antibody fragments, such as multispecific (e.g., bispecific) antibodies or antigen-binding antibody fragments.

[0034] In one aspect, provided herein are variant immunoglobulin CLκ or CLλ domain polypeptides (also referred to herein as variant CLκ or CLλ domain polypeptides, variant CLκ or variant CLλ, or equivalents), and also provided herein are light chain polypeptides comprising such variant CL domain polypeptides.

[0035] In some embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, may preferentially pair with the variant CHI domain polypeptide rather than with another given CHI domain (such as a WT CHI domain polypeptide or another variant CHI domain polypeptide), and / or may preferentially pair with a heavy chain polypeptide comprising the variant CHI domain polypeptide rather than with another heavy chain polypeptide comprising a wild-type or another given variant CHI domain polypeptide.

[0036] In some embodiments, a variant CLκ or CLλ domain polypeptide may comprise at least one amino acid substitution (eg, relative to a parent, e.g., wild-type sequence, such as SEQ ID NO: 2 or 9).

[0037] In some embodiments, a variant CLκ or CLλ domain polypeptide may comprise at least one amino acid substitution, which may comprise or consist of an amino acid substitution at one or more of the following amino acid positions (CL positions): 114, 120, 124, 127, 129, 133, 135, 137, 138, 178, and / or 180, according to EU numbering.

[0038] In some embodiments, a variant CLκ or CLλ domain polypeptide may comprise one or more additional amino acid substitutions at a CLκ position outside of positions 114, 120, 124, 127, 129, 133, 135, 137, 138, 178, and / or 180. In some cases, such additional positions may optionally be selected from the CLκ or CLλ positions listed in Table 1.

[0039] In some embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, may optionally preferentially pair with a variant CH1 domain polypeptide or a heavy chain comprising a variant CH1 domain polypeptide. In such embodiments, a variant CH1 domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, to which a heavy chain comprising a variant CLκ or CLλ domain polypeptide, or a CH1 domain polypeptide, preferentially pairs, may comprise at least one amino acid substitution which may comprise or consist of an amino acid substitution at one or more of the following positions according to EU numbering: 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and 187, provided that such a variant CLκ domain polypeptide may not be part of an existing CH1-CLκ set (i.e., a set other than CTL3l) listed in Table 1, and such a variant CLλ domain polypeptide may not be part of an existing CH1-CLλ set (i.e., a CTL3l set) listed in Table 1.

[0040] In some embodiments, the amino acid substitution in the variant CLκ or CLλ domain polypeptide may comprise or consist of an amino acid substitution at: (I) position 135, (II) position 124, (III) position 129, (IV) position 133, (V) position 137 and / or 138, (VI) position 178 and / or 180, or (VII) position 127.

[0041] In some embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, may preferentially pair with a variant CH1 domain polypeptide or a heavy chain comprising a variant CH1 domain polypeptide. In such embodiments, the amino acid substitution in the variant CH1 domain polypeptide may comprise or consist of an amino acid substitution at: (I) 185 and / or 187, (II) 145, 147, and / or 148, (III) 147 or 148, (IV) 145, (V) 166 and / or 187, (VI) 145 and / or 147, or (VII) 124 and / or 147.

[0042] In some embodiments, the combination of substitution positions in the CH1-CL set may include any one of the CH1-CLκ sets in Table 2 and / or any one of the CH1-CLλ sets in Table 28.

[0043] In some embodiments, the amino acid substitutions in the variant CLκ or CLλ domain polypeptide are at: (i) positions 129, 178, and 180; (ii) positions 124, 133, and 178, or positions 133 and 178; (iii) position 135; (iv) positions 135 and 178; (v) positions 124 and 129; (vi) positions 114, 135, and 138; (vii) positions 137 and 138; or (xiv) 129 and 180; (xv) 133 and 180; (xi) 120, 178, and 180; (xii) 127, 129, and 178; (xiii) 114, 137, and 138; (xiv) 129 and 180; (xv) 133 and 180; or (xvi) 129.

[0044] In some embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, may preferentially pair with a variant CH1 domain polypeptide or a heavy chain comprising a variant CH1 domain polypeptide. In such embodiments, the amino acid substitutions in the variant CH1 domain polypeptide are at (i) positions 145, 147, and 181, or 147 and 175, (ii) positions 128 and 147, (iii) positions 168 and 185, or 168, 185, and 187, (iv) positions 147 and 185, (v) position 148, (vi) positions 139, 141, and 187, (vii) positions 166 and 187, or (viii) positions 124 and 147 or (xiv) 147 and 185 or 147, 175 and 181; (xv) 145 and 147; or (xvi) 147 and 185.

[0045] In further embodiments, the variant CLκ or CLλ domain polypeptide may comprise one or more of the following amino acid substitutions: 114D, 114Q, 120S, 124E, 124S, 127D, 127R, 127T, 129D, 129E, 129R, 133Q, 133Y, 135R, 135S, 137S, 137T, 138E, 138R, 178E, 178H, 178R, and 180H, 180Q, 180R, and / or 180S.

[0046] In yet further embodiments, the amino acid substitutions in the variant CLκ or CLλ domain polypeptide are (i) 129R, 178R, and 180Q, (ii) 124E, 133Q, and 178E, or 133Q and 178E, (iii) 135R, (iv) 135S and 178R, (v) 124S and 129E, (vi) 114D, 135S, and 138R, (vii) 137S and 138E, or 138E, (viii) 135S, (ix) 127D and 129E, (x) 124S and 129E, (xiv) 120S, 178H, and 180Q; (xv) 127T, 129D, and 178R; (xvi) 114Q, 137T, and 138E; (xvii) 129D, 178R, and 180H; (xviii) 129D and 180Q; (xix) 133Y and 180R; or (xx) 129R and 180S, or 129R.

[0047] In certain embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain comprising such a variant CLκ or CLλ domain polypeptide, may preferentially pair with a variant CH1 domain polypeptide or a heavy chain comprising a variant CH1 domain polypeptide. In such embodiments, the amino acid substitutions in such variant CH1 domain polypeptides are: (i) 168S, 185S, and 187D, (ii) 128R and 147R, (iii) 145Q, 147E, and 181E, (iv) 147T and 185Q, (v) 148R, (vi) 139R, 141Q, and 187Q, (vii) 166K and 187K, (viii) 168R and 185E, or (ix) 124R and 147R. , (x) 147H and 148E, (xi) 145S, (xii) 145S and 181Q, (xiii) 145S, (xiv) 145Q and 181E, (xv) 124R, 145S, and 147Q, (xvi) 166K and 187K, (xvii) 147R and 175D, (xviii) 147R, 175E, and 181Q, (xix) 145S and 147N, or (xx) 147N and 185Y.

[0048] In some preferred embodiments, the amino acid substitutions in the variant CLκ or CLλ domain polypeptide may consist of: (i) 129R, 178R, and 180Q; (ii) 124E, 133Q, and 178E, 133Q and 178E; (iii) 135R; or (iv) 135S and 178R.

[0049] In some embodiments, a variant CLκ or CLλ domain polypeptide, or a light chain polypeptide comprising such a variant CLκ or CLλ domain polypeptide, preferentially pairs with a variant CH1 domain polypeptide or a heavy chain polypeptide comprising a variant CH1 domain polypeptide. In such cases, the amino acid substitutions in the variant CH1 domain polypeptide can include or consist of: (i) 168S, 185S, and 187D; (ii) 128R and 147R; (iii) 145Q, 147E, and 181E; or (iv) 147T and 185Q.

[0050] In certain embodiments, the variant CLκ or CLλ domain polypeptide may comprise an amino acid sequence selected from one of SEQ ID NOs: 32, 22, 12, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202, or any one of SEQ ID NOs: 59, 99, 39, 199, 89, 49, 29, 19, 69, 79, 109, 119, 129, 139, 149, 159, 169, 179, 189, or 209.

[0051] In some preferred embodiments, a variant CLκ or CLλ domain polypeptide may comprise an amino acid sequence selected from one of SEQ ID NOs: 12, 22, 32, 42, or one of SEQ ID NOs: 59, 99, 39, 199, 89, 49, or 29. In some embodiments, a light chain polypeptide according to the present disclosure may comprise any of the variant CL domain polypeptides described above.

[0052] Another object of the present invention is to provide a set of variant CH1 domain polypeptides and variant CLκ or CLλ domain polypeptides that preferentially pair with each other (such a set is a "variant CH1-CL set," "CH1-CL variant set," "CH1-CL design," "designed CH1-CL," "network," or the like). One or more CH1-CL sets according to the present invention can be incorporated into a polypeptide, molecule, or multispecific (e.g., bispecific) antibody or antigen-binding antibody fragment.

[0053] In one aspect, provided herein is a CH1-CL set (which may be a kit comprising a CH1 domain polypeptide and a CL polypeptide), which may comprise a variant CH1 domain polypeptide and / or a variant CLκ or CLλ domain polypeptide.

[0054] In some embodiments, a CH1-CL set according to the invention may comprise any of the variant CH1 domain polypeptides described above, and / or any of the variant CLκ or CLλ domain polypeptides described above.

[0055] In some embodiments, the CH1-CL set can be any of the CH1-CLκ sets listed in Table 2 or any of the CH1-CLλ sets listed in Table 28.

[0056] In certain embodiments, the variant CH1 domain polypeptides and variant CLκ or CLλ domain polypeptides of the CH1-CL set are set forth in SEQ ID NOs: 31 and 32, respectively, SEQ ID NOs: 21 and 22, respectively, SEQ ID NOs: 11 and 12, respectively, SEQ ID NOs: 41 and 42, respectively, SEQ ID NOs: 51 and 52, respectively, SEQ ID NOs: 61 and 62, respectively, SEQ ID NOs: 71 and 72, respectively, SEQ ID NOs: 81 and 82, respectively, SEQ ID NOs: 91 and 92, respectively, SEQ ID NOs: 101 and 102, respectively, SEQ ID NOs: 111 and 112, respectively, SEQ ID NOs: 121 and 122, respectively, SEQ ID NOs: 131 and 132, respectively, SEQ ID NOs: 141 and 142, respectively, SEQ ID NOs: 151 and 152, respectively, SEQ ID NOs: 161 and 162, respectively, SEQ ID NOs: 171 and 172, respectively, SEQ ID NOs: 181 and 182, respectively, SEQ ID NOs: 191 and 192, respectively, SEQ ID NOs: 201 and 202, respectively, SEQ ID NOs: 211 and 212, respectively, SEQ ID NOs: 213 and 214, respectively, SEQ ID NOs: 215 and 216, respectively, SEQ ID NOs: 217 and 218, respectively, SEQ ID NOs: 219 and 220, respectively, SEQ ID NOs: 221 and 222, respectively, SEQ ID NOs: 222 and 223, respectively, SEQ ID NOs: 22 91 and 192, or SEQ ID NOs:201 and 202, respectively, SEQ ID NOs:51 and 59, respectively, SEQ ID NOs:91 and 99, respectively, SEQ ID NOs:31 and 39, respectively, SEQ ID NOs:191 and 199, respectively, SEQ ID NOs:81 and 89, respectively, SEQ ID NOs:21 and 29, respectively, SEQ ID NOs:41 and 49, respectively, SEQ ID NOs:11 and 19, respectively, SEQ ID NOs:61 and 69, respectively, SEQ ID NOs:71 and 79, respectively, SEQ ID NOs:101 and 109, respectively, SEQ ID NOs:111 and 119, respectively, SEQ ID NOs:121 and 129, respectively, SEQ ID NOs:131 and 139, respectively, SEQ ID NOs:141 and 149, respectively, SEQ ID NOs:151 and 159, respectively, SEQ ID NOs:161 and 169, respectively, SEQ ID NOs:171 and 179, respectively, SEQ ID NOs:181 and 189, respectively, or SEQ ID NOs:201 and 209, respectively.

[0057] In certain embodiments, the variant CH1 domain polypeptides and variant CL domain polypeptides of the CH1-CL set may comprise the amino acid sequences of SEQ ID NOs: 31 and 32, respectively, SEQ ID NOs: 21 and 22, respectively, SEQ ID NOs: 11 and 12, respectively, SEQ ID NOs: 41 and 42, respectively, SEQ ID NOs: 51 and 59, respectively, SEQ ID NOs: 91 and 99, respectively, SEQ ID NOs: 31 and 39, respectively, SEQ ID NOs: 191 and 199, respectively, SEQ ID NOs: 81 and 89, respectively, SEQ ID NOs: 21 and 29, respectively, or SEQ ID NOs: 41 and 49, respectively.

[0058] In another aspect, provided herein is an immunoglobulin polypeptide comprising: (i) at least one variant CH1 domain polypeptide, or at least one heavy chain polypeptide comprising a variant CH1 domain polypeptide, and / or (ii) at least one variant CLκ or CLλ domain polypeptide, or a light chain polypeptide comprising a variant CLκ or CLλ domain polypeptide.

[0059] In some embodiments, the immunoglobulin polypeptide may comprise at least one variant CH1 domain polypeptide or a heavy chain polypeptide comprising a variant CH1 domain polypeptide, which may be any of the variant CH1 domain polypeptides described above.

[0060] In some embodiments, the immunoglobulin polypeptide may comprise at least one variant CLκ or CLλ domain polypeptide, or a light chain polypeptide comprising a variant CLκ or CLλ domain polypeptide, which may be any of the variant CLκ or CLλ domain polypeptides described above.

[0061] In some embodiments, immunoglobulin polypeptides according to the invention may comprise one or more of: (i) an antigen-binding domain; (ii) a CH1 domain or variant CH1 domain polypeptide; (iii) an immunoglobulin heavy chain constant region 2 ("CH2") domain or variant CH2 domain polypeptide; (iv) an immunoglobulin heavy chain constant region 3 ("CH3") domain or variant CH3 domain polypeptide; and / or (v) a light chain constant region (CL) domain or variant CL (e.g., variant CLκ or CLλ) domain polypeptide.

[0062] In certain embodiments, the antigen-binding domain may comprise an immunoglobulin heavy chain variable region ("VH") domain, an immunoglobulin light chain variable region ("VL") domain, a single-chain fragment variable ("scFv"), an antigen-binding fragment (Fab), F(ab'), F(ab')2, F(ab')2, or a combination thereof. In certain embodiments, the CH1 domain may comprise a wild-type CH1 amino acid sequence or comprise one or more amino acid substitutions relative to the wild-type CH1 amino acid sequence. In certain embodiments, the CH2 domain may comprise a wild-type CH2 amino acid sequence or comprise one or more amino acid substitutions relative to the wild-type CH2 amino acid sequence. In certain embodiments, the CH3 domain may comprise a wild-type CH3 amino acid sequence or comprise one or more amino acid substitutions relative to the wild-type CH3 amino acid sequence. In certain embodiments, the CL domain may comprise a wild-type CL amino acid sequence or comprise one or more amino acid substitutions relative to the wild-type CL amino acid sequence.

[0063] In certain embodiments, an immunoglobulin polypeptide may comprise a VH domain and may be bound to or paired with another polypeptide comprising a VL domain, and the VH and VL domains may form an antigen-binding site. In certain embodiments, a polypeptide may comprise a VL domain and may be bound to or paired with another polypeptide comprising a VH domain, and the VL and VH domains may form an antigen-binding site.

[0064] In another aspect, provided herein is a molecule comprising at least a first polypeptide comprising at least one variant CH1 domain polypeptide, or a heavy chain comprising a variant CH1 domain polypeptide, and a second polypeptide comprising at least one variant CLκ or CLλ domain polypeptide, or a heavy chain comprising a variant CLκ or CLλ domain polypeptide.

[0065] In some embodiments, the first and second polypeptides of such molecules can be linked to each other or paired with each other, optionally via a disulfide bond.

[0066] In some embodiments, the variant CH1 domain polypeptide of such a molecule may be any of the variant CH1 domain polypeptides according to the invention.

[0067] In some embodiments, the variant CLκ or CLλ domain polypeptide of such a molecule may be any of the variant CLκ or CLλ domain polypeptides according to the invention.

[0068] In some embodiments, the first polypeptide and the second polypeptide may be any of the variant CH1 domain-containing polypeptides described above and the variant CLκ or CLλ domain-containing polypeptides described above, respectively.

[0069] In certain embodiments, the first polypeptide comprises an antigen-binding domain and / or the second polypeptide comprises an antigen-binding domain.

[0070] In some cases, the antigen-binding domain of the first polypeptide and the antigen-binding domain of the second polypeptide of such molecules can optionally comprise a VH and a VL, or a VL and a VH, respectively, and optionally form an antigen-binding site specific for a first epitope. In some cases, the antigen-binding domain of the first polypeptide can optionally comprise an scFv or nanobody specific for the first epitope, and / or the antigen-binding domain of the second polypeptide can comprise an scFv or nanobody specific for a second epitope, respectively, and optionally, the first epitope is the same as or different from the second epitope.

[0071] In other embodiments, the molecule may further comprise a third polypeptide comprising at least one variant CH1 domain polypeptide or a heavy chain polypeptide comprising a variant CH1 domain polypeptide, and a fourth polypeptide comprising at least one variant CLκ or CLλ domain polypeptide or a light chain polypeptide comprising a variant CLκ or CLλ domain polypeptide. In such embodiments, the variant CH1 domain polypeptide may be any of the variant CH1 domain polypeptides according to the invention, and / or the variant CLκ or CLλ domain polypeptide may be any of the variant CLκ or CLλ domain polypeptides according to the invention.

[0072] In certain embodiments, the third polypeptide and the fourth polypeptide may be linked to each other or paired with each other, optionally via a disulfide bond.

[0073] In some embodiments, the variant CH1 domain polypeptide of the third polypeptide may be the same as or different from the variant CH1 domain polypeptide of the first polypeptide, and / or the variant CLκ or CLλ domain polypeptide of the fourth polypeptide may be the same as or different from the variant CLκ or CLλ domain polypeptide of the second polypeptide.

[0074] In some embodiments, the third polypeptide and the fourth polypeptide may be any of the variant CH1 domain-containing polypeptides described above, and any of the variant CLκ or CLλ domain-containing polypeptides described above, respectively.

[0075] In some embodiments, the third polypeptide may comprise an antigen-binding domain and / or the fourth polypeptide may comprise an antigen-binding domain.

[0076] In some cases, the antigen-binding domain of the third polypeptide and the antigen-binding domain of the fourth polypeptide may comprise a VH and a VL, or a VL and a VH, respectively, and optionally form an antigen-binding site specific for a third epitope, which optionally may be the same as or different from the first and / or second epitope. In some cases, the antigen-binding domain of the third polypeptide may comprise an scFv or nanobody specific for the third epitope, and / or the antigen-binding domain of the fourth polypeptide may comprise an scFv or nanobody specific for a fourth epitope, respectively, which optionally may be the same as or different from the fourth epitope, and optionally may be the same as or different from the first and / or second epitope.

[0077] In certain embodiments, a molecule according to the present disclosure may be a multispecific antibody or antigen-binding antibody fragment, optionally a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific antibody or antigen-binding antibody fragment.

[0078] In further embodiments, the molecule may optionally include a structure depicted in any one of Figures 2-7.

[0079] In further embodiments, the molecule may optionally comprise IgG, and even more optionally, IgG1, IgG2, IgG3, or IgG4.

[0080] In certain embodiments, in such molecules, the variant CH1 domain polypeptide of the third polypeptide may differ from the variant CH1 domain polypeptide of the first polypeptide, and / or the variant CLκ or CLλ domain polypeptide of the fourth polypeptide may differ from the variant CLκ or CLλ domain polypeptide of the second polypeptide. In such embodiments, the CH1 and variant CLκ or CLλ domain polypeptides of the first and second polypeptides may be referred to as a first CH1-CL set, and the CH1 and variant CLκ or CLλ domain polypeptides of the third and fourth polypeptides may be referred to as a second CH1-CL set.

[0081] In certain embodiments, the first CH1-CL set and the second CH1-CL set can be independently selected from the CH1-CLκ sets listed in Table 2 and the CH1-CLλ sets listed in Table 28.

[0082] In some preferred embodiments, the first CH1-CL set and the second CH1-CL set are network 1443 and network 1993, respectively; network 1039 and network 1993, respectively; network 1443 and network 964, respectively; network 1443 and network 1039, respectively; network 1443 and network 367, respectively; network 1443 and network 2366, respectively; network 1039 and network 367, respectively; network 1039 and network 2529, respectively; network 1039 and network 742, respectively; network 1039 and network 2529, respectively; and network 2366, respectively, network 1993 and network 1443, respectively, network 1993 and network 1039, respectively, network 964 and network 1443, respectively, network 1039 and network 1443, respectively, network 367 and network 1443, respectively, network 2366 and network 1443, respectively, network 367 and network 1039, respectively, network 2529 and network 1039, respectively, network 742 and network 1039, respectively, or two CH1-CLκ sets of network 2366 and network 1039, respectively.

[0083] In some exemplary embodiments, the first CH1-CL set and the second CH1-CL set may be two CH1-CLλ sets of network 367 and network 1621, respectively, network 964 and network 1443, respectively, network 367 and network 2529, respectively, network 964 and network 1621, respectively, network 367 and network 1443, respectively, network 964 and network 2529, or network 1443 and network 1993, respectively.

[0084] In some specific embodiments, the first CH1-CLκ set and the second CH1-CLκ set may be network 1443 and network 1993, respectively, or two CH1-CLκ sets of network 1993 and network 1443, respectively.

[0085] In some specific embodiments, the first CH1-CL set and the second CH1-CL set may be two CH1-CLλ sets of network 367 and network 1621, respectively, or network 964 and network 1443, respectively.

[0086] In some specific embodiments, the amino acid substitutions in the variant CH1 domain of a first polypeptide may comprise or consist of 145Q, 147E, and 181; the amino acid substitutions in the variant CLκ domain of a second polypeptide may comprise or consist of 129R, 178R, and 180Q; the amino acid substitutions in the variant CH1 domain of a third polypeptide may comprise or consist of 128R and 147R; and the amino acid substitutions in the variant CLκ domain of a fourth polypeptide may comprise or consist of 124E, 133Q, and 178E.

[0087] In some specific embodiments, the amino acid substitutions in the variant CH1 domain of a first polypeptide may comprise or consist of 128R and 147R; the amino acid substitutions in the variant CLκ domain of a second polypeptide may comprise or consist of 124E, 133Q, and 178E; the amino acid substitutions in the variant CH1 domain of a third polypeptide may comprise or consist of 145Q, 147E, and 181E; and the amino acid substitutions in the variant CLκ domain of a fourth polypeptide may comprise or consist of 129R, 178R, and 180Q.

[0088] In some specific embodiments, the amino acid substitution in the variant CH1 domain of the first polypeptide may comprise or consist of 148R, the amino acid substitution in the variant CLλ domain of the second polypeptide may comprise or consist of 124S and 129E, the amino acid substitution in the variant CH1 domain of the third polypeptide may comprise or consist of 145S and 147N, and the amino acid substitution in the variant CLλ domain of the fourth polypeptide may comprise or consist of 133Y and 180R.

[0089] In some specific embodiments, the amino acid substitutions in the variant CH1 domain of the first polypeptide may comprise or consist of 145S and 147N, the amino acid substitutions in the variant CLλ domain of the second polypeptide may comprise or consist of 133Y and 180R, the amino acid substitutions in the variant CH1 domain of the third polypeptide may comprise or consist of 148R, and the amino acid substitutions in the variant CLλ domain of the fourth polypeptide may comprise or consist of 124S and 129E.

[0090] In some specific embodiments, the amino acid substitutions in the variant CH1 domain of the first polypeptide may comprise or consist of 124R and 147R, the amino acid substitutions in the variant CLλ domain of the second polypeptide may comprise or consist of 127D and 129E, the amino acid substitutions in the variant CH1 domain of the third polypeptide may comprise or consist of 145Q, 147E, and 181E, and the amino acid substitutions in the variant CLλ domain of the fourth polypeptide may comprise or consist of 129R, 178R, and 180Q.

[0091] In some specific embodiments, the amino acid substitutions in the variant CH1 domain of the first polypeptide may comprise or consist of 145Q, 147E, and 181E; the amino acid substitutions in the variant CLλ domain of the second polypeptide may comprise or consist of 129R, 178R, and 180Q; the amino acid substitutions in the variant CH1 domain of the third polypeptide may comprise or consist of 124R and 147R; and the amino acid substitutions in the variant CLλ domain of the fourth polypeptide may comprise or consist of 127D and 129E.

[0092] In some specific embodiments of the molecules, the variant CH1 domain of the first polypeptide, the variant CL domain of the second polypeptide, the variant CH1 domain of the third polypeptide, and the variant CL domain of the fourth polypeptide comprise the amino acid sequences of: (A) SEQ ID NOs: 31, 32, 21, and 22, respectively; (B) SEQ ID NOs: 21, 22, 31, and 32, respectively; (C) SEQ ID NOs: 51, 59, 191, and 199, respectively; (D) SEQ ID NOs: 191, 199, 51, and 59, respectively; (E) SEQ ID NOs: 91, 99, 31, and 39, respectively; or (F) SEQ ID NOs: 31, 39, 91, and 99, respectively.

[0093] In some embodiments, when such a molecule is a multispecific antibody or fragment thereof, the molecule may be specific for two different antigens. In yet another aspect, provided herein are polynucleotides.

[0094] In some embodiments, one or more polynucleotides according to the invention may encode (i) any of the variant CH1 domain polypeptides described above, or any heavy chain polypeptide comprising any of the variant CH1 domains described above; (ii) any of the variant CLκ or CLλ domain polypeptides, or any light chain polypeptide comprising any of the variant CLκ or CLλ domains described above; (iii) any of the polypeptides described above; and / or (iv) any of the molecules or vectors containing above.

[0095] In some embodiments, one or more vectors according to the invention may comprise one or more of the polynucleotides described above.

[0096] In yet another aspect, provided herein is a cell comprising: (i) any of the variant CH1 domain polypeptides described above, or any heavy chain polypeptide comprising any of the variant CH1 domains described above; (ii) any of the variant CLκ or CLλ domain polypeptides described above, or any light chain polypeptide comprising any of the variant CLκ or CLλ domains; (iii) any of the immunoglobulin polypeptides described above; (iv) any of the molecules described above; (v) any of the polynucleotides described above; and / or (vi) any of the vectors described above.

[0097] In some embodiments, such cells are mammalian cells, optionally Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells, such as HEK293 cells, hi some embodiments, such cells are yeast cells.

[0098] In yet another aspect, provided herein are (I) compositions comprising (i) any of the variant CH1 domain polypeptides described above, or any heavy chain polypeptide comprising any of the variant CH1 domains described above, (ii) any of the variant CLκ or CLλ domain polypeptides described above, or any light chain polypeptide comprising any of the variant CLκ or CLλ domains described above, (iii) any of the immunoglobulin polypeptides described above, (iv) any of the molecules described above, (v) any of the polynucleotides described above, and / or (vi) any of the vectors described above, and / or (vii) any of the cells described above, and (II) a pharmaceutically or diagnostically acceptable carrier.

[0099] In yet another aspect, provided herein is a method for generating a CH1 domain library. Such a library can be a CH1 domain-encoding polynucleotide library or a CH1 domain polypeptide library.

[0100] In some embodiments, such methods for generating a CH1 domain-encoding polynucleotide library include incorporating mutations in silico or in vitro or randomizing nucleic acids at one or more predetermined nucleotide positions in a plurality of CH1 domain-encoding polynucleotides, wherein at least one of the one or more predetermined nucleotide positions can be within a codon encoding an amino acid at at least one of the predetermined CH1 domain amino acid positions.

[0101] In certain embodiments, one or more of the predetermined CH1 domain amino acid positions may be within or adjacent to the interface of the CH1 and CL domains.

[0102] In certain embodiments, one or more of the predetermined CH1 domain amino acid positions can be predicted to affect CH1-CL domain interactions. In some cases, the interaction can be a hydrogen-bond-mediated interaction. In some cases, the prediction can be performed in silico or in vitro. In certain cases, the prediction can be performed in silico using a Rosetta Monte Carlo (MC) hydrogen-bonding network (HBNet).

[0103] In certain embodiments, at least one of the one or more predetermined nucleotide positions can be within a codon encoding an amino acid at one or more of the predetermined CH1 domain amino acid positions selected from: 145, 147, 181, 128, 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and 187 according to EU numbering.

[0104] In certain embodiments, incorporating mutations and / or randomizing nucleic acids may use 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.

[0105] In certain embodiments, such a variant CH1 domain library may be designed to identify one or more CH1 domain polypeptides that preferentially pair with a given CL (CLκ or CLλ) domain or variant CL (CLκ or CLλ) domain polypeptide rather than with a wild-type CL (CLκ or CLλ) domain polypeptide, rather than with a given CL (CLκ or CLλ) domain polypeptide, or rather than with another given variant CL (CLκ or CLλ) domain polypeptide.

[0106] Also provided is a CH1 domain-encoding polynucleotide library generated using such a method.

[0107] In some embodiments, such a method for generating a CH1 domain polypeptide library may include obtaining, in silico or in vitro, a plurality of CH1 domain polypeptides corresponding to a plurality of CH1 domain-encoding polynucleotides contained in such a CH1 domain-encoding polynucleotide library.

[0108] Alternatively, in some embodiments, a method for generating a CH1 domain polypeptide library can include incorporating substitutions in silico or in vitro at one or more predetermined CH1 domain amino acid positions in a plurality of CH1 domain polypeptides.

[0109] In certain embodiments, one or more of the one or more predetermined CH1 domain amino acid positions (i) are located within or proximal to the interface of the CH1 and CL domains; (ii) are predicted to affect CH1-CL domain interactions, optionally hydrogen-bond-mediated interactions; optionally, the prediction is performed in silico or in vitro; further optionally, the prediction is performed in silico using RosettaMC HBNet; and / or (iii) may be selected from positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering.

[0110] In some embodiments, such a CH1 domain polypeptide library may be for identifying one or more variant CH1 domain polypeptides that preferentially pair with a given or variant CL domain polypeptide rather than with a wild-type or another given variant CL domain polypeptide.

[0111] In some embodiments, such a CH1 domain polypeptide library may comprise a predetermined number of CH1 substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0112] Also provided is a CH1 domain polypeptide library generated using such a method.

[0113] In yet another aspect, provided herein is a method for generating a CLκ and / or CLλ domain library, which may be a CLκ and / or CLλ domain-encoding polynucleotide library or a CLκ and / or CLλ domain polypeptide library.

[0114] In some embodiments, such methods for generating a CLκ and / or CLλ domain-encoding polynucleotide library may comprise incorporating mutations in silico or in vitro or randomizing nucleic acids at one or more predetermined nucleotide positions in a plurality of CLκ and / or CLλ domain-encoding polynucleotides, wherein at least one of the one or more predetermined nucleotide positions is within a codon encoding an amino acid at at least one of the predetermined CLκ and / or CLλ domain amino acid positions.

[0115] In certain embodiments, one or more of a given CLκ and / or CLλ domain amino acid positions may be within or adjacent to the interface of the CH1 domain and the CLκ and / or CLλ domain.

[0116] In certain embodiments, one or more of the predetermined CLκ and / or CLλ domain amino acid positions may be predicted to affect CH1-CL domain interactions. In some cases, the interaction may be a hydrogen-bond-mediated interaction. In some cases, the prediction may be performed in silico or in vitro. In certain cases, the prediction may be performed in silico using a Rosetta Monte Carlo (MC) hydrogen-bonding network (HBNet).

[0117] In certain embodiments, at least one of the one or more predetermined nucleotide positions may be within a codon encoding an amino acid at one or more of the predetermined CLκ and / or CLλ domain amino acid positions selected from: 129, 178, 180, 124, 133, 114, 120, 124, 127, 129, 133, 135, 137, and 138, 178, and 180 according to EU numbering.

[0118] In certain embodiments, incorporating mutations and / or randomizing nucleic acids may use 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.

[0119] In certain embodiments, a variant CLκ and / or CLλ domain library may comprise only CL domains of the κ isotype, only CL domains of the λ isotype, or at least one CL domain of the κ isotype and at least one CL domain of the λ isotype.

[0120] In certain embodiments, such variant CLκ and / or CLλ domain libraries may be designed to identify one or more variant CLκ and / or CLλ domain polypeptides that preferentially pair with a given or variant CH1 domain polypeptide rather than with a wild-type CH1 domain polypeptide or another given variant CH1 domain polypeptide.

[0121] In yet another aspect, provided herein are variant CLκ and / or CLλ domain libraries.

[0122] Further provided is a CLκ and / or CLλ domain-encoding polynucleotide library generated using the methods described above.

[0123] In some embodiments, such methods for generating a CLκ and / or CLλ domain polypeptide library may include obtaining, in silico or in vitro, a plurality of CLκ and / or CLλ domain polypeptides corresponding to a plurality of CLκ and / or CLλ domain-encoding polynucleotides included in the CLκ and / or CLλ domain-encoding polynucleotide library described above.

[0124] Alternatively, in some embodiments, a method for generating a CLκ and / or CLλ domain polypeptide library may comprise incorporating substitutions in silico or in vitro at one or more predetermined CLκ and / or CLλ domain amino acid positions in a plurality of CLκ and / or CLλ domain polypeptides.

[0125] In certain embodiments, one or more of the one or more predetermined CLκ and / or CLλ domain amino acid positions may be within or adjacent to the interface of the CH1 and CL domains.

[0126] In certain embodiments, one or more of the one or more predetermined CLκ and / or CLλ domain amino acid positions may be predicted to affect CH1-CL domain interactions, optionally hydrogen bond-mediated interactions, optionally with the prediction performed in silico or in vitro, further optionally with the prediction performed in silico using RosettaMC HBNet.

[0127] In certain embodiments, one or more of the one or more predetermined CLκ and / or CLλ domain amino acid positions may be selected from positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering.

[0128] In some embodiments, the library may be for identifying one or more variant CLκ and / or CLλ domain polypeptides that preferentially pair with a given or variant CH1 domain polypeptide rather than with a wild-type or another given variant CH1 domain polypeptide.

[0129] In some embodiments, the CLκ and / or CLλ domain polypeptides of the library may comprise a predetermined number of CLκ and / or CLλ substitution positions. In certain embodiments, the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0130] Further provided are CLκ and / or CLλ domain polynucleotide libraries generated using the methods described above.

[0131] In yet another aspect, provided herein is a method for generating a CH1-CL domain set library, which may be a CH1-CL domain-encoding polynucleotide set library or a CH1-CL domain polypeptide set library.

[0132] In some embodiments, such methods for generating a CH1-CL domain-encoding polynucleotide set library may include incorporating mutations in silico or in vitro or randomizing nucleic acids at one or more predetermined nucleotide positions in a plurality of CH1-CL domain-encoding polynucleotide sets, wherein at least one of the one or more predetermined nucleotide positions may be within a codon encoding an amino acid at at least one of the predetermined CH1 and / or CL domain amino acid positions.

[0133] In certain embodiments, one or more of the CH1 and / or CL domain amino acid positions of interest may be within or adjacent to the interface of the CH1 and CL domains.

[0134] In certain embodiments, one or more of the predetermined CH1 and / or CL domain amino acid positions can be predicted to affect CH1-CL domain interactions. In some cases, the interactions can be hydrogen-bond mediated interactions. In some cases, the predictions can be performed in silico or in vitro. In certain cases, the predictions can be performed in silico using a Rosetta Monte Carlo (MC) hydrogen bond network (HBNet).

[0135] In certain embodiments, one or more of the predetermined CH1 domain amino acid positions may be selected from CH1 positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering; and / or

[0136] In certain embodiments, one or more of the predetermined CL domain amino acid positions may be selected from CL positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering.

[0137] In some embodiments, one or more mutations may be generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups.

[0138] In some embodiments, the library may be for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CH1 domain rather than with wild-type or another given variant CH1 domain polypeptide, and / or for identifying one or more variant CH1 domain polypeptides that preferentially pair with a given or variant CL domain rather than with wild-type or another given variant CL domain polypeptide, or for identifying one or more sets of variant CH1 domains and variant CL domains that preferentially pair with each other.

[0139] In some embodiments, the CL domains encoded by the CH1-CL domain-encoding polynucleotide set library may include a CLκ domain and / or a CLλ domain.

[0140] Also provided is a CH1-CL domain-encoding polynucleotide set library generated using such a method.

[0141] In some embodiments, such a method for generating a CH1-CL domain polypeptide set library may include obtaining, in silico or in vitro, a plurality of CH1-CL domain polypeptide sets corresponding to a plurality of CH1-CL domain-encoding polynucleotides included in the CH1-CL domain-encoding polynucleotide set library described above.

[0142] Alternatively, in some embodiments, such methods for generating a CH1-CL domain polypeptide set library may include incorporating substitutions in silico or in vitro at one or more predetermined CH1 and / or CL domain amino acid positions in a plurality of CH1-CL domain polypeptide sets.

[0143] In certain embodiments, one or more of the one or more predetermined CH1 and / or CL domain amino acid positions may be within or adjacent to the interface of the CH1 and CL domains.

[0144] In certain embodiments, one or more of the one or more predetermined CH1 and / or CL domain amino acid positions may be predicted to affect CH1-CL domain interactions, optionally hydrogen-bond-mediated interactions, optionally with the prediction performed in silico or in vitro, and further optionally with the prediction performed in silico using a Rosetta Monte Carlo (MC) hydrogen-bonding network (HBNet).

[0145] In certain embodiments, one or more of the one or more predetermined CH1 and / or CL domain amino acid positions may be selected from CH1 domain amino acid positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering, and / or may be selected from CL domain amino acid positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering.

[0146] In some embodiments, the library may be for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CH1 domain rather than with wild-type or another given variant CH1 domain polypeptide, and / or for identifying one or more variant CH1 domain polypeptides that preferentially pair with a given or variant CL domain rather than with wild-type or another given variant CL domain polypeptide, or for identifying one or more sets of variant CH1 domains and variant CL domains that preferentially pair with each other.

[0147] In some embodiments, the CL domains encoded by the CH1-CL domain-encoding polynucleotide set library may include a CLκ domain and / or a CLλ domain.

[0148] In some embodiments, the CH1 domain polypeptides of the CH1-CL domain polypeptide set library may comprise a predetermined number of CH1 substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0149] In some embodiments, the CL domain polypeptides of the CH1-CL domain polypeptide set library may comprise a predetermined number of CL substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0150] In some embodiments, a method for generating a CH1-CL domain polypeptide set library includes a first step of providing a plurality of CH1-CL domain polypeptide sets; and a second step of calculating CH1-CL inter-domain interaction strengths for one or more of the plurality of CH1-CL domain polypeptide sets, where optionally the calculating is (a) in silico or in vitro, optionally in silico using a Rosetta Monte Carlo (MC) hydrogen bond network (HBNet), and / or (b) based on the strength of CH1-CL inter-domain hydrogen bonds and / or CH1-CL inter-domain binding energies; and (a) optionally a reference CH1-CL domain polypeptide set, which is a WT CH1-CL domain polypeptide set or a known CH1-CL domain polypeptide set, or (b) optionally a reference CH1-CL domain polypeptide set, which is a WT CH1-CL domain polypeptide set or a known CH1-CL domain polypeptide set. and a third step of selecting one or more sets of CH1-CL domain polypeptides calculated to have stronger CH1-CL domain interactions compared to a reference CH1-CL domain interaction strength, which is the CH1-CL domain interaction strength of a CH1-CL domain set or a known CH1-CL domain polypeptide set.

[0151] In some embodiments, a CH1-CL domain polypeptide library may be provided to identify one or more variant CL domain polypeptides that preferentially pair with a variant CH1 domain polypeptide rather than with a wild-type or another given variant CH1 domain polypeptide.

[0152] In some embodiments, the CL domain in the CH1-CL domain polypeptide set library may comprise a CLκ domain and / or a CLλ domain.

[0153] In some embodiments, the CH1 domain polypeptides of the CH1-CL domain polypeptide set library comprise a predetermined number of CH1 substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5; and / or

[0154] In some embodiments, the CL domain polypeptides of the CH1-CL domain polypeptide set library may comprise a predetermined number of CL substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0155] In yet another aspect, provided herein is a CH1-CL domain polypeptide set library.

[0156] In some embodiments, such libraries may be produced by any of the methods for generating CH1-CL domain polypeptide set libraries described herein.

[0157] In some embodiments, the CH1-CL domain set library may be a CH1-CLκ domain set library, a CH1-CLλ domain set library, or a CH1-CL domain set library, in which the CL domains of the library comprise one or more CLκ domains and one or more CH1-CLλ domains.

[0158] In another aspect, provided herein is a method of identifying one or more sets of variant CH1 domain polypeptides and variant CLκ and / or CLλ domain polypeptides, wherein the variant CH1 domain polypeptides and variant CLκ or CLλ domain polypeptides preferentially pair with each other.

[0159] In some embodiments, such a method may include three steps (steps (a) to (c)).

[0160] In some cases, step (a) may include providing (a-1) a first polypeptide comprising a wild-type or variant CH1 domain polypeptide, and (a-2) a second polypeptide comprising a wild-type or variant CLκ or CLλ domain polypeptide. Optionally, the sets of (a-1) and (a-2) are provided in silico or in vitro.

[0161] In certain cases, (i) the first polypeptide in step (a) can be derived from any CH1 domain polypeptide library described herein or expressed from any variant CH1 domain-encoding polynucleotide library described herein.

[0162] In certain cases, (ii) the second polypeptide in step (b) may be derived from any CLκ and / or CLλ domain polypeptide library or expressed from any CLκ and / or CLλ domain-encoding polynucleotide library described herein.

[0163] In certain cases, (iii) the first polypeptide in step (a) and the second polypeptide in step (b) may be derived from any CH1-CL domain polypeptide set library described herein or expressed from any CH1-CL domain-encoding polynucleotide set library described herein.

[0164] In certain cases, (iv) the first polypeptide in step (a) and the second polypeptide in step (b) may be expressed from a CH1-CL domain set library in which the CH1 and / or CL domains comprise one or more random amino acid modifications.

[0165] In some cases, step (b) may comprise quantifying binding preference between the variant CH1 domain polypeptide and the variant CLκ or CLλ domain polypeptide.

[0166] In certain cases, binding preference may be based on the strength of CH1-CL interdomain hydrogen bonds and / or CH1-CL interdomain binding energy, and optionally, quantification is performed in silico or in vitro.

[0167] In some cases, step (c) may include selecting one or more sets of variant CH1 domain polypeptides and variant CLκ or CLλ domain polypeptides that provide preferential CH1-CL pairing. In some cases, the preferential CH1-CL pairing may be an equal or higher preferential pairing relative to a reference set of CH1-CL domain polypeptides. In certain cases, the reference set of CH1-CL domain polypeptides may include a wild-type CH1 domain, a wild-type CLκ or CLλ domain, any of the variant CH1 domain polypeptides described above, and / or any of the variant CLκ or CLλ domain polypeptides described above. In certain cases, the reference set of CH1-CL domain polypeptides may be a set of CH1-CL domain polypeptides set shown in Table 1.

[0168] In some embodiments, the method of identification may utilize a combination of amino acid substitutions in CH1 and / or CLκ or CLλ identified herein as affecting light-heavy pairing.

[0169] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of 145, 147, and / or 181, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of 129, 178, and / or 180.

[0170] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 128 and / or 147, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 124, 133, and / or 178.

[0171] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 168, 185, and / or 187, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of position 135.

[0172] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 147 and / or 185, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 135 and / or 178.

[0173] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of position 148, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 124 and / or 129.

[0174] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of 139, 141, and / or 187, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of 114, 135, and / or 138.

[0175] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 166 and / or 187, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 137 and / or 138.

[0176] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 168 and / or 185, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of position 135.

[0177] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 124 and / or 147, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 127 and / or 129.

[0178] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 147 and / or 148, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 127 and / or 129.

[0179] In certain embodiments, one or more predetermined CH1 domain amino acid positions may include or consist of position 145, and / or one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of position 133.

[0180] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 145 and / or 181, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of position 133.

[0181] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of position 145, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 124 and / or 133.

[0182] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 145 and / or 181, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 120, 178, and / or 180.

[0183] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of 124, 145, and / or 147, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of 127, 129, and / or 178.

[0184] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 166 and / or 187, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 114, 137, and / or 138.

[0185] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of 147 and / or 175, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of 129, 178, and / or 180.

[0186] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 147, 175, and / or 181, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 129 and / or 180.

[0187] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 145 and / or 147, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 133 and / or 180.

[0188] In certain embodiments, the one or more predetermined CH1 domain amino acid positions may include or consist of positions 147 and / or 185, and / or the one or more predetermined CLκ or CLλ domain amino acid positions may include or consist of positions 129 and / or 180.

[0189] In some embodiments of the method, the first polypeptide may comprise or be attached to a first label and / or the second polypeptide may comprise or be attached to a second label.

[0190] In such embodiments, the quantifying step (b) may comprise detecting the first label and / or the second label.

[0191] In some embodiments of the method, in step (a), the first polypeptide and the second polypeptide may be provided in silico (e.g., computationally modeled in a complex) and, in such cases, in step (b), quantifying may include calculating a score indicative of the binding energy between the CH1 domain and the CLκ domain, such as, but not limited to, total energy or energy from hydrogen bonds.

[0192] In such embodiments, the score is optionally ΔΔG, ΔΔG cognate total score , ΔΔG cognate hbond_all ,RBPP,RBPP total score ,RBPP hbond_all , and / or RBPP bond elec backrub 18k may be selected from:

[0193] In some embodiments of the method, in step (a), the first polypeptide and the second polypeptide may be provided in silico.

[0194] In such embodiments, the quantifying in step (b) may be performed in silico using Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet).

[0195] In some embodiments of the method, in step (a), the first polypeptide and the second polypeptide may be provided in vitro (e.g., recombinantly co-expressed), and in such cases, in step (b), quantifying comprises measuring the amount of the CH1-CLκ pair via liquid chromatography-mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, and / or flow cytometry.

[0196] In some embodiments, the method of identification may further comprise selecting one or more sets of CH1-CL domains based on one or more properties of the antibody comprising the first and second sets of polypeptides selected in step (c).

[0197] In some embodiments, the one or more properties include: (i) (i-1) production yield, optionally assessed in one or more cell types, optionally mammalian cells such as CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) suitability for one or more antibody purification methods, optionally including Protein A affinity purification; (ii) degree of aggregation, optionally the presence of multimers of full-sized antibodies; (iii) correct pairing rate, optionally between CH1 domains and / or between CH1 domains and CL domains; (iv) melting temperature (Tm) and / or aggregation temperature (Tagg), optionally Tagg266, (v) isoelectric point (pI), (vi) level of interaction with the multispecific reagent ("PSR"), (vii) hydrophobic interactions of the antibody, (viii) self-interactions, (ix) stability against high or low pH stress, (x) solubility, (xi) production cost and / or time, (xii) other stability parameters, (xiii) shelf life, (xiv) in vivo half-life, and / or immunogenicity.

[0198] Such antibody properties can be measured or assessed using any suitable method used in the art.

[0199] In certain embodiments, the degree of aggregation, optionally the presence of multimers of full-sized antibodies, may be quantified using chromatography, optionally size exclusion chromatography (SEC), or electrophoresis, optionally SDS-PAGE.

[0200] In certain embodiments, the rate of correct pairing, optionally between CH1 domains and / or between CH1 and CL domains, may be assessed using LC-MS.

[0201] In certain embodiments, Tm and / or Tagg, optionally Tagg266, may be measured using differential scanning fluorimetry (DSF) and / or differential scanning calorimetry (DSC) and / or using an instrument, optionally an Uncle®.

[0202] In certain embodiments, the level of interaction with PSR" can be measured by the methods described in WO2014 / 179363.

[0203] In certain embodiments, the hydrophobic interactions of antibodies may be measured using hydrophobic interaction chromatography ("HIC"), optionally as described in Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561.

[0204] In certain embodiments, self-interaction can be measured by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92.

[0205] In certain embodiments, self-interactions can be measured by dynamic light scattering (DLS).

[0206] Thus, in another aspect, provided herein is a method of screening combinations of (i) a first set of a first variant CH1 domain polypeptide and a first variant CL domain polypeptide (a "first CH1-CL domain polypeptide set"), and (ii) a second set of a second variant CH1 domain polypeptide and a second variant CL domain polypeptide (a "second CH1-CL domain polypeptide set"), for a combination suitable for a desired antigen specificity, having a desired antibody or antibody fragment structure (e.g., any of the structures described herein, including the structures of Figures 2-7, and / or optionally an IgG, and even more optionally an IgG1, IgG2, IgG3, or IgG4) and / or optionally having a desired variable region sequence.

[0207] Such a method may include: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments comprising different combinations of (i) a first candidate set of CH1-CL domain polypeptides and (ii) a second candidate set of CH1-CL domain polypeptides; and (b) selecting one or more combinations of (i) the first set of CH1-CL domain polypeptides and (ii) the second set of CH1-CL domain polypeptides based on one or more properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a).

[0208] In certain embodiments, at least one of the one or more properties may be selected from properties (i)-(xv) above.

[0209] In some embodiments, the plurality of multispecific antibodies and / or antigen-binding antibody fragments comprises: (I) a first polypeptide comprising a first variant CH1 domain polypeptide and a first antigen-binding domain polypeptide; (II) a second polypeptide comprising a second variant CH1 domain polypeptide and a second antigen-binding domain polypeptide; (III) a third polypeptide comprising a first variant CL domain polypeptide and a third antigen-binding domain polypeptide; and (IV) a fourth polypeptide comprising a second variant CL domain polypeptide and a fourth antigen-binding domain polypeptide, optionally wherein the first and third polypeptides preferentially pair with each other and the second and fourth polypeptides preferentially pair with each other.

[0210] In certain embodiments, the plurality of multispecific antibodies and / or antigen-binding antibody fragments may comprise a structure depicted in any of Figures 2-7.

[0211] In some cases, (i) the first variant CH1 domain polypeptide may be any of the variant CH1 domain polypeptides described herein, (ii) the second variant CH1 domain polypeptide may be any of the variant CH1 domain polypeptides described herein, (iii) the first CLκ or CLλ domain polypeptide may be any of the variant CLκ or CLλ domain polypeptides described herein, and / or (iv) the second CLκ or CLλ domain polypeptide may be any of the variant CLκ or CLλ domain polypeptides described herein.

[0212] In certain cases, the first antigen-binding domain and the third antigen-binding domain may form a first antigen-binding site specific for a first epitope of interest, and the second antigen-binding domain and the fourth antigen-binding domain may form a second antigen-binding site specific for a second epitope of interest, optionally wherein the first epitope of interest and the second epitope of interest are different from each other.

[0213] In certain cases, the first antigen-binding domain and the third antigen-binding domain may form a first antigen-binding site specific for a first epitope of interest, the second antigen-binding domain may form a second antigen-binding site specific for a second epitope of interest, and the fourth antigen-binding domain may form a third antigen-binding site specific for a third epitope of interest, optionally where the first epitope of interest is different from the second and / or third epitope of interest.

[0214] In certain cases, the first antigen-binding domain may form a first antigen-binding site specific for a first epitope of interest, the second antigen-binding domain and the fourth antigen-binding domain may form a second antigen-binding site specific for a second epitope of interest, and the third antigen-binding domain may form a third antigen-binding site specific for a third epitope of interest, optionally wherein the second epitope of interest is different from the first and / or third epitope of interest.

[0215] In certain cases, the first antigen-binding domain may form a first antigen-binding site specific for a first epitope of interest, the second antigen-binding domain may form a second antigen-binding site specific for a second epitope of interest, the third antigen-binding domain may form a third antigen-binding site specific for a third epitope of interest, and the fourth antigen-binding domain may form a fourth antigen-binding site specific for a fourth epitope of interest, optionally wherein the first and / or third epitopes are different from the second and / or fourth epitopes.

[0216] In some embodiments, at least one of the one or more properties may be selected from properties (i)-(xv) described above.

[0217] Also provided herein are libraries and methods for identifying one or more sets of first and second polypeptides that can preferentially pair with each other.

[0218] In one aspect, provided herein is a method of generating a library of a set of first and second candidate polypeptide-encoding polynucleotides, wherein (i) the first candidate polypeptide is the same as or a variant of a first parent polypeptide, and (ii) the second candidate polypeptide is the same as or a variant of a second parent polypeptide.

[0219] In some embodiments, the method comprises: (a) providing a set of polynucleotides encoding a first parent polypeptide and a set of polynucleotides encoding a second parent polypeptide; and (b) incorporating mutations in silico or in vitro or randomizing nucleic acids at one or more predetermined nucleotide positions in the set of polynucleotides of step (a), wherein at least one of the one or more predetermined nucleotide positions is within a codon encoding an amino acid at one or more of the predetermined amino acid positions of the first and / or second parent polypeptide.

[0220] In some embodiments, one or more of the predetermined amino acid positions of the first and / or second parent polypeptide may be within or adjacent to an interface between the first parent polypeptide and the second parent polypeptide, optionally, the amino acid positions within or adjacent to the interface are predicted in silico or in vitro, and / or

[0221] In some embodiments, one or more of the predetermined amino acid positions of the first and / or second parent polypeptides may be predicted to affect the interaction between the first parent polypeptide and the second parent polypeptide, optionally, inter-polypeptide hydrogen bond-mediated interactions and / or inter-polypeptide binding energy, optionally, the prediction is performed in silico or in vitro, further optionally, the prediction is performed in silico using a Rosetta Monte Carlo (MC) hydrogen bond network (HBNet).

[0222] In some embodiments, one or more mutations may be generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups.

[0223] In some embodiments, the library can optionally be for identifying first and second polypeptides that preferentially pair with each other for a set of first and second parent polypeptides.

[0224] Also provided herein are libraries of sets of first and second candidate polypeptide-encoding polynucleotides generated using the methods described herein.

[0225] In another aspect, provided herein is a method of generating a library of a set of first and second candidate polypeptides, wherein (i) the first candidate polypeptide is the same as or a variant of a first parent polypeptide, and (ii) the second candidate polypeptide is the same as or a variant of a second parent polypeptide.

[0226] In some embodiments, the method may comprise obtaining, in silico or in vitro, a plurality of sets of first and second candidate polypeptides corresponding to the first and second candidate polypeptide-encoding polynucleotides contained in the polynucleotide library described above; or

[0227] In some embodiments, the method may involve incorporating substitutions in silico or in vitro at one or more predetermined amino acid positions of the first and / or second parent polypeptide.

[0228] In certain embodiments, one or more of the one or more predetermined amino acid positions may be within or adjacent to the interface of the first parent polypeptide and the second parent polypeptide, optionally, the amino acid positions within or adjacent to the interface are predicted in silico or in vitro; and / or

[0229] In certain embodiments, one or more of the one or more predetermined amino acid positions may be predicted to affect the interaction between a first parent polypeptide and a second parent polypeptide, optionally, inter-polypeptide hydrogen bond-mediated interactions and / or inter-polypeptide binding energy, optionally, the prediction is performed in silico or in vitro, further optionally, the prediction is performed in silico using a Rosetta Monte Carlo (MC) hydrogen bond network (HBNet).

[0230] In some embodiments, the library can optionally be for identifying first and second polypeptides that preferentially pair with each other for a set of first and second parent polypeptides.

[0231] In some embodiments, a first candidate polypeptide in the library may comprise a predetermined number of substitutions relative to a first parent polypeptide, optionally where the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0232] In some embodiments, second candidate polypeptides in the library may comprise a predetermined number of substitutions relative to the second parent polypeptide, optionally where the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0233] Further provided herein is a library of sets of first and second candidate polypeptides generated using the methods described above.

[0234] In another aspect, provided herein is a method of identifying one or more sets of first and second candidate polypeptides, wherein (i) the first candidate polypeptide is the same as or a variant of a first parent polypeptide, (ii) the second polypeptide is the same as or a variant of a second parent polypeptide, (iii) the first polypeptide is a variant of the first parent polypeptide and / or the second polypeptide is a variant of the second parent polypeptide, and (iv) the first and second polypeptides pair preferentially with each other, optionally more preferentially than the first and second parent polypeptides.

[0235] In some embodiments, the method comprises: (a) providing a plurality of sets of first and second candidate polypeptides, optionally wherein the providing is performed in silico or in vitro; (b) quantifying binding preference between the first and second candidate domain polypeptides, optionally wherein the binding preference is based on the strength of inter-polypeptide hydrogen bonds and / or inter-polypeptide binding energy, further optionally wherein the quantifying is performed in silico or in vitro; and (c) selecting one or more sets of first and second polypeptides that provide preferential inter-polypeptide pairing, optionally equal to or higher preferential pairing relative to a reference polypeptide set, further optionally wherein the reference polypeptide set is a set of (I) a first parent polypeptide or variant thereof and (II) a second parent polypeptide or variant thereof.

[0236] In some embodiments, at least one set of first and second candidate polypeptides in step (a) may be (i-1) derived from a library of sets of first and second candidate polypeptides described above, or (i-2) expressed from a library of sets of first and second candidate polypeptide-encoding polynucleotides described above, and / or (ii) derived from a library of sets of first and second candidate polypeptides (ii-1) in which the first and / or second candidate polypeptides comprise one or more random amino acid modifications, or (ii-2) expressed from a library of sets of first and / or second candidate polypeptide-encoding polynucleotides in which the first and / or second candidate polypeptide-encoding polynucleotides comprise one or more random mutations.

[0237] In some embodiments, the first polypeptide may comprise or be attached to a first label and / or the second polypeptide may comprise or be attached to a second label, and optionally, the quantifying step (b) comprises detecting the first label and / or the second label.

[0238] In some embodiments, in step (a), providing may be performed in silico, and in step (b), quantifying may optionally include calculating a score selected from ΔΔΡ, ΔΔΡcognate total score, ΔΔΡcognate hbond_all, RBPP, RBPPtotal score, RBPPhbond_all, and / or RBPPbond elec backrub 18k, and / or quantifying may be performed in silico using Rosetta Multi-Carlo (MC) Hydrogen Bond Network (HBNet).

[0239] In some embodiments, in step (a), providing may be performed in vitro, optionally recombinantly, and in step (b), quantifying comprises measuring the amount of CH1-CL pairs via liquid chromatography-mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, and / or flow cytometry. [Brief explanation of the drawings]

[0240] [Figures 1A-1D] Schematics are provided to generally illustrate the advantages of preferential pairing of CH1 domains with CL domains in various multispecific antibody designs. In Figures 1A-1D, a bispecific antibody of interest (boxed) comprises (a) a half-antibody specific for epitope A, comprising (a-1) a heavy chain ("heavy chain A") comprising a VH (brick pattern) specific for epitope A, and (a-2) a light chain ("light chain A") comprising a VL (horizontal stripes) specific for epitope A, and (b) a half-antibody specific for epitope B, comprising (b-1) a heavy chain ("heavy chain B") comprising a VH (checkerboard pattern) specific for epitope B, and (a-2) a light chain ("light chain B") comprising a VL (vertical stripes) specific for epitope B.

[0241] [Figure 1A] An exemplary production of such a bispecific antibody is shown when heavy chain A, light chain A, heavy chain B, and light chain A all contain wild-type constant domains. When such four chains are co-expressed, co-delivered, or mixed in a ratio of approximately 1:1:1:1, and there is complete promiscuity in heavy-light-chain pairing and heavy-heavy chain pairing, 10 different antibody products can be produced, with each ratio as shown. Approximately 12.5% ​​of the products will correspond to the desired bispecific antibody (boxed).

[0242] [Figure 1B]Figure 1A shows an exemplary production of a bispecific antibody, including heavy chain heterodimerization techniques in heavy chains A and B. Any suitable heavy chain heterodimerization technique can be used, including, but not limited to, "knobs-into-holes" technique (see, e.g., U.S. Patent No. 5,731,168), which is a CH3 domain modification that promotes CH3 heterodimerization. Figure 1B depicts heavy chain-heavy chain heterodimerization technique in the CH3 domain only (paired CH3s, where a triangle is added to one CH3 and removed from the other), although heterodimerization modifications can be present in the hinge, CH2, and / or CH3 domains. When such heavy chain A, light chain A, heavy chain B, and light chain B are co-expressed, co-delivered, or mixed in a ratio of approximately 1:1:1:1, and the heavy chain heterodimerization technology allows exclusively heavy chain-heavy chain heteropairing, four different antibody products can be produced, each in the proportions shown. Approximately 25% of the products will correspond to the desired bispecific antibody (boxed).

[0243] [Figure 1C]1 shows an exemplary production of such a bispecific antibody, where heavy chain A comprises a variant CH1 domain (solid) that preferentially pairs with the variant CL domain (dot) of light chain A rather than the CL domain of light chain B. The variant CH1 domain (solid) can be a variant CH1 domain according to the present disclosure, and / or the variant CL domain (dot) can be a variant CL domain according to the present disclosure. The variant CH1 domain and variant CL domain can be a variant CH1-CL domain set according to the present disclosure. The CH1 domain of heavy chain B and the CL domain of light chain B can be wild-type or any suitable modified CH1 and CL domain (another variant CH1-CL domain set according to the present disclosure, e.g., a variant CH1-CLκ domain set or a variant CH1-CLλ domain set). When such heavy chain A, light chain A, heavy chain B, and light chain B are co-expressed, co-delivered, or mixed in a ratio of approximately 1:1:1:1, if the variant CH1 domains (solid) and variant CL domains (dots) pair exclusively with each other and there is complete promiscuity in inter-heavy chain pairing, three distinct antibody products can be generated at the respective proportions shown. Approximately 50% of the products will correspond to the desired bispecific antibody (boxed). When the pairing preference between the CH1 domains (solid) and variant CL domains (dots) is closer to an exclusive preference, the product types and proportions of individual products will be more similar to those shown in Figure 1C. When the most ideal CH1-CH domain set is used, approximately 50% of the product will be the desired bispecific antibody. However, even if it does not reach 50%, a CH1-CH domain set that provides the desired bispecific antibody in more than 12.5% ​​(i.e., higher than when the corresponding wild-type CH1-CL set is used without heavy chain heterodimerization technology) will facilitate efficient production of bispecific antibodies.

[0244] [Figure 1D]Figure 1C shows an exemplary production of a bispecific antibody, further including heavy chain heterodimerization techniques in heavy chains A and B. Any suitable heavy chain heterodimerization technique can be combined with bispecific antibodies comprising variant CH1 domains and / or variant CL domains according to the present disclosure. Various heterodimerization techniques are available, including, but not limited to, "knob-into-hole" techniques (see, e.g., U.S. Patent No. 5,731,168), which are CH3 domain modifications that promote CH3 heterodimerization. Figure 1D depicts heavy chain-heavy chain heterodimerization techniques in the CH3 domain only (paired CH3s, where a triangle is added to one CH3 and removed from the other), although heterodimerization modifications can be present in the hinge, CH2, and / or CH3 domains. When such heavy chain A, light chain A, heavy chain B, and light chain B are co-expressed, co-delivered, or mixed in a ratio of approximately 1:1:1:1, the intended antibody product (inside the box) can only be produced if the variant CH1 domains (solid) and variant CL domains (dots) pair exclusively with each other and the heavy chain dimerization technology exclusively allows heavy chain-heavy chain heteropairing, i.e., 100%. Using the most ideal CH1-CH domain set, approximately 100% of the product will be the desired bispecific antibody. However, even if it does not reach 100%, a CH1-CHκ domain set that provides the desired bispecific antibody in more than 50% (i.e., higher than when the corresponding wild-type CH1-CL set is used with heavy chain heterodimerization technology) will facilitate efficient production of bispecific antibodies.

[0245] [Figure 2-8]Exemplary, non-limiting embodiments of various multispecific antibody structures with which the variant CH1 and / or variant CL domain variants disclosed herein may be used are provided. In Figures 2-8, unless otherwise indicated, the following applies: (1) each domain is presented as a rectangle with text therein indicating the domain name (e.g., CH1, CL, etc.); (2) a set of interconnected domains represents a polypeptide (e.g., heavy chain polypeptide, light chain polypeptide, etc.); (3) the orientation of the domains within a polypeptide follows the orientation of the text indicating the domain name, from N-terminus to C-terminus; (4) even when a diagram does not explicitly show linkers, hinges, and / or disulfide bonds, linkers or hinges may be used between domains as needed, and disulfide bonds may exist between polypeptides and / or between domains, presumably to enable correct formation of antibody binding sites; (5) CH2 and / or CH3 domains shown in diagrams may be omitted whenever possible and replaced with hinges or linkers, if appropriate; (6) rectangles without a pattern (i.e., open) may individually contain the corresponding wild-type sequence or may contain one or more amino acid substitutions relative to the wild-type sequence. , domains; (7) the CH1, CH2, and CH3 domains, individually, can be of any (heavy chain) isotype; (8) when more than one CH1 domain is present in a structure, the CH1 domains may or may not be of the same isotype; when more than one CH2 domain is present in a structure, the CH2 domains may or may not be of the same isotype; when more than one CH3 domain is present in a structure, the CH3 domains may or may not be of the same isotype; (9) the hinge, CH2, and / or CH3 domains can include modifications such as those that promote heteropairing between two different hinges, two different CH2 domains, and / or two different CH3 domains; (10) a "CL" domain can be a kappa or lambda CL domain unless otherwise specified; (11) a CH1 domain with a number separated by a hyphen (e.g., CH1-1, CH1-2, CH1-3, etc.) can be a CL domain with the matching number (e.g.,(12) Filled CH1 domains with numbers separated by hyphens (e.g., "CH1-1," "CH1-2," "CH1κ-3," etc.) are variant CH1 domains that preferentially or non-preferentially pair with dotted CLs with matching numbers (e.g., "CL-1," "CL-2," "CL-3," etc.), which may be variant CLκ or CLλ domains according to the present disclosure; and pairs of filled CH1 domains and dotted CL pairs are variant CH1 domains that preferentially or non-preferentially pair with dotted CLs with matching numbers (e.g., "CL-1," "CL-2," "CL-3," etc.), which may be variant CLκ or CLλ domains according to the present disclosure. (13) CH1-CL sets with the same numbering (e.g., the set of CH1-1 and CL-1 and the set of CH1κ-1 and CLκ-1) represent two of the same CH1-CLκ sets (i.e., the substitutions are the same between the sets), and CH1-CL sets with different numbering (e.g., the set of CH1-1 and CL-1 and the set of CH1-2 and CL-2) are two CH1-CL sets that may be the same or different from each other (the substitutions in the two sets may or may not be the same). (14) Pairing between an open CH1 domain having a number (e.g., CH1-1, CH1-2, CH1-3, etc.) and an open CL domain having a matching number (e.g., CL-1, CL-2, CL-3, etc.) may or may not be a preferential pairing; (15) When such pairing between an open CH1 domain and an open CL domain is a preferential pairing, the CH1-CL set may be different from any CH1-CL set that preferentially pairs with each other, e.g., different CH1-CL sets used within the same structure. (16) VH-1 and VL-1 form an antigen-binding site for a first epitope, VH-2 and VL-2 form an antigen-binding site for a second epitope, VH-3 and VL-3 form an antigen-binding site for a third epitope, VH-4 and VL-4 form an antigen-binding site for a fourth epitope, and VH-5 and VL-5 form an antigen-binding site for a fifth epitope;VH-6 and VL-6 form the antigen-binding site for the sixth epitope; (17) all of the first through sixth epitopes may be different from each other, or all of the first through sixth epitopes may not be different from each other; (18) for a given VH-VL pair, if the VH alone confers sufficient specificity for the cognate antigen (e.g., nanobody), the VL may be omitted even if it is shown in a diagram.

[0246] [Figure 2A] Exemplary, non-limiting embodiments of various multispecific antibody structures with which the variant CH1 and / or CL disclosed herein can be used are provided. The antibody on the top left (boxed) is an exemplary basic full-size bispecific antibody, with no hinges or disulfide bonds explicitly shown. The antibody in the box may, for example, include hinges between CH1-1 and CH2-1, and between CH1-2 and CH2-2, with disulfide bonds present between the hinges, between the CH1-1 and CL-1 domains, and between the CH1-2 and CL-2 domains (center). Alternatively, the antibody in the box may, for example, include hinges between CH1-1 and CH2-1, and between CH1-2 and CH2-2, with disulfide bonds present between the hinges, between the CL-1 and hinge, and between the CL-2 and hinge (right). Hinge and disulfide bonds, such as those shown in the center and right antibody structures, may be present in any structure shown in the figures and described herein, even if not explicitly shown.

[0247] [Figure 2B]Exemplary variations of the antibody structure shown in Figure 2A are provided. In some variants of the boxed antibodies, the CH3 domain may be absent (top left) and the CH2 domain may be absent (top right). In some variants, both the CH2 and CH3 domains may be absent (center and bottom). In such cases, hinge and disulfide bonds may be present, as shown in the center. Alternatively, multispecificity may be provided by a mixture of two different Fab fragments of different specificities (bottom left) or a mixture of two different Fab' fragments of different specificities (bottom right). Even when not explicitly shown, any constant domain may be omitted in any of the structures or variations thereof in Figures 2-7, as appropriate.

[0248] [Figure 2C] An exemplary variation of the antibody structure shown in the box in Figure 2A is provided. In addition to the first CH1-CL set, a second CH1-CL set is used. The first and second sets are distinct sets, and the CH1 (i.e., CH1-1) of the first set preferentially binds to the CL (i.e., CL-1) of the first set rather than the CL (i.e., CL-2) of the second set, and the CH1 (i.e., CH1-2) of the second set preferentially binds to the CL (i.e., CL-2) of the second set rather than the CL (i.e., CL-1) of the first set. The structure facilitates improved efficiency in manufacturing, even when not explicitly shown. The equivalent modification depicted in Figure 2C (additional preferentially paired CH1-CL sets) can be applied to any of the other structures or variations thereof in Figures 2-7, as appropriate or desired.

[0249] [Figure 2D]Exemplary variations of the antibody structure shown in the box in Figure 2A are provided. The structures include heavy-heavy chain heteropairing modifications (pairing domains depicted as a triangle added on one domain and removed from the other) within the CH3 (left), CH2 (center), and / or CH3 (right) domains. Two different modification orientations (top vs. bottom) are depicted. Even when not explicitly shown, the equivalent modifications depicted in Figure 2D (addition of heavy-heavy chain heteropairing techniques) can be applied to any of the structures or variations thereof in Figures 2-7, as appropriate.

[0250] [Figure 3] Further exemplary variations of the antibody structure shown in the box in Figure 2A are provided. The VH and VL positions are varied relative to the structure in the box in Figure 2A. Even when not explicitly shown, the equivalent modifications depicted in Figure 3 (switching the VH and VL positions) can be applied to any of the structures or variations thereof in Figures 2-7, as appropriate.

[0251] [Figures 4A-4D] Further exemplary variations of the antibody structure in the box shown in Figure 2A and the variations in Figure 3 are provided. The CH1 and CL positions are varied relative to the structures in Figures 2-3. The equivalent variations shown in Figures 4A-4D (switching the CH1 and CL positions) can also be applied to any of the structures shown in Figures 2-7 or variations thereof, as appropriate, even if not explicitly shown.

[0252] [Figure 5A]Exemplary variations of the antibody structure shown in the box in Figure 2 are provided. Specifically, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added to the N-terminus of the heavy and light chains in different orientations. While both a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are depicted, only one pair (either the third epitope-specific pair or the fourth epitope-specific pair) can be added if desired. The third or fourth additional VH-VL pair may or may not be identical in paratope sequence composition and epitope specificity to the first or second VH-VL pair, respectively. The equivalent variation depicted in Figure 5A (the addition of one or more VH-VL pairs) can also be applied to any of the structures shown in Figures 2-7 or variations thereof, as appropriate, even if not explicitly shown.

[0253] [Figure 5B] An exemplary variation of the antibody structure shown in the box in Figure 5A is provided. Specifically, a constant domain is further added between the two variable domains within the same polypeptide. In other words, a Fab (or Fab-like) fragment specific for a third epitope and a Fab (or Fab-like) fragment specific for a fourth epitope are added to the N-terminal side of the antibody structure shown in the box in Figure 2. Although both a Fab (or Fab-like) fragment specific for a third epitope and a Fab (or Fab-like) fragment specific for a fourth epitope are depicted, if desired, only one Fab (or Fab-like) fragment (only a Fab (or Fab-like) fragment specific for the third epitope or only a Fab (or Fab-like) fragment specific for the fourth epitope) can be added (see, for example, Figure 2C in Klein C. et al., Methods. 2019 Feb 1;154:21-31). The third or fourth additional Fab (or Fab-like) fragments may or may not be identical in paratope sequence composition and epitope specificity to the first or second Fab (or Fab-like) fragment, respectively.

[0254] [Figure 5C]Additional variations of the antibody structures in the boxes in Figure 2 are provided. Similar to the structure in Figure 5A, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added in a different orientation, and the order of VH and VL on the light chain differs from that in Figure 5A. The equivalent variation depicted in Figure 5C (the addition of one or more VH-VL pairs) can also be applied to any of the structures shown in Figures 2-7 or variations thereof, as appropriate, even if not explicitly shown.

[0255] [Figures 6A-6E] Further variations of the antibody structures in the boxes in Figure 2 are provided. Specifically, in Figures 6A-6D, an scFv specific for a third epitope and an scFv specific for a fourth epitope are added. Although two scFvs are depicted, only one scFv can be added if desired. In Figure 6A, the scFvs are added to the C-terminus of the heavy chain. The four structures in Figure 6A differ in the VH-VL order within each scFv. In Figure 6B, the scFvs are added to the C-terminus of the light chain. The four structures in Figure 6B differ in the VH-VL order within each scFv. In Figure 6C, the scFvs are added to the N-terminus of the heavy chain. The four structures in Figure 6C differ in the VH-VL order within each scFv. In Figure 6D, the scFvs are added to the N-terminus of the light chain. The four structures in Figure 6D differ in the VH-VL order within each scFv. Although not shown in Figures 6A-6D, two scFvs can be added at different positions (e.g., one at the C-terminus of the heavy chain and one at the N-terminus of the light chain). In Figure 6E, four scFvs are added to the N-terminus of the heavy and light chains. Although not explicitly shown, the VH-VL order within any one or more of the scFvs can be switched in the same manner as in Figures 6A-6D. Furthermore, more than one scFv can be added at any appropriate location and combination of locations (e.g., at the light chain N-terminus, light chain C-terminus, heavy chain N-terminus, and / or heavy chain C-terminus). The equivalent modification depicted in Figure 6 (addition of one or more scFvs) can also be applied to any of the structures shown in Figures 2-7 or modifications thereof, as appropriate, even if not explicitly shown.

[0256] [Figures 7A-7D]Further variations of the antibody structure in the box in Figure 2 are provided. Specifically, a Fab fragment specific for a third epitope and a Fab fragment specific for a fourth epitope are added to the C-terminus of the heavy chain. Although two Fab fragments are depicted, only one Fab can be added if desired. In Figure 7A, the structure includes at least one CH1-CL set, which may or may not be identical to a variant CH1-CL set according to the present disclosure. As shown in Figure 7B, at least two preferentially pairing CH1-CL sets, the same or different, each of which may or may not be identical to a CH1-CL set according to the present disclosure, can be used. For example, in the case of the central bispecific antibody structure, when the two heavy chains are identical to each other and the two light chains are identical to each other, and the CH1 and CL domains of the CH1-CL set pair exclusively with each other, the use of a CH1-CL set would enable excellent production efficiency and provide only the intended bispecific antibody (i.e., approximately 100% of the product) without the need for heavy-heavy chain heteropairing techniques. Alternatively, as shown in Figure 7C, at least three CH1-CL sets, each of which may or may not be identical to a CH1-CL set according to the present disclosure, may be incorporated, and as shown in Figure 7D, at least four CH1-CL sets, each of which may or may not be identical to a CH1-CL set according to the present disclosure, may be incorporated, and each of which may or may not be identical to a CH1-CL set according to the present disclosure, may be incorporated, and the equivalent modifications (addition of one or more Fab fragments) depicted in Figures 7A-7D may also be applied to any of the structures shown in Figures 2-7 or modifications thereof, as appropriate, even if not explicitly shown.

[0257] [Figures 8A-8D]The results and overall scheme of the screening in Example 1 are shown. Figure 8A provides a graph showing the number of amino acid substitutions within the CH1 and CLκ domains in each of the unique CH1-CLκ pairs identified in the first stage of Example 1 (N=3164). Figure 8B provides a matrix of the number of CLκ-substituted CH1 substitutions in each of the unique CH1-CLκ pairs identified in the first stage of Example 1 (N=3164). Figure 8C provides a plot generated in the second stage of Example 1 showing the Rosetta side chain hydrogen bond score term (ΔGhbond_sc_total) as a function of the total number of substitutions in each of the CH1-CLκ sets. Figure 8D provides a schematic diagram of the screening in Example 1. In the first stage, MC HBNet was used to sample sequence space with side chain rotamer flexibility and a rigid protein backbone, resulting in a set of 3164 unique CH1+CLκ sequences (the results are shown in Figures 8A-8B). In the second stage, the Rosetta optimization step tested whether the HBNet-predicted hydrogen bonds persisted under optimization with both backbone and side-chain flexibility (the results are shown in Figure 8C). The CH1-CLκ set selected in Example 1 underwent in silico screening based on the interface binding energy in Example 2.

[0258] [Figures 9A-9C]The overall scheme and results of the screening in Example 2 are shown. Figure 9A provides a scheme of the screening steps in Example 2. The 20 CH1-CLκ sets selected in Example 2 were subjected to experimental characterization as a single interface design (SID) format in Example 3. Figure 9B provides a graph comparing the interface binding energy change ΔΔGtotal score backrub 18k before (left) and after (right) the WT reversion substitution in the CH1-CLκ sets (individual sets referred to as "individual networks" in Figure 9B), where it was determined in step 6 of Example 6 that the WT reversion set would be carried forward rather than the non-reversion set. For example, when substitution 145Q in CH1 was returned to the WT amino acid residue and substitution 137Q in CLκ was returned to the WT amino acid residue, the CH1-CLκ set referred to as "Network_2529" showed better interface binding energy. Therefore, a WT reversion set including the WT amino acid residue at position 145 in CH1 and the WT amino acid residue at position 129 in CLκ was selected for experimental characterization in Example 3. The graph in Figure 9B further compares the ΔΔGtotal score backrub18k of the designed CH1-CLκ set with that of a mismatched CH1-CLκ set (i.e., a set in which either CH1 or CLκ is designed (i.e., not WT), but the other is WT). Figure 9C provides a graph comparing the interface binding energy change ΔΔGtotal score backrub18k of the carried-forward CH1-CLκ set without reversion.

[0259] [Figure 10]This figure provides a scheme for LC-MS used in the examples to evaluate bsAb production products. The schematic on the left illustrates the workflow. A portion of the produced IgG can be used for truncated full-length LC-MS, which can be used to confirm the sequence and / or quantify the relative expression of different antibody chains. A portion of the produced IgG can be subjected to digestion to produce Fab fragments. A portion of the Fab fragments can be used for truncated LC-MS, and another portion of the Fab fragments can be used for non-truncated LC-MS. The non-truncated LC-MS results provide the correct pairing ratio (correct pairing between heavy and light chains), while the truncated LC-MS results can be used to quantify the relative amounts of different antibody chains after digestion. On the right are exemplary LC-MS data showing different peaks corresponding to different heavy and light chain pairs.

[0260] [Figure 11] Figure 1 shows a matrix providing the calculated RBPPhbond+electrostatic backrun 18k scores for Abs containing two different CH1-CLκ sets. Negative values ​​indicate preferential pairing between the indicated CH1 and CLκ domains, with more negative values ​​indicating more preferential pairing. For example, when network 1443 and network 1993 are used as the two CH1-CLκ sets in a DID Ab, the RBPPhbond+electrostatic backrun 18k score is as low as -6.1.

[0261] [Figure 12]The wild-type CH1-CLκ interface and its electron density (shown in a comparative orientation to that of the human Fab designated ADI-64596 in Figure 13) are shown. Representative electron density within the region of interest for the panitumumab variable fragment (Fv) and Fab crystal structures of the WT CH1 domain of IgG1 paired to the WT CLκ domain are shown. Carbon atoms of the heavy chain (HC) are colored light gray, carbon atoms of the kappa light chain (κLC) are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a gray mesh drawn at 1.0σ with a resolution of 2.0 Å. Data for this crystal structure extend to a near-atomic resolution of 2.6 Å.

[0262] [Figure 13] Figure 1 shows the CH1-CLκ interface of ADI-64596 and its electron density. Representative electron density within the region of interest for the crystal structure of ADI-64596, which contains panitumumab Fv and a variant CH1 domain (of IgG) containing L145Q, K147E, and S181E paired with a CLκ domain containing T129R, T178R, and T180Q (i.e., the CH1-CLκ set of network 1443). HC carbon atoms are colored light gray, κLC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a gray mesh drawn at 1.0σ with a resolution of 2.0 Å. Data for this crystal structure extend to a near-atomic resolution of 2.35 Å.

[0263] [Figure 14]The wild-type CH1-CLκ interface and its electron density (shown in a comparative orientation) paired with a human Fab named ADI-64597 in FIG. 15 are shown. Representative electron density within the region of interest for the panitumumab Fv and Fab crystal structures of the WT CH1 domain of IgG1 paired with the WT CLκ domain are shown. H-C carbon atoms are colored light gray, kappa LC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a gray mesh drawn at 1.0σ at 2.0 Å resolution. Data for this crystal structure extend to near-atomic resolution of 2.6 Å.

[0264] [Figure 15] The ADI-64597 CH1-CLκ interface and its electron density are shown. Representative electron density within the region of interest for the crystal structure of ADI-64597, which contains panitumumab Fv and a variant CH1 domain (of IgG) containing L128R and K147R paired with a CLκ domain containing Q124E, V133Q, and T178E (i.e., the CH1-CLκ set of network 1993). HC carbon atoms are colored light gray, κLC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a gray mesh drawn at 1.0σ with a resolution of 2.0Å. Data for this crystal structure extend to near-atomic resolution of 2.2Å.

[0265] [Figure 16]Figure 1 shows substitutions at the CH1-CLκ interface (network 1443) present in ADI-64596 Fab that involve approximately 12 additional polar contacts not present in the panitumumab WT CH1-CLκ interface. Representation of the novel hydrogen bond network at the ADI-64596 CH1-CLκ interface. HC carbon atoms are colored light gray, LC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are shown in stick representation, while the main chain is shown as a cartoon. Hydrogen bonds are shown as black dotted lines, while salt bridges are shown as light gray dotted lines.

[0266] [Figure 17] Figure 1 shows substitutions at the CH1-CLκ interface (Network 1993) present in ADI-64597 Fab that are involved in several additional polar contacts not present in the panitumumab WT CH1-CLκ interface. Representation of novel polar contacts at the ADI-64597 CH1-CLκ interface. HC carbon atoms are colored light gray, LC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Main chains are shown as cartoon or stick representations, while side chains are shown in stick representations. Hydrogen bonds are shown as black dotted lines, while salt bridges are shown as light gray dotted lines.

[0267] [Figure 18]Several substitutions in the ADI-64597 CH1 domain (i.e., Network 1993 CH1 domain) and the orthogonal ADI-64596 CLκ domain (i.e., Network 1443 CLκ domain) that are predicted to sterically clash with each other and reduce the propensity for mispairing are shown. (a-c) Representation of the pairing interface surrounding the region of interest. Alignment of the ADI-64597 and ADI-64596 constant regions reveals steric clashes at the CH1-CLκ interface of several substituted and unsubstituted positions for this mispairing construct, including (a) L128R in CH1 and V at position 133 in CLκ, (b) K147R in CH1 and T129R in CLκ, and (c) S at position 183 in CH1 and T178R in CLκ. HC carbon atoms are colored light gray, LC carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are shown in stick representation, side chains involved in collisions are shown with transparent molecular surfaces, and main chain atoms are shown in cartoon representation.

[0268] [Figure 19] Figure 1 shows a matrix providing the calculated RBPPhbond+electrostatic backrun 18k scores for Abs containing two different CH1-CLκ sets. Negative values ​​indicate preferential pairing between the indicated CH1 and CLλ domains, with more negative values ​​indicating more preferential pairing. For example, when network 367 and network 1612 are used as the two CH1-CLλ sets in a DID Ab, the RBPPhbond+electrostatic backrun 18k score is as low as -4.8. DETAILED DESCRIPTION OF THE INVENTION

[0269] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0270] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 95 and 105, and all values ​​therebetween (e.g., 96, 99, 99.5, 100.5, 104, etc.).

[0271] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0272] The terms "antibody" or "Ab" are used in the broadest sense and encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably, fragments that exhibit the desired antigen-binding activity, also referred to as "antigen-binding antibody fragments"). A "complete antibody," "complete Ab," "full-size antibody," "full-size Ab," "full-length antibody," "intact antibody," or "whole antibody," or equivalents, encompass molecules having substantially the same structure as a natural antibody. For example, an intact IgG (or IgD or IgE) antibody contains two heavy immunoglobulin chains and two light immunoglobulin chains. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to an intact antibody, or a combination of portions from multiple intact antibodies, that binds to the antigen bound by one or more intact antibodies.

[0273] In some cases, a full-size antibody, e.g., a full-size IgG or IgG-like antibody, comprises four polypeptide chains: two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC comprises a variable region, such as a heavy chain variable region ("VH"), and a heavy chain constant region ("CH"). In the case of an intact antibody, the CH comprises a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. In the case of an antibody fragment, when the fragment comprises a CH, the CH may comprise a CH1 domain, a hinge, a CH2 domain, and / or a CH3 domain; in some preferred embodiments, the CH comprises at least a CH1 domain. The variant CH1 domains disclosed herein can be used in combination with wild-type CH2 and / or CH3 domains, or CH2 and / or CH3 domains containing one or more amino acid substitutions, e.g., that alter or improve antibody stability and / or effector function and / or that promote CH3 heterodimerization. Optionally, a hinge can also be used. Each LC comprises a variable region, such as a light chain variable region ("VL"), and a light chain constant region ("CL"). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be naturally or artificially modified. A consensus sequence of amino acids can be defined based on a side-by-side comparative analysis of two or more CDRs. Thus, the three CDRs in the heavy chain are designated "CDRH1," "CDRH2," and "CDRH3," and the three CDRs in the light chain are designated "CDRL1," "CDRL2," and "CDRL3." In other cases, the antibody may include multimers thereof (e.g., IgM) or antigen-binding fragments thereof.

[0274] The light chain constant region (CL) domain of an antibody refers to the constant domain of the antibody light chain located at the C-terminus of the variable region of the light chain. There are two major CL isotypes, kappa ("k") and lambda ("λ"), and such CL domains are referred to herein as kappa CL domains ("CLκ" domains) and lambda CL domains ("CLλ" domains). Unless specified, a CL domain may be a CLκ or a CLλ. In some cases, a CLκ domain may have an amino acid sequence encoded by any of the functional IGKC genes listed by the IGMT. In some cases, a CLλ domain may have an amino acid sequence encoded by any of the functional IGLC genes listed by the IGMT.

[0275] Numbering of amino acid residues in antibody variable and constant domains may be performed according to the EU index or EU numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Unless otherwise specified, the EU numbering system is used herein.

[0276] An "antigen-binding fragment of an antibody" or "antigen-binding antibody fragment" includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that comprises an antibody domain (e.g., a VH domain or a CH3 domain) and specifically binds to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fvs, antigen-binding fragment ("Fab") fragments, Fab' fragments, Fab' containing a free sulfhydryl group ("Fab'-SH"), F(ab')2 fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain variable fragments ("scFv"), nanobodies, or VHHs, or only the 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 or nanobody. In some embodiments, the antigen-binding fragment comprises a variant CH1 domain, a variant CLκ domain, and / or a variant CH1-CLκ set that preferentially forms a CH1-CLκ pair rather than another CH1-CL pair. In some preferred embodiments, the antigen-binding fragment comprises a variant CH1 domain, a variant CLλ domain, and / or a variant CH1-CLλ set that preferentially forms a CCH1-CLλ pair rather than another CH1-CL pair.

[0277] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). A multispecific antigen-binding fragment of an antibody can comprise at least two antigen-binding sites (each comprising at least one variable region, such as a VH or VL), capable of specifically binding to different antigens or epitopes.

[0278] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations and / or substitutions or arising during production of the monoclonal antibody preparation), which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0279] A "multispecific antibody," which may also be referred to herein as a "multispecific compound," refers to an antibody comprising at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens and / or at least two different epitopes. In some embodiments, a multispecific antibody comprises (1) a first heavy chain and a first light chain that form a cognate pair and bind to a first antigen, and (2) a second heavy chain and a second light chain that form a cognate pair and bind to a second antigen.

[0280] A "bispecific antibody," which may also be referred to herein as a "bispecific compound," is a type of multispecific antibody and refers to an antibody that contains two different antigen-binding domains that recognize and specifically bind to at least two different antigens or at least two epitopes. The at least two epitopes may or may not be within the same antigen. A bispecific antibody may, for example, target two different surface receptors, two different cytokines / chemokines, receptors, and ligands on the same or different cells (e.g., an immune cell and a cancer cell).

[0281] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes within any of the following antigens): CD3, 0772P (CA125, MUC16; Genbank Accession Number AF36148), adipophilin (perilipin-2, adipose differentiation-related protein, ADRP, ADFP, MGC10598; NCBI Reference Sequence: NP-001113.2), AIM-2 (absent in melanoma 2, PYHIN4, interferon-inducible protein AIM2; NCBI Reference Sequence: NP-004824.1), ALDH1 A1 (aldehyde dehydrogenase 1 family, member A1, ALDH1, PUMB1, retinaldehyde dehydrogenase 1, ALDC, ALDH-E1, ALHDII, RALDH 1, EC 1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1, acetaldehyde dehydrogenase 1, aldehyde dehydrogenase 1, soluble aldehyde dehydrogenase, hepatic cytosolic ALDH class 1, epididymal luminal protein 12, epididymal luminal protein 9, epididymal secretory sperm-binding protein Li 53e, retinal dehydrogenase 1, RaIDH1, aldehyde dehydrogenase family 1 member A1, aldehyde dehydrogenase, cytoplasmic, EC 1.2.1; NCBI Reference Number: NP-000680.2); alpha-actinin-4 (ACTN4, actinin, alpha 4, FSGS1, focal segmental glomerulosclerosis 1, non-muscle alpha-actinin 4, F-actin cross-linking protein, FSGS, actinin-4, actinin alpha 4, alpha-actinin-4; NCBI Reference Sequence: NP-004915.2); alpha-fetoprotein (AFP, HPAFP, FETA, alpha-1-fetoprotein, alpha-fetoglobulin, alpha-1-fetoprotein, alpha-fetoglobulin, HP; GenBank: AAB58754.1); amphiregulin (AREG, SDGF, schwannoma-derived growth factor, colorectal cell-derived growth factor, AR, CRDGF; GenBank: AAA51781.1); ARTC1 (ART1, ADP-ribosyltransferase 1, mono(ADP-ribosyl)transferase 1, ADP-ribosyltransferase C2 and C3 toxin-like 1, ART2, CD296, RT6, ADP-ribosyltransferase 2, GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1, EC 2.4.2.31, CD296 antigen; NP); ASLG659; ASPHD1 (aspartate beta-hydroxylase domain containing 1, aspartate beta-hydroxylase domain-containing protein 1, EC1.14.11, GenBank: AAI44153.1); B7-H4 (VTCN1, V-Set domain-containing T-cell activation inhibitor 1, B7H4, B7 superfamily member 1, immune costimulatory protein B7-H4, B7h.5, T-cell costimulatory molecule B7x, B7S1, B7X, VCTN1, H4, B7 family member; PRO1291, B7 family member, H4, T-cell costimulatory molecule B7x, V-Set domain-containing T-cell activation inhibitor 1, protein B7S1; GenBank: AAZ17406.1 ); BAFF-R (TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, BAFFR, B-cell activating factor receptor, BAFF receptor, BLyS receptor 3, CVID4, BROMIX, CD268, B-cell activating factor receptor, prolixin, tumor necrosis factor receptor superfamily member 13C, BR3, CD268 antigen; NCBI sequence NP-443177); BAGE-1; BCLX(L); BCR-ABL fusion protein (b3a2); beta-catenin (CTNNB1, catenin (cadherin-associated protein), beta 1, 88 kDa, CTNNB, MRD19, catenin (cadherin-associated protein), beta 1 (88 kDa), armadillo, catenin beta-1; GenBank: CAA61107.1); BING-4 (WDR46, WD repeat domain 46, C6orf11, BING4, WD repeat-containing protein BING4, chromosome 6 open reading frame 11, FP221, UTP7, WD repeat-containing protein 46; NP); BMPR1 B (bone morphogenetic protein receptor type IB, GenBank accession number NM-00120; NP); B-RAF (Brevican (BCAN, BEHAB, GenBank accession number AF22905); brevican (BCAN, chondroitin sulfate proteoglycan 7, brain-enriched hyaluronan-binding protein, BEHAB, CSPG7, brevican proteoglycan, brevican core protein, chondroitin sulfate proteoglycan BEHAB; GenBank: AAH27971.1)); CALCA (calcitonin-related polypeptide alpha, CALC1, cal Cytokinin 1, calcitonin, alpha-type CGRP, calcitonin gene-related peptide I, CGRP-I, CGRP, CGRP1, CT, KC, calcitonin / calcitonin-related polypeptide, alpha, katacalcin; NP); CASP-5 (CASP5, caspase 5, apoptosis-related cysteine ​​peptidase, caspase 5, apoptosis-related cysteine ​​protease, protease ICH-3, protease TY, ICE(rel)-111, ICE(rel)III, ICEREL-III, ICH-3, caspase-5, TY protease, EC 3.4.22.58, ICH3, EC 3.4.22; NP); CASP-8; CD19 (CD19 - B-lymphocyte antigen CD19 isoform 2 precursor, B4, CVID3 [Homo sapiens], NCBI Reference Sequence: NP-001761.3); CD20 (CD20 - B-lymphocyte antigen CD20, transmembrane 4 domain, subfamily A, member 1, B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7; NCBI Reference Sequence: NP-690605.1); CD21 (CD21 (CR2 (complement receptor) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 Genbank accession number M2600); (CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, LybB, SIGLEC-2, FLJ22814, Genbank accession number AK02646); CD22; CD33 (CD33 molecule, CD33 antigen (Gp67), sialic acid-binding Ig-like lectin 3, sialic acid-binding Ig-like lectin 3, SIGLEC3, gp67, SIGLEC-3, myeloid cell surface antigen CD33, p67 , Siglec-3, CD33 antigen; GenBank: AAH28152.1); CD45; CD70 (CD70 tumor necrosis factor (ligand) superfamily, member 7; surface antigen CD70, Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factor ligand superfamily member 7; NCBI reference sequence for Homo sapiens species: NP-001243.1); CD72 (CD72 (B cell differentiation antigen CD72, Lyb-; 359 aa, μl:8.66, MW:40225, TM:1[P] gene chromosome:9p13.3, Genbank accession number NP-001773.); CD79a (CD79a (CD79A, CD79a, immunoglobulin-related alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B), forms a complex with Ig M molecules on the surface, and transduces signals involved in B cell differentiation), μl:4.84, MW:25028 TM:2[P] gene chromosome: 19q13.2, GenBank accession number NP-001774.1; CD79b (CD79b (CD79B, CD79b, IGb (immunoglobulin-related beta), B29, GenBank accession number NM-000626 or 1103867); Cdc27 (cell division cycle 27, D0S1430E, D17S978E, anaphase-promoting complex subunit 3, anaphase-promoting complex subunit 3, ANAPC3, APC3, C DC27Hs, H-NUC, CDC27 homolog, cell division cycle 27 homolog (S. Cerevisiae), HNUC, NUC2, anaphase-promoting complex, protein 3, cell division cycle 27 homolog, cell division cycle protein 27 homolog, Nuc2 homolog; GenBank: AAH11656.1), CDK4 (cyclin-dependent kinase 4, cell division protein kinase 4, PSK-J3, EC2.7.11.22, CMM3, EC2.7.11;NCBI Reference Sequence: NP-000066.1); CDKN2A (cyclin-dependent kinase inhibitor 2A, MLM, CDKN2, MTS1, cyclin-dependent kinase inhibitor 2A (inhibits melanoma, P16, CDK4), cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK4A, P14, P14ARF, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16, CDK4 inhibitor P16-INK4, cell cycle negative regulator beta, p14ARF, p16-INK4, p16-INK4a, p16INK4A, p19ARF, NP); CEA; CLL1 (CLL-1 (CLEC12A, MICL, DCAL, It encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is tightly linked to other CTL / CTLD superfamily members within the natural killer gene complex region on chromosome 12p13 (Drickamer, K et al., 2013). Curr.Opin.Struct.Biol.9:585-90

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[2004] , Marshall AS, et al J.Biol.Chem.279:14792-80,2004.CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing ITIM motifs. CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit, endopeptidase Clp, EC 3.4.21.92, PRLTS3, ATP-dependent protease ClpAP (E. coli), ClpP (caseinolytic protease, ATP-dependent, proteolytic subunit, E. coli) homolog, ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli), ClpP caseinolytic protease, ATP-dependent, proteolytic subunit homolog (E. coli), human, proteolytic subunit, ATP-dependent protease ClpAP, proteolytic subunit, human, ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit, ClpP caseinolytic peptidase, ATP-dependent, proteolytic, proteolysis subunit homolog, ClpP caseinolytic protease, ATP-dependent, proteolytic subunit homolog, putative ATP-dependent Clp protease proteolytic subunit, mitochondrial; NP; COA-1; CPSF; CRIPTO (CRPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP-003203 or NM-00321); Cw6; CXCR5 (Burkitt's lymphoma receptor 1, G protein-coupled receptor activated by CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly AIDS, lymphoma, myeloma, and leukemia development); 372 aa, μl: 8.54, MW: 41959, TM: 7 [P] gene chromosome: 11q23.3, GenBank accession number NP-001707.); CXORF61 CXORF61 - Chromosome X open reading frame 61 [Homo sapiens], NCBI reference sequence: NP-001017978.1); cyclin D1 (CCND1, BCL1, PRAD1, D11S287E, B-cell CLL / lymphoma 1, B-cell lymphoma 1 protein, BCL-1 oncogene, PRAD1 oncogene, cyclin D1 (PRAD1: parathyroid adenomatosis 1), G1 / S-specific cyclin D1, parathyroid adenomatosis 1, U21B31, G1 / S-specific Cyclin-D1, BCL-1; NCBI reference sequence: NP-444284.1), cyclin-A1 (CCNA1, CT146, cyclin A1; GenBank: AAH36346.1); dek-can fusion protein; DKK1 (Dickkopf WNT signaling pathway inhibitor 1, SK, hDkk-1, Dickkopf (Xenopus laevis) homolog 1, Dickkopf 1 homolog (Xenopus Laevis), DKK-1, Dickkopf 1 homolog, Dickkopf-related protein-1, Dickkopf-1-like, Dickkopf-like protein 1, Dickkopf-related protein 1, Dickkopf-1, Dkk-1; GenBank: AAQ89364.1); DR1 (downregulator of transcription 1, TBP-binding (negative cofactor 2), cofactor 2-beta, TATA-binding protein-associated phosphoprotein, NC2, NC2-beta, protein Dr1, NC2-beta, downregulator of transcription 1; NCBI Reference Sequence: NP-001929.1); DR13 (major histocompatibility complex, class II, DR beta 1, HLA-DR1B, DRw10, DW2.2 / DR2.2, SS1, DRB1, HLA-DRB, HLA class II histocompatibility antigen, DR-1 beta chain, human leukocyte antigen DRB1, lymphocyte antigen DRB1, MHC class II antigen, MHC class II antigen, MHC class II HLA-DR beta 1 chain, MHC class II HLA-DR-beta cell surface glycoprotein, MHC class II HLA-DRw10-beta DR-1, DR-12, DR-13, DR-14, DR-16, DR-4, DR-5, DR-7, DR-8, DR-9, DR1, DR12, DR13, DR14, DR16, DR4, DR5, DR7, DRB, DR9, DRw11, DRw8, HLA-DRB2, clone P2-beta-3, MHC class II antigen DRB1*1, MHC class II antigen DRB1*10, MHC class II antigen DRB1*11, MHC class II antigen DRB1*12, MHC class II antigen DRB1*13, MHC class II antigen DRB1*14, MHC class II antigen DRB1*15, MHC class II antigen DRB1*16, MHC class II antigen DRB1*3, MHC class MHC class II antigen DRB1*4, MHC class II antigen DRB1*7, MHC class II antigen DRB1*8, MHC class II antigen DRB1*9; NP); E16 (E16 (LAT1, SLC7A5, Genbank accession number NM-00348); EDAR (EDAR tumor necrosis factor receptor superfamily member EDAR precursor; EDA-A1 receptor; Downless homolog; Ectodysplasin-A receptor; Ectodermal dysplasia receptor; Anhidrotic ectodysplasin receptor 1, DL; ECTD10A; ECTD10B; ED1R; ED3; ED5; EDA-A1R; EDA1R; EDA3; HRM1 [Homo sapiens], NCBI reference sequence: NP-071731.1); EFTUD2 (elongation factor Tu GTP-binding domain-containing 2, elongation factor Tu GTP-binding domain-containing protein 2, hSNU114, SNU114 homolog, U5 SnRNP-specific protein, 116 KDa, MFDGA, KIAA0031, 116 KD, U5 SnRNP-specific protein, 116 KDa U5 small nuclear ribonucleoprotein component, MFDM, SNRNP116, Snrp116, Snu114, U5-116 KD, SNRP116, U5-116 KDa, GenBank:AAH02360.1); EGFR (epidermal growth factor receptor, ERBB, proto-oncogene C-ErbB-1, receptor tyrosine-protein kinase ErbB-1, ERBB1, HER1, EC 2.7.10.1, epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog), erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian), PlG61, avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog, cell proliferation inhibitory protein 40, cell proliferation inducer protein 61, mENA, EC 2.7.10; GenBank: AAH94761.1); EGFR-G719A; EGFR-G719C; EGFR-G719S; EGFR-L858R; EGFR-L861 Q; EGFR-57681; EGFR-T790M; elongation factor 2 (EEF2, eukaryotic translation elongation factor 2; EF2, polypeptidyl-tRNA translocase; EF-2; SCA26; EEF-2; NCBI reference sequence: NP-001952.1); ENAH (hMena) (corresponding homolog (Drosophila); MENA, mammalian counterpart; ENA, NDPP1; protein corresponding homolog; GenBank: AAH95481.1) - results for only "ENAH" and not "ENAH (hMena)"; EpCAM (epithelial cell adhesion molecule; M4S1; MIC18; tumor-associated Transient calcium signaling receptor 1, TACSTD1, TROP1, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, major gastrointestinal tumor-associated protein GA733-2, EGP314, KSA, DIAR5, HNPCC8, antigen identified by monoclonal antibody AUA1, EGP-2, EGP40, ESA, KS1 / 4, MK-1, human epithelial glycoprotein-2, membrane component, chromosome 4, surface marker (35 kD glycoprotein), EGP, Ep-CAM, GA733-2, M1S2, CD326 antigen, epithelial cell surface antigen, hEGP314, KS 1 / 4 antigen, ACSTD1; GenBank: AAH14785.1); EphA3 (EPH receptor A3, ETK1, ETK; TYRO4, HEK, Eph-like tyrosine kinase 1, tyrosine-protein kinase receptor ETK1; EK4, EPH-like kinase 4, EC2.7.10.1, EPHA3, HEK4, ephrin type A receptor 3, human embryonic kinase 1, TYRO4 protein tyrosine kinase, hEK4, human embryonic kinase, tyrosine-protein kinase TYRO4, EC 2.7.10; GenBank: AAH63282.1);. EphB2R; epiregulin (EREG, ER, propylegulin; GenBank: AAI36405.1); ETBR (EDNRB, type B endothelin receptor; HSCR2, HSCR, nonselective endothelin receptor; ET-B, ET-BR; ETRB, ABCDS; WS4A; ETB, endothelin B receptor; NP); ETV6-AML1 fusion protein; EZH2 (enhancer of zest homolog 2 (Drosophila)); lysine N-methyltransferase 6; ENX-1; KMT6 EC 2.1.1.43, EZH1, WVS, enhancer of zest (Drosophila) homolog 2, ENX1, EZH2b, KMT6A, WVS2, histone-lysine N-methyltransferase EZH2, enhancer of zest homolog 2, EC2.1.1; GenBank: AAH10858.1); FcRH1 (FCRL1, Fc receptor-like 1; FCRH1, Fc receptor homolog 1, FcR-like protein 1, immunoreceptor translocation-associated protein 5; IFGP1, IRTA5, hIFGP1, IFGP family protein 1; CD307a, Fc receptor-like protein 1, immunoglobulin superfamily Fc receptor; Gp42; FcRL1; CD307a Antigen; GenBank: AAH33690.1; FcRH2 (FCRL2, Fc receptor-like 2, SPAP1, SH2 domain-containing phosphatase anchor protein 1, Fc receptor homolog 2, FcR-like protein 2, immunoglobulin receptor translocation-associated protein 4, FCRH2, IFGP4, IRTA4, IFGP family protein 4, SPAP1A, SPAP1 B, SPAP1C, CD307b, Fc receptor-like protein 2, immunoreceptor translocation-associated protein 4, immunoglobulin superfamily Fc receptor, Gp42, SH2 domain-containing phosphatase anchor protein 1, FcRL2, CD307b antigen; GenBank: AAQ88497.1; FcRH5 (FCRL5, Fc receptor-like 5; IRTA2, F receptor homolog 5, FcR-like protein 5, immunoreceptor translocation-associated protein 2; BXMAS1, FCRH5, CD307, CD307e, PRO820, Fc receptor-like protein 5, immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), FCRL5, CD307e antigen; GenBank: AAI01070.1); FLT3-ITD; FN1 (fibronectin 1, cold-insoluble globulin, FN, migration-stimulating factor, CIG, FNZ, GFND2, LETS, ED-B, FINC, GFND, MSF, fibronectin; GenBank: AAI43764.1); G250 (MN, CAIX, carbonic anhydrase IX, carbonic anhydrase, RCC-associated protein G250, carbonic anhydrase IX, membrane antigen MN, Renal cell carcinoma-associated antigen G250, CA-IX, P54 / 58N, pMW1, RCC-associated antigen G250, carbonic anhydrase 9 (NP); alias results for "G250" instead of "G250 / MN / CAIX"; GAGE-1, 2, 8; GAGE-3, 4, 5, 6, 7; GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1 L; GDNFR-alpha; GFR-alpha-; U95847; BC014962; NM-145793NM-005264; GEDA (Genbank accession number AY26076); GFRA1 - GDNF family receptor alpha-1; GDNF receptor alpha-1; GDNFR-alpha-1; GFR-alpha-1; RET ligand 1; TGF-beta-related neurotrophic factor receptor 1 [Homo sapiens], ProtKB / Swiss-Prot:P56159.2; glypican-3 (GPC3, glypican 3, SDYS, glypican proteoglycan 3, intestinal protein OCI-5, GTR2-2, MX R7, SGBS1, DGSX, OCI-5. SGB, SGBS, heparan sulfate proteoglycan, secreted glypican-3, OCI5; GenBank: AAH35972.1); GnTVf; gp100 (PMEL, pre-melanosome protein; SILV, D12S53E; PMEL17; SIL, melanocyte protein; Pmel17, melanocyte lineage-specific antigen; GP100, melanoma-associated ME20 antigen, silver locus protein homolog; ME20-M, ME20M; P1, P100, silver (mouse homolog)-like, silver homolog (mouse); ME20; SI, melanoma protein Mel 17, melanoma protein; PMEL, melanosome matrix protein 17; silver, mouse, homolog; GenBank: AAC60634.1); GPC; GPNMB (glycoprotein (transmembrane) Nmb, glycoprotein NMB, glycoprotein Nmb-like protein, osteoactivin, transmembrane glycoprotein HGFIN, HGFIN, NMB, transmembrane glycoprotein, transmembrane glycoprotein NMB; GenBank: AAH32783.1); GenBank: AAH32783.1); GPR172A (G protein-coupled receptor 17 2A; GPCR41; FLJ11856; D15Ertd747e; NP-078807.1; NM-024531.3); GPR19 (G protein-coupled receptor 19; Mm.478; NP-006134.1; NM-006143.2); GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR1; NP-115940.2; NM-032551.4); HA VCR1 (hepatitis A virus cellular receptor 1, T-cell immunoglobulin mucin family member 1, kidney injury molecule 1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1, T-cell immunoglobulin mucin receptor 1, T-cell membrane protein 1, HAVCR, HAVCR-1, T-cell immunoglobulin domain and mucin domain protein 1, HAVcr-1, T-cell immunoglobulin and mucin domain-containing protein 1, GenBank: AAH13325.1); HER2 (ERBB2, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2, NGL, NEU, neuro / glioblastoma-derived oncogene homolog, metastatic lymph node gene 19 protein, proto-oncogene C-ErbB-2, proto-oncogene Neu, tyrosine kinase cell surface receptor HER2, MLN 19, p185erbB2, EC 2.7.10.1, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2 (neuronal / glioblastoma-derived oncogene homolog), CD340, HER-2, HER-2 / neu, TKR1, C-Erb B2 / Neu protein, herstatin, neuroblastoma / glioblastoma-derived oncogene homolog, receptor tyrosine-protein kinase ErbB-2, V-Erb-B2 erythroblastic leukemia viral oncogene homolog 2, neuronal / glioblastoma-derived oncogene homolog, MLN19, CD340 antigen, EC 2.7.10;NP);HER-2 / neu-alias as above;HERV-K-MEL;HLA-DOB (beta subunit of MHC class II molecules (la antigens) that bind peptides and present them to CD4+ T lymphocytes);273 aa, μl: 6.56, MW: 30820.TM: 1 [P] Gene chromosome: 6p21.3, GenBank accession number NP-002111); hsp70-2 (HSPA2, heat shock 70 kDa protein 2, heat shock 70 kDa protein 2, HSP70-3, heat shock associated 70 KDa protein 2, heat shock 70 KDa protein 2; GenBank: AAD21815.1); IDO1 (indoleamine 2,3-dioxygenase 1, IDO, INDO, indoleamine-pyrrole 2,3-dioxygenase, IDO-1, indoleamine-pyrrole 2,3 dioxygenase, indoleamine 2,3 dioxygenase, indole 2,3 dioxygenase, EC 1.13.11.52; NCBI Reference Sequence: NP-002155.1); IGF2B3; IL13Ralpha2 (IL13RA2, interleukin-13 receptor, alpha 2, cancer / testis antigen 19, interleukin-13-binding protein, IL-13R-alpha-2, IL-13RA2, IL-13 receptor subunit alpha-2, IL-13R subunit alpha-2, CD213A2, CT19, IL-13R, IL13BP, interleukin-13 binding protein, interleukin-13 receptor alpha 2 chain, interleukin-13 receptor subunit alpha IL-2, IL13R, CD213a2 antigen; NP); IL20Ra; intestinal carboxylesterase; IRTA2 (another name for FcRH5); kallikrein 4 (KLK4, kallikrein-related peptidase 4, PRSS17, EMSP1, enamel matrix serine proteinase 1, kallikrein-like protein 1, serine protease 17, KLK-L1, PSTS, AI2A1, kallikrein 4 (protase, enamel matrix, prostate), ARM1, EMSP, androgen-regulated message 1, enamel matrix serine protease 1, kallikrein, kallikrein-4, prostase, EC 3.4.21.-, prostase, EC 3.4.21; GenBank: AAX30051.1); KIF20A (kinesin family member 20A, RAB6KIFL, RAB6 interacting, kinesin-like (rabkine 6), mitosis a; LAGE-1; LDLR-fucosyltransferase AS fusion protein; lengusin (LGSN, lengusin, lens protein with glutamine synthetase domain); GLULD1, glutamate-ammonia ligase domain-containing protein 1; LGS, glutamate-ammonia ligase (glutamine synthetase) domain-containing 1, glutamate-ammonia ligase Glutamine synthase domain-containing 1, lens glutamine synthase-like; GenBank: AAF61255.1; LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR6; NP-003658.1; NM-003667.2); LY64 (lymphocyte antigen 64 (RP10, a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa, μl: 6.20, MW: 74147 TM:1[P] gene chromosome: 5q12, GenBank accession number NP-005573.; Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-; NP-002337.1; NM-002346.2); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT; NP-067079.2; NM-021246.2); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ3522; NP-059997.3; NM-017527.43); LyPD1-LY6 / PL AUR domain-containing 1, PHTS [Homo sapiens], GenBank: AAH17318.1; MAGE-A1 (melanoma antigen family A, 1 (antigen MZ2-E, MAGE1, melanoma antigen family A1, MAGEA1, melanoma antigen MAGE-1, melanoma-associated antigen 1, melanoma-associated antigen MZ2-E, antigen MZ2-E, cancer / testis antigen 1.1, CT1.1, MAGE-1 antigen, cancer / testis antigen family 1, member 1, cancer / testis antigen family 1, member 1, MAGE1A); NCBI reference sequence: NP-004979.3); MAGE-A10 (MAGEA10, melanoma antigen family A, 10; MAGE10, MAGE-10 antigen, melanoma-associated antigen 10; cancer / testis antigen 1.10; CT1.10, cancer / testis antigen family 1, member 10; cancer / testis antigen family 1, member 10; NCBI reference sequence: NP-001238757.1); MAGE-A12 (MAGEA12, melanoma antigen family A, 12; MAGE12, cancer / testis antigen 1.12; CT1.12, MAGE12F antigen, cancer / testis antigen family 1, member 12; cancer / testis antigen family 1, member 12; Focal antigen family 1, member 12, melanoma-associated antigen 12, MAGE-12 antigen; NCBI reference sequence: NP-001159859.1); MAGE-A2 (MAGEA2, melanoma antigen family A, 2, MAGE2, cancer / testis antigen 1.2, CT1.2; MAGEA2A, MAGE-2 antigen, cancer / testis antigen family 1, member 2, cancer / testis antigen family 1, member 2, melanoma antigen 2, melanoma-associated antigen 2, NCBI reference sequence: NP-001269434.1); MAGE-A3 (MAGEA3, melanoma antigen family A, 3, MAGE3, MAGE-3 antigen, antigen MZ2-D, melanoma-associated antigen 3, cancer / testis antigen 1.3, CT1.3, cancer / testis antigen family 1, member 3, HIPS, HYPD, MAGEA6, cancer / testis antigen family 1, member 3; NCBI reference sequence: NP-005353.1); MAGE-A4 (MAGEA4, melanoma antigen family A, 4); MAGE4, ​​melanoma-associated antigen 4, cancer / testis antigen 1.4, CT1.4, MAGE-4 antigen, MAGE-41 antigen, MAGE-X2 antigen, MAGE4A, MAGE4B, cancer / testis Antigen family 1, member 4, MAGE-41, MAGE-X2, cancer / testis antigen family 1, member 4; NCBI reference sequence: NP-001011550.1); MAGE-A6 (MAGEA6, melanoma antigen family A, 6); MAGE6, MAGE-6 antigen, melanoma-associated antigen 6; cancer / testis antigen 1.6, CT1.6; MAGE3B antigen, cancer / testis antigen family 1, melanoma antigen family A6, member 6; MAGE-3b, MAGE3B, cancer / testis antigen family 1, member 6; NCBI reference sequence: NP-787064.1); MAGE-A9 (MAGEA9, melanoma antigen family A, 9; MAGE9, MAGE-9 antigen, melanoma-associated antigen 9; cancer / testis antigen 1.9; CT1.9, cancer / testis antigen family 1, member 9; cancer / testis antigen family 1, member 9; MAGEA9A; NCBI reference sequence: NP-005356.1); MAGE-C1 (MAGEC1, melanoma antigen family C, 1; cancer / testis antigen 7.1; CT7.1, MAGE-C1 antigen, cancer / testis antigen family 7, member 1; CT7, cancer / testis antigen family 7, member 1; melanoma-associated antigen C1; NCBI reference sequence: NP-005453.2); MAGE-C2 (MAGEC2, melanoma antigen family C, 2; MAGEE1, cancer / testis antigen 10, CT10; HCA587, melanoma antigen family E, 1, cancer / testis specific; hepatocellular carcinoma-associated antigen 587; MAGE-C2 antigen; MAGE-E1 antigen; hepatocellular carcinoma antigen 587; melanoma-associated antigen C2; NCBI reference sequence: NP-057333.1); mammaglobin-A (SCGB2A2, secretoglobin, family 2A, member 2; MGB1, mammaglobin 1; UGB2, mammaglobin A; mammaglobin-A, mammaglobin-1, secretoglobin family 2A member 2; NP); MART2 (H HAT, hedgehog acyltransferase; SKI1, T cell-recognized melanoma antigen 2; skinny hedgehog protein 1; Skn, T cell-recognized melanoma antigen 2; protein-cysteine ​​N-palmitoyltransferase HHAT, EC 2.3.1.-; GenBank: AAH39071.1); M-CSF (CSF1, colony-stimulating factor 1 (macrophage); MCSF, CSF-1; ranimostim, macrophage colony-stimulating factor 1; ranimostim; GenBank: AAH21117.1); MCSP (SMCP, sperm mitochondrial-associated cysteine-rich protein; MCS, mitochondrial sheath selenoprotein; HSMCSGEN1, sperm mitochondrial-associated cysteine-rich protein; NCBI reference sequence: NP-109588.2); XAGE-1b / GAGED2a; WT1 (Wilms tumor 1, WAGR, GUD, WIT-2, WT33, amino-terminal domain of EWS, NPHS4, last three zinc fingers of the DNA-binding domain of WT1, AWT1, Wilms tumor protein, EWS-WT1; GenBank: AAB33443.1); VEGF; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP; NP-000363.1; NM-000372.4; GenBank: AAB60319.1); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, GenBank accession number NM-01763); TRP2-INT2; TRP-2; TRP-1 / gp75 (tyrosinase-related protein 1, 5,6-dihydroxyindole-2-carboxylic acid oxidase, CAS2, CATB, TYRP, OCAS, catalase B, b-protein, glycoprotein 75, EC 1.14.18., melanoma antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11, DHICA oxidase, EC 1.14.18, GP75, EC 1.14.18.1; triosephosphate isomerase (triosephosphate isomerase 1, TPID, triosephosphate isomerase, HEL-S-49, TIM, epididymal secretory protein Li 49, TPI, triosephosphate isomerase, EC 5.3.1.1); TRAG-3 (CSAG family member 2, cancer / testis antigen family 24, CSAG3B, member 2, CSAG family member 3B, cancer / testis antigen family 24 member 2, cancer / testis antigen 24.2, chondrosarcoma-associated gene 2 / 3 protein, taxol resistance gene 3 protein, chondrosarcoma-associated gene 2 / 3 protein-like, CT24.2, taxol resistance-associated gene 3, TRAG-3, CSAG3A, TRAG3; TMEM46 (cisa homolog 2 (Xenopus laevis); SHISA; NP-001007539.1; NM-001007538.1; TMEM118 (RING finger protein, transmembrane 2; RNFT2; FLJ1462; NP-001103373.1; NM-001109903.1; TMEFF1 (transmembrane protein 1 with an EGF-like domain and two follistatin-like domains; tomoregulin-; H7365; C9orf2; C9ORF2; U19878; X83961; NM-080655; NM-003692; TGF-beta RII (TGFBR2, transforming growth factor, beta receptor II (70 / 80 kDa), TGF-beta-RII), WMFS2, tbeta R-II, TGFR-2, TGF-beta receptor type IIB, TGF-beta type II receptor, TGF-beta receptor type 2, EC 2.7.11.30, transforming growth factor beta receptor type IIC, AAT3, tbeta R-II, transforming growth factor, beta receptor II (70-80 kD), TGF-beta receptor type II, FAA3, transforming growth factor-beta receptor type II, LDS1 B, HNPCC6, LDS2B, LDS2, RITC, EC 2.7.11, TAAD2; TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI reference sequence: NP-057276, NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5 Genbank accession numbers AF179274, AY358907, CAF85723, CQ782436; TAG-2; TAG-1 (contactin 2 (axonal), TAG-1, AXT, axonin-1 cell adhesion molecule, TAX, contactin 2 (transiently expressed), TAXI, contactin-2, axonal glycoprotein; TAG-1, transiently expressed axonal glycoprotein, transient axonal glycoprotein, axonin-1, TAX-1, TAG1, FAMES; PRF:444868); SYT-SSX1 or -SSX2 fusion protein; survivin; STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial antigen 2 of the prostate, six-transmembrane prostate protein, Genbank accession number AF45513; STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM-01244); SSX-4; SSX-2 (SSX2, synovial sarcoma, X Breakpoint 2, X Breakpoint 2, SSX, X Breakpoint 2B, cancer / testis antigen 5.2, X chromosome-linked 2, tumor antigen HOM-MEL-40, CT5.2, HD21, cancer / testis antigen family 5, HOM-MEL-40, isoform B, cancer / testis antigen family 5 member 2a, member 2a, protein SSX2, sarcoma, sarcoma, synovium, X chromosome-linked 2, synovium, synovial sarcoma, X Breakpoint 2B, synovial sarcoma, SSX2A, Sp17; SOX10 (SRY (sex-determining region Y)-Box 10, mouse, PCWH, DOM, WS4, WS2E, WS4C, dominant megacolon, mouse, human homolog, dominant megacolon, SRY-related HMG-Box gene 10, human homolog, transcription factor SOX-10; GenBank: CAG30470.1); SNRPD1 (small nuclear ribonucleoprotein D1, small nuclear ribonucleoprotein D1, polypeptide 16 kDa, polypeptide (16 kD), SNRPD, HsT2456, Sm-D1, SMD1, Sm-D autoantigen, small nuclear ribonucleoprotein D1 polypeptide 16 kDa pseudogene, SnRNP core protein D1, small nuclear ribonucleoprotein Sm D1); SLC35D3 (solute carrier family 35, member D3, FRCL1, Fringe-binding-like protein 1, bA55K22.3, Frc, Fringe-like 1, solute carrier family 35 member D3 NCBI GenBank: NC-000006.11 NC-018917.2NT-025741.16); SIRT2 (sirtuin 2, NAD-dependent deacetylase sirtuin-2, SIRL2, silent signaling regulator 2, regulatory protein SIR2 homolog 2, Sir2-related protein type 2, SIR2-like protein 2, sirtuin type 2, sirtuin (silent mating-type regulation 2 homolog) 2 (S. cerevisiae), sirtuin-2, sirtuin (silent mating-type regulation 2, S. cerevisiae, homolog) 2, EC 3.5.1., SIR2; GenBank: AAK51133.1); Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane Domain™ and short cytoplasmic domain, (Semaphorin) 5B, GenBank accession number AB04087; secretin 1 (SCRN1, SES1, KIAA0193, secretin-1; GenBank: EAL24458.1); SAGE (SAGE1, sarcoma antigen 1, cancer / testis antigen 14, CT14, putative tumor antigen; NCBI reference sequence: NP-061136.2); RU2AS (KAAG1, kidney-associated antigen 1, RU2AS, RU2 antisense gene protein, kidney-associated antigen 1; GenBank: AAF23613.1); RNF43-E3 ubiquitin-protein ligase RNF43 precursor [Homo sapiens], RNF124; URCC; NCBI reference sequence: NP-060233.3; RhoC (RGS5 (regulator of G protein signaling 5, MSTP032, regulator of G protein signaling 5, MSTP092, MST092, MSTP106, MST106, MSTP129, MST129; G enBank:AAB84001.1); RET (ret oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE; NP-066124.1; NM-020975.4); RBAF600 (UBR4, ubiquitin-protein ligase E3 component N-recognin 4, zinc finger, type 1 UBR1, ZUBR1, E3 ubiquitin-protein ligase UBR4, RBAF600, 600 KDa retinoblastoma protein-associated factor, zinc finger UBR1 type protein 1, EC 6.3.2., N-recognin-4, KIAA0462, p600, EC 6.3.2, KIAA1307; GenBank: AAL83880.1); RAGE-1 (MOK, MOK protein kinase, renal tumor antigen, RAGE, MAPK / MAK / MRK overlapping kinase, renal tumor antigen 1, renal cell carcinoma antigen, RAGE-1, EC 2.7.11.22, RAGE1; UniProtKB / Swiss-Prot: Q9UQ07.1); RAB38 / NY-MEL-1 (RAB38, NY-MEL-1, RAB38, member of the RAS oncogene family, melanoma antigen NY-MEL-1, Rab-related GTP-binding protein, Ras-related protein Rab-38, rrGTPbp; GenBank: AAH15808.1); PTPRK (DJ480J14.2.1 (protein tyrosine phosphatase, receptor type, K R-PTP-kappa, protein tyrosine phosphatase kappa, protein tyrosine phosphatase kappa), protein tyrosine phosphatase, receptor type, K, protein-tyrosine phosphatase kappa, protein tyrosine phosphatase, receptor type, kappa, R-PTP-kappa, receptor type tyrosine-protein phosphatase kappa, EC 3.1.3.48, PPTK; GenBank: AAI44514.1); PSMA; PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628; PSCA (prostate stem cell antigen precursor, Genbank accession number AJ29743); PRDX5 (peroxiredoxin 5, EC 1.11.1.15, type VI TPx); B166, antioxidant enzyme B166; HEL-S-55, liver tissue 2D-Page Spot 71B; PMP20, peroxisomal antioxidant enzyme; PRDX6, thioredoxin peroxidase PMP20; PRXV; AOEB166, epididymal secretory protein Li 55, Alu co-repressor 1, peroxiredoxin-5, mitochondria, peroxiredoxin V, prx-V, thioredoxin reductase, Prx-V, ACR1, Alu co-repressor, PLP; GenBank: CAG33484.1); PRAME (antigen preferentially expressed in melanoma, preferentially expressed antigen in melanoma, MAPE, O1P-4, OIPA, CT130, cancer / testis antigen 130, melanoma antigen preferentially expressed in tumors, Opa-interacting protein 4, Opa-interacting protein O1P4; GenBank: CAG30435.1); pml-RAR alpha fusion protein; PMEL17 (Silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp10 BC001414; BT007202; M32295; M77348; NM-006928; PBF (ZNF395, zinc finger protein 395; PRF-1, Huntington's disease regulatory region binding protein; HD gene regulatory region binding protein 2; protein 2, papillomavirus regulatory factor 1; HD regulatory region binding protein 2; papillomavirus regulatory factor; PRF1; HDBP-2; Si-1-8-14; HDBP2, Huntington's disease gene regulatory region binding protein 2; HDRF-2, papillomavirus regulatory factor PRF-1; PBF; GenBank: AAH01237.1); PAX5 (synaptic box 5, synaptic box homeotic gene 5, BSAP, synaptic box protein Pax-5, B cell lineage-specific activator; synaptic domain gene 5, synaptic box gene 5 (B cell lineage-specific activator protein), B cell-specific transcription factor; synaptic box gene 5 (B cell lineage-specific activator); PAP (REG3A, regenerating islet-derived 3 alpha, INGAP, PAP-H, liver-intestine-pancreas protein; PBBCGF, human islet peptide; REG-Ill, pancreatitis-associated protein 1; Regi, Reg III-Alpha, liver-intestine-pancreas, regenerating islet-derived protein III-alpha, pancreatic beta-cell growth factor, HIP, PAP homologous protein, HIP / PAP, proliferation-inducing protein 34, PAP1, proliferation-inducing protein 42, REG-3-alpha, regenerating islet-derived protein 3-alpha; GenBank: AAH36776.1); p53 (TP53, tumor protein P53, TPR53, P53, cellular tumor antigen P53, antigen NY-CO-13, mutated tumor protein 53, phosphoprotein P53, P53 tumor suppressor, BCC7, transformation-associated protein 53, LFS1, tumor protein 53, Li-Fraumeni syndrome, tumor suppressor P53); P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP that may be involved in synaptic transmission and neurogenesis, and defects may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), μl: 7.63, MW:47206 TM:1[P] gene chromosome:17p13.3, Genbank accession number NP-002552.;. OGT (O-linked N-acetylglucosamine (GlcNAc) transferase, O-GlcNAc transferase P110 subunit, O-linked acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-peptide N-acetylglucosamine transferase 110 kDa subunit, UDP-N-acetylglucosamine:polypeptide-N-acetylglucosamine transferase, uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase, O-GlcNAc transferase subunit P110, EC 2.4.1.255, O-linked acetylglucosamine transferase 110 kDa subunit, EC 2.4.1, HRNT1, EC 2.4.1.186, O-GLCNAC GenBank:AAH38180.1); 0A1 (osteoarthritis QTL 1, OASD; GenBank:CAA88742.1), NY-ESO-1 / LAGE-2 (cancer / testis antigen 1B, CTAG1 B, NY-ESO-1, LAGE-2, ESO1, CTAG1, CTAG, LAGE2B, cancer / testis antigen 1, autoimmune cancer / testis antigen NY-ESO-1, Ancer antigen 3, cancer / testis antigen 6.1, New York esophageal squamous cell carcinoma 1, L antigen family member 2, LAGE2, CT6.1, LAGE2A; GenBank: AAI30365.1); NY-BR-1 (ANKRD30A, ankyrin repeat domain 30A, breast cancer antigen NY-BR-1, serologically defined breast cancer antigen NY-BR-1, ankyrin repeat domain-containing protein 30A; NCBI reference sequence: NP-443723.2); N-ras (NRAS, neuroblastoma RAS viral (V-Ras) oncogene homolog, NRAS1, transforming protein N-Ras, GTPase NRAS, ALPS4, N-Ras protein part 4, NS6, oncogene homolog, HRAS1; GenBank:AAH05219.1); NFYC (nuclear transcription factor Y, gamma; HAP5, HSM, nuclear transcription factor Y subunit C; transactivator HSM-1 / 2; CCAAT-binding factor subunit C; NF-YC, CCAAT transcription binding factor subunit gamma; CAAT box DNA-binding protein subunit C; histone H1 transcription factor large subunit 2A; CBFC, nuclear transcription factor Y subunit gamma; CBF-C, transactivator HSM-1; H1TF2A, transcription factor NF-Y, C subunit; neo-PAP (PAPOLG, poly(A) polymerase gamma; neopoly(A) polymerase; nuclear poly(A) polymerase gamma; polynucleotide adenyltransferase gamma; SRP RNA 3' adenylyltransferase / Pap2; PAP-gamma; Neo-PAP; SRP RNA 3'-adenylyltransferase, PAP2, EC2.7.7.19, PAPG; NCBI reference sequence: NP-075045.2); NCA (CEACAM6, Genbank accession number M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM-00642); myosin class I; MUM-3; MUM-2 (TRAPPC1, transport Protein particle complex 1, BETS, BETS homolog, MUM2, ubiquitin-mutated melanoma 2, multiple myeloma protein 2, transport protein particle complex subunit 1; MUM-1f; mucins (MUC1, mucin 1, cell surface associated; PEMT, PUM, CA15-3; MCKD1, ADMCRO, medullary cystic kidney disease 1 (autosomal dominant); ADMCKD1, mucin 1, transmembrane; CD227, breast cancer-associated antigen DF3; MAM6, cancer antigen 15-3; MCD, cancer-associated mucin; MCRO, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, epicillin, EMA, H23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 antigen; UniProtKB / Swiss-Prot:P15941.3); MUCSAC (mucin SAC, oligomeric mucus / gel forming, tracheobronchial mucin 'MUC5, TBM, mucin 5, subtypes A and C, tracheobronchial / gastric, leB, gastric mucin, mucin SAC, oligomeric mucus / gel forming pseudogene, Lewis B blood group antigen, LeB, major airway glycoprotein, MUC-SAC, mucin-5 subtype AC, tracheobronchial; MUC1 (mucin 1, cell surface associated, PEMT, PUM, CA15-3, MCKD1, ADMCRO, medullary cystic kidney disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer associated mucin, MCKD, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC-1 / X, polymorphic epithelial mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, epicillin, EMA, h23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 Antigen; MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM-01776; MRP4 - multidrug resistance-associated protein 4 isoform 3, MOAT-B; MOATB [Homo sapiens], NCBI reference sequence: NP-001288758.1); MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM-00582); MMP-7 (MMP7, matrilysin, MPSL1, matrine, matrix metalloproteinase 7 (matrilysin, uterine), matrix metalloproteinase-7, EC 3.4.24.23, Pump-1 protease, matrine, uterine metalloproteinase, PUMP1, MMP-7, EC 3.4.24, PUMP-1;GenBank:AAC37543.1);. MMP-2 (MMP2, matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase), MONA, CLG4A, matrix metalloproteinase 2 (gelatinase A, 72 kD gelatinase, 72 kD type IV collagenase), CLG4, 72 kDa gelatinase, 72 kDa type IV collagenase), matrix metalloproteinase-2, MMP-II, 72 kDa gelatinase, collagenase IV-A type, MMP-2, matrix metalloproteinase-II, TBE-1, neutrophil gelatinase, EC 3.4.24.24; GenBank: AAH02576.1); and Meloe;

[0282] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes within any of the following antigens): 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO , CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL 14, CCL15, CCL16, CCLl 7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR 10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, C D21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, C D61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, degradation-promoting factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, interferon (INF)-beta, interferon (INF) ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, Sigma, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, Tf R, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL) Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSFllB(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFFR), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSFIA(TNF RI CD120a, p55~60), TNFRSFIB(TNF RII CD120b, p75~80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-lBB) CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSFIA (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTA, TNFSF1), TNFSF3 (LTb TNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, signaling receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression LewisY-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6 , WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0283] In certain embodiments, a multispecific (e.g., bispecific) antibody according to the present disclosure comprises a first antigen-binding domain having specificity for CD3 and a second antigen-binding domain having specificity for 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO , CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL 14, CCL15, CCL16, CCLl 7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR 10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, C D21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, C D61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, degradation-promoting factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, interferon (INF)-beta, interferon (INF) ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, Sigma, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, Tf R, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL) Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSFllB(OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFFR)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA) TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55~60)、TNFRSFIB(TNF RII). CD120b, p75~80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18(GITR LINK, AITR TL6, TNFSFIA (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTA、TNFSF1)、TNFSF3(LTbTNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, signaling receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WN and a second binding domain having specificity for a second antigen selected from the group consisting of T8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0284] In certain embodiments, the combination of antigens that can be targeted by the bispecific antibody can be any combination of antigens, as the present invention is universally applicable to various bsAbs with different cognate antigen combinations. Non-limiting examples include: CD3 and Her2, CD3 and Her3, CD3 and EGFR, CD3 and CD19, CD3 and CD20, CD3 and EpCAM, CD3 and CD33, CD3 and PSMA, CD3 and CEA, CD3 and gp100, CD3 and gpA33, CD3 and B7-H3, CD64 and EGFR, CEA and HSG, TRAIL-R2 and LT beta R, EGFR and IGFR, VEGFR2 and VEGFR3, VEGFR2 and PDGFR alpha, PDGFR alpha and PDGFR beta, EGFR and TGF-beta, EGFR and IFN-alpha, EGFR and IL-12p40, 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, CD20 and IFN-alpha, CD20 and TGF-beta, CD30 and CD16A, FceRI and CD32B, CD32B and CD79B, BCMA and HEL MP65 and SAP-2, IL-17A and IL-23, IL-1 alpha and IL-1 beta, IL-12 and IL-18, VEGF and osteopontin, VEGF and Ang-2, VEGF and PDGFR beta, VEGF and Her2, VEGF and DLL4, FAP and DR5, FcgRII and IgE, PD-1 and PD-L1, CEA and DTPA, CEA and IMP288, and LukS-PV and LukF-PV.

[0285] "Different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, as well as different and / or distinct epitopes, which may be contained within one protein, polypeptide, or other molecule. Consequently, a bispecific antibody may bind to two epitopes on the same polypeptide.

[0286] The term "epitope" is used herein in the broadest sense to encompass one or more regions of an antigen that interact with a corresponding paratope. A protein or peptide epitope can include amino acid residues that directly interact with the paratope (e.g., via hydrogen bonds or hydrophobic interactions) and non-interacting amino acid residues (e.g., residues that generally contribute to the conformation of the epitope). Epitopes can be defined as structural or functional. A functional epitope is generally an epitope that has residues that directly contribute to a function of the antigen (e.g., affinity for another protein or enzymatic activity). A structural epitope is an epitope that has residues that contribute to the antigen structure that may not significantly contribute to antigen function. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include determinants that are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, an epitope may have specific three-dimensional structural characteristics and / or specific charge characteristics. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and 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 bound by an antibody.

[0287] According to IMGT (International ImMunoGeneTics Information System for Immunoglobulins or Antibodies, T-cell Receptors, MH, Immunoglobulin Superfamilies IgSF, and MhSF), the CH1 domain is amino acid positions (or simply referred to herein as "positions") 118-215 (EU numbering), and the hinge region is amino acid positions 216-230 (EU numbering). The term "CH1 domain" is used broadly herein to refer to a heavy chain region that includes at least seven consecutive amino acid positions of heavy chain positions 118-215 (EU numbering), and in some cases also includes a portion of the hinge region (a portion of heavy chain positions 216-230 (EU numbering)) (e.g., up to position 218). The CH1 domain reference sequence corresponding to amino acid positions 118-220 according to EU numbering is provided herein as SEQ ID NO: 1, which corresponds to the CH1 domain sequence of the human IgG1 allotypes "IGHG1*01 (J00228)," "IGHG1*04 (JN582178)," or "IGHG1*07," and is an exemplary amino acid sequence of a wild-type (WT) CH1 domain.

[0288] CH1 domain reference sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (positions 118-220 according to EU numbering) (SEQ ID NO: 1).

[0289] Alternative CH1 domain reference sequences for human IgG1 may include, but are not limited to, SEQ ID NO: 3, which corresponds to the CH1 domain sequence of human IgG1 allotypes "IGHG1*03(Y14737)" or "IGHG1*08".

[0290] Alternative CH1 domain reference sequence (214R relative to SEQ ID NO: 6):

number

[0291] These CH1 domain reference sequences are intended to be exemplary, as applicants intend that a "CH1 domain" sequence encompasses any naturally occurring CH1 domain allotype or allelic variant.

[0292] Thus, the amino acid modifications in the variant CH1 domain polypeptides according to the present disclosure can be relative to and / or incorporated into any parent CH1 domain polypeptide, for example, a wild-type sequence such as, but not limited to, SEQ ID NO: 1, or any allelic variant thereof, such as, but not limited to, SEQ ID NO: 3.

[0293] According to IMGT, the CH2 domain is amino acid positions (or simply referred to herein as "positions") 231-340 (EU numbering). The term "CH2 domain" is used broadly herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of heavy chain positions 231-340 (EU numbering). A CH2 domain reference sequence corresponding to amino acid positions 231-340 according to EU numbering is provided herein as SEQ ID NO: 7, which is an exemplary amino acid sequence of a wild-type (WT) CH2 domain.

[0294] CH2 domain reference sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7).

[0295] This CH2 domain reference sequence is intended to be exemplary, as applicants intend that the "CH2 domain" sequence encompasses any naturally occurring CH2 domain allotype or allelic variant.

[0296] According to IMGT, the CH3 domain is amino acid positions (or simply referred to herein as "positions") 341-446 (EU numbering). The term "CH3 domain" is used broadly herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of heavy chain positions 341-446 (EU numbering). The CH3 domain reference sequence corresponding to amino acid positions 341-446 according to EU numbering corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*01 (J00228)" or "IGHG1*08" and is provided herein as SEQ ID NO: 1, which is an exemplary amino acid sequence of a wild-type (WT) CH3 domain.

[0297] CH3 domain reference sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8).

[0298] Alternative CH3 domain reference sequences for human IgG1 may include, but are not limited to, SEQ ID NO: 2, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*03 (Y14737)", SEQ ID NO: 5, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*04 (JN582178)", and SEQ ID NO: 6, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*07".

[0299] Alternative CH3 domain reference sequences (356E and 358M relative to SEQ ID NO: 1):

number

[0300] Alternative CH3 domain reference sequence (422I relative to SEQ ID NO: 1):

number

[0301] Alternative CH3 domain reference sequence (431G relative to SEQ ID NO: 1):

number

[0302] Again, it is expressly noted that these CH3 domain reference sequences are intended to be exemplary, as applicants intend that "CH3 domain" sequences include any naturally occurring CH1 domain allotypes or allelic variants.

[0303] There are two major CL isotypes, κ and λ, and such CL domains are referred to herein as CLκ domains and CLλ domains.

[0304] According to IMGT, the CLκ domain is amino acid positions 108-214 (EU numbering). The term "CLκ domain" is used broadly herein to refer to a light chain region comprising at least seven consecutive amino acid positions of kappa light chain positions 108-214 (EU numbering). A CLκ domain reference sequence corresponding to amino acid positions 108-214 (EU numbering) is provided herein as SEQ ID NO: 2, which is an exemplary amino acid sequence of a wild-type (WT) CLκ domain.

[0305] CLκ domain reference sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (positions 108 to 214 according to EU numbering) (SEQ ID NO: 2).

[0306] According to IMGT, the CLλ domain is amino acid positions 107-215 (EU numbering). The term "CLλ domain" is used broadly herein to refer to a light chain region comprising at least seven consecutive amino acid positions of lambda light chain positions 107-215 (EU numbering). A CLλ domain reference sequence corresponding to amino acid positions 107-215 (EU numbering) is provided herein as SEQ ID NO: 9, which is an exemplary amino acid sequence of a wild-type (WT) CLλ domain.

[0307] CLλ domain reference sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (positions 107 to 215 according to EU numbering) (SEQ ID NO: 9).

[0308] Various naturally occurring sequences (corresponding to different allotypes) of the constant domains of human IgG1, IgG2, IgG3, and IgG4 are known in the art and can be found, for example, in Vidarsson et al., Front Immunol. 2014 Oct 20;5:520 and U.S. Patent No. 9,150,663, the disclosures of which are incorporated herein by reference in their entireties. These constant domain reference sequences are intended to be exemplary, as applicants intend that constant domain sequences include any naturally occurring CH1 domain allotype or allelic variant.

[0309] The terms "cognate," "cognate pair," or "cognate pairing," as used herein when referring to the relationship between a CH1 domain and a CL domain, mean that at least one of the CH1 and CL domains contains an amino acid substitution such that the CH1 and CL domains preferentially pair with each other.

[0310] As used herein, the term "cognate pair" or "cognate pairing" when referring to antigen binding or epitope binding refers to a pairing or association of two antibody chains (e.g., heavy and light chains), each comprising a variable region (e.g., VH and VL, respectively), such that the combination of the variable regions provides the intended binding specificity for the epitope or antigen. As used herein, the term "non-cognate pair" or "non-cognate pairing" refers to a pairing or association of two antibody chains (e.g., heavy and light chains), each comprising a variable region (e.g., VH and VL, respectively), such that the combination of the variable regions does not provide the intended binding specificity for the epitope or antigen.

[0311] Provided herein are engineered variant CH1 and CL domains that include at least one amino acid substitution that promotes pairing between the CH1 and CL domains, such that such pairing may be formed more preferentially compared to another CH1-CL set, e.g., compared to a WT CH1-CL set or another variant CH1-CL set.

[0312] As used herein, the term "variant CH1 domain" (also referred to as a CH1 domain variant) refers to a CH1 domain (which may also include a portion of the hinge region described above, such as SEQ ID NO: 1) having an amino acid sequence in which one or more amino acid substitutions are made in the CH1 domain sequence. CH1 sequences in which such amino acid substitutions are made include, but are not limited to, the CH1 domain reference sequence SEQ ID NO: 1. In the library screened to identify the described variant CH1 domains, a nucleic acid sequence encoding SEQ ID NO: 1 was mutated. When one or more amino acid substitutions in a variant CH1 domain promote pairing with a specific CL domain, e.g., a variant CLκ domain, such a variant CH1 domain may also be referred to as a CH1 design, a designed CH1 domain, or the like, and the term "designed" as used in this manner indicates that the CH1 domain has been designed (i.e., modified) to pair with a specific CL domain.

[0313] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. A constant domain according to the present disclosure can be of any antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. A CH3 domain as used herein can be derived from the CH1 of an antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. CH1 substitutions according to the present disclosure can be made to any CH1 domain sequence, such as, but not limited to, the CH1 reference sequence SEQ ID NO: 1. SEQ ID NO: 1 is a human IgG1 CH1 domain sequence, but given the sequence similarity between, for example, human IgG1 and human IgG2 or human IgG4, CH1 substitutions according to the present disclosure can also be incorporated into human IgG2 or IgG4 CH1 sequences, with similar preferential CH1-CL pairing still being expected. When CH2 and / or CH3 domains are used with variant CH1 domains of the present disclosure, the CH2 and / or CH3 domains can be derived from any antibody isotype, and the CH2 and / or CH3 domain isotype does not necessarily have to be the same as the CH1 domain isotype. In addition, the CH2 and / or CH3 domains used with the variant CH1 domain can be wild-type, e.g., germline, or a variant thereof.

[0314] As used herein, the term "variant CLκ domain" (also referred to as a CLκ domain variant) refers to a CLκ domain having an amino acid sequence in which one or more amino acid substitutions are made to the CLκ domain sequence. CLκ sequences in which such amino acid substitutions are made include, but are not limited to, the CLκ domain reference sequence SEQ ID NO: 2. In the library screened to identify the described variant CLκ domains, a nucleic acid sequence encoding SEQ ID NO: 2 was mutated. When one or more amino acid substitutions in a variant CLκ domain promote pairing with a specific CH1 domain, e.g., a variant CH1 domain, such a variant CLκ domain may also be referred to as a CLκ design, a designed CLκ domain, or the like, and the term "designed" as used in this manner indicates that the CLκ domain has been designed (i.e., modified) to pair with a specific CH1 domain.

[0315] As used herein, the term "variant CLλ domain" (also referred to as a CLλ domain variant) refers to a CLλ domain having an amino acid sequence in which one or more amino acid substitutions have been made to the CLλ domain sequence. CLλ sequences in which such amino acid substitutions have been made include, but are not limited to, the CLλ domain reference sequence SEQ ID NO: 9. In the library screened to identify the described variant CLλ domains, a nucleic acid sequence encoding SEQ ID NO: 9 was mutated. When one or more amino acid substitutions in a variant CLλ domain promote pairing with a specific CH1 domain, e.g., a variant CH1 domain, such a variant CLλ domain may also be referred to as a CLλ design, a designed CLλ domain, or the like, and the term "designed" as used in this manner indicates that the CLλ domain has been designed (i.e., modified) to pair with a specific CH1 domain.

[0316] The term "variant CL domain" (also referred to as CL domain variant) is used herein to encompass variant CLκ domains and variant CLλ domains.

[0317] The terms "CH1-CL domain set," "CH1-CL set," or "CH1-CL pair" refer to a combination of a CH1 domain and a CL domain (kappa or lambda). The term "CH1-CL domain polypeptide set" may be used to emphasize that the CH1 and CL domains are polypeptides. A "CH1-CL domain set" may refer to a combination of a CH1 domain and a CLκ domain, and may be a "CH1-CLκ domain set" (also referred to as a "CH1-CLκ set" or "CH1-CLκ pair"), or a "CH1-CLλ domain set" (also referred to as a "CH1-CLλ set" or "CH1-CLλ pair"), and may refer to a combination of a CH1 domain and a CLλ domain. The term "CH1-CL domain-encoding polynucleotide set" refers to a combination of a CH1 domain-encoding polynucleotide and a CL domain-encoding polynucleotide (the CL domain may be kappa or lambda).

[0318] A set name may be given to each CH1-CL set. As described in more detail herein below (e.g., in connection with Tables 2 and 28), a "CH1-CLκ set name" may be given to each "CH1-CLκ set" based on specific amino acid substitutions at specific positions in the CH1 and CLκ domains of the set (the substitutions are relative to the WT CH1 and CLκ sequences), and a "CH1-CLλ set name" may be given to each "CH1-CLλ set" based on amino acid substitutions at specific positions in the CH1 and CLλ domains of the set (the substitutions are relative to the WT CH1 and CLλ sequences). When a CH1-CL set includes a non-wild-type CH1 domain and / or a non-wild-type CL domain, such a set may also be referred to as a variant CH1-CL domain set or variant CH1-CL set (the terms "variant CH1-CLκ domain set," "variant CH1-CLκ set," "variant CH1-CLλ domain set," or "variant CH1-CLλ set" may also be used to identify a CL isotype). When a CH1 domain in a CH1-CL set contains one or more amino acid substitutions to promote specific pairing with a given CL domain, such a CH1 domain can also be referred to as a CH1 designed domain or a designed CH1 domain. When a CL domain in a CH1-CL set contains one or more amino acid substitutions to promote specific pairing with a given CH1 domain, such a CL domain can also be referred to as a CL designed domain or a designed CL domain (the terms "CLκ designed domain," "designed CLκ domain," "CLλ designed domain," or "designed CLλ domain" can also be used to identify a CL isotype).

[0319] When amino acid substitutions in the CH1 and / or CL domains in a CH1-CL set promote specific pairing with one another (relative to pairing with other similar domains), such CH1-CL sets may also be referred to as CH1-CL designs, CH1-CL design sets, designed CH1-CL sets, designed CH1-CL, or the like (the terms "CH1-CLκ design," "CH1-CLκ design set," "designed CH1-CLκ set," "designed CH1-CLκ," "CH1-CLλ design," "CH1-CLλ design set," "designed CH1-CLλ set," and "designed CH1-CLλ" may also be used to identify a CL isotype). As used in this manner, the term "designed" indicates that the CH1 and / or CL domains have been designed (i.e., modified) to pair with one another.

[0320] The term "CH1-CL design set" encompasses CH1-CL design sets referred to herein by the "network" designation. While the networks were initially identified by applicants by screening the CH1-CLκ set as described in Examples 1-2, the same "network" designation is also used to refer to the corresponding CH1-CLλ set. A "network" defines that the designed CLκs and designed CLλ domains belonging to the network contain the same specified amino acid residues at the specified positions. However, because the WT CLκ and WT CLλ sequences are not identical, even if a designed CLκ domain may contain the specified amino acid residue at the specified position due to a substitution into the WT CLκ domain sequence, the designed CLλ domain may contain the same specified amino acid residue because the specified amino acid residue is the WT residue, not necessarily due to a substitution into the WT CLλ domain sequence.

[0321] For example, "Network 1993" defines that, regardless of the light chain isotype, the CH1 domain of the CH1-CL set belonging to "Network 1039" contains 128R and 147R (R at position 128 and R at position 147), and the CL domain of the CH1-CL set belonging to "Network 1039" contains 124E, 133Q, and 178E (E at position 124, Q at position 133, and E at position 178). When network 1993 refers to the CH1-CLκ set, the CH1-CLκ design set has the CH1-CLκ set name "H_128R_147R-L_124E_133Q_178E" and includes a variant CH1 domain containing 128R and 147R, which may be the result of two substitutions, L128R and K147R (substitutions relative to sequence 1), and a variant CLκ domain containing 124E, 133Q, and 178E, which may be the result of three substitutions, Q124E, V133Q, and T178E (substitutions relative to sequence 2). An exemplary variant CH1 domain sequence for network 1993 is provided by SEQ ID NO:21, and an exemplary variant CLκ domain sequence for network 1993 is provided by SEQ ID NO:22. When network 1993 refers to the CH1-CLλ design set, the CH1 domain includes an R at position 128 and an R at position 147, and the CLλ domain includes an E at position 124, a Q at position 133, and an E at position 178. The R at position 128 and the R at position 147 in CH1 may again be the result of two substitutions, L128R and K147R (substitutions relative to sequence 1), but for the CLλ domain, the E at position 124 may not be due to a substitution because the WT amino acid residue at position 124 is E in the case of the λ isotype (unlike the κ isotype), while the Q at position 133 and the E at position 178 may again be the result of substitutions V133Q and T178E. Thus, the CH1-CLλ set name for network 1993 is "H_128R_147R-L_133Q_178E."An exemplary variant CH1 domain sequence of network 1993 is provided by SEQ ID NO:21, and an exemplary variant CLλ domain sequence of network 1993 is provided by SEQ ID NO:29.

[0322] A CH1 domain or variant CH1 domain that "preferentially" pairs with a CL domain or variant CL domain, a variant CH1 domain that provides "preferential" pairing with a CL domain or variant CL domain, or a "preferential" CH1-CL pairing means that a CH1 domain or variant CH1 domain pairs with a given CL domain or variant CL domain rather than with another CL domain, such as a wild-type CL (CLκ or CLλ) domain, another variant CL (CLκ or CLλ), CL domain, or variant CL domain of a different light chain isotype. A CL domain or variant CL domain that "preferentially" pairs with a CH1 domain or variant CH1 domain, a CL domain or variant CL domain that provides "preferential" pairing with a CH1 domain or variant CH1 domain, or a "preferential" CH1-CL pairing means that a CL domain or variant CL domain pairs with a given CH1 domain or variant CH1 domain rather than with another CH1 domain or another variant CH1 domain, such as a wild-type CH1 domain or another variant CH1 domain.

[0323] Such preferential CH1-CL pairing can be indicated, for example, by the formation of a pair between a given CH1 domain or variant CH1 domain and a given CL domain or variant CL domain other than a CH1-CL pair when the given CH1 domain or variant CH1 domain is computationally or recombinantly mixed, co-expressed, or co-provided with an approximately 1:1 mixture of the given CL domain or variant CL domain and another CL domain (wild-type or another variant), and / or when the given CL domain or variant CL domain is computationally or recombinantly mixed, co-expressed, or co-provided with an approximately 1:1 mixture of the given CH1 domain or variant CH1 domain and another CH1 domain (wild-type or variant). Such preferential pairing or the degree of preferential pairing between a given CH1 domain or variant CH1 domain and a CL domain or variant CL domain can be indicated, for example, by a computationally calculated score (ΔΔG, ΔΔG cognate total score , ΔΔG cognate hbond_all ,RBPP,RBPP total score ,RBPP hbond_all , and / or RBPP bond elec backrub 18k or by the percentage of intended CH1-CL pairs among all CH1-CL pairs formed (e.g., "% CH1-CL pairs" or "% CH1-CL pair," or "% CH1-CL" (e.g., "% CH1-CLκ pairs" or "% CH1-CLλ pairs"); or by a direct comparison of the amount of intended CH1-CL pairs and other CH1-CL pairs. In some cases, "preferential" CH1-CL pairings can be quantified by expressing full-sized bispecific antibodies having structures such as the structure shown in FIG. 2A (boxed); in certain cases, full-sized bispecific antibodies may be produced using, for example, heavy chain heterodimerization techniques (such as the "knob-in-hole" technique) shown in FIG. 2D and comparing the relative amount of intended bispecific antibodies among all full-sized antibodies produced.

[0324] The degree of preferential CH1-CL pairing may be quantified by any available computational method, such as Rosetta scoring, and / or any available laboratory assay, such as, but not limited to, liquid chromatography-mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, or flow cytometry. For example, full-size bispecific anti-antibodies designed to include heavy chain heterodimerization technology (e.g., having the structure shown in FIG. 2D ) by co-expressing a first and second heavy chain in an approximately 1:1 ratio and a first and second light chain in an approximately 1:1 ratio (the first and second heavy chains and first and second light chains described in the detailed description of FIG. 2D ), and the % of intended bispecific antibodies (i.e., correctly paired pairs (also referred to herein as “PC”)) among all full-size antibodies can be quantified. In such cases, the % of PCs when a variant CH1 domain disclosed herein and / or a variant CL domain disclosed herein is used can be about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some preferred embodiments, In some preferred embodiments, the % of PC may be about 70% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 75% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 80% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 85% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 90% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 95% or greater. In some more preferred embodiments, the % of CH1-CL pairs may be about 100%. Full-size bispecific antibodies containing similar but two different CH1-CL sets (e.g., having the structure shown in Figure 2C) and designed to further include heavy chain heterodimerization technology can be produced in the same manner, and the % of PC can be measured and assessed in the same manner.

[0325] Antibodies and antibody fragments comprising variant CH1 domains, variant CL domains, and / or variant CH1-CL domain sets, or such variant CH1 domains and / or such variant CH1-CL domain sets, can be characterized by, for example, chromatography, such as size exclusion chromatography (SEC), or electrophoresis, such as SDS-PAGE, for example, the degree of aggregation (e.g., the presence of multimers of intact antibodies) (also referred to herein as purity), the melting temperature (Tm), which can be measured, for example, by differential scanning fluorometry (DSF), the production yield in an appropriate cell type (e.g., HEK293 cells or yeast cells), the "pI", the isoelectric point ("pI"), the level of interaction with a multispecific reagent ("PSR"), which can be measured as in WO 2014 / 179363, e.g., Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561, may be further evaluated based on one or more additional properties, such as, but not limited to, antibody hydrophobic interaction, solubility, production cost and / or time, stability, shelf life, in vivo half-life, and / or immunogenicity, which may be measured by hydrophobic interaction chromatography ("HIC"), as measured in May-Jun;7(3):553-561. Any of these or other properties may be used as evaluation criteria in addition to the CH1-CL% value when evaluating a given variant CH1 domain or CH1-CL set. Thus, a variant CH1 domain or variant CH1-CL set of interest that yields a relatively low correctly paired ("PC")% value of CH1-CL pairs may be as ideal as another variant CH1 domain or CH1-CL set with a relatively high PC% value, if the variant CH1 domain or variant CH1-CL set of interest offers a good profile in one or more of the above-mentioned properties. For example, a variant CH1 domain or variant CH1-CL set that yields 80% PC with 3% aggregation (3% of the expression products are multimers of whole antibodies) may be as ideal as another variant CH1 domain or variant CH1-CL set that yields 90% PC with 10% aggregation.

[0326] The term "library" is used herein to encompass any collection of biological materials, such as nucleic acids, peptides, proteins, and their sequence information. For example, a "CH1 domain-encoding polynucleotide library" refers to a collection of polynucleotides encoding different CH1 domain polypeptides or their polynucleotide sequences, and a "CH1 domain polypeptide library" refers to a collection of different CH1 domain polypeptides or their amino acid sequences. Similarly, a "CL domain-encoding polynucleotide library" refers to a collection of polynucleotides encoding different CL domain polypeptides or their polynucleotide sequences, and a "CL domain polypeptide library" refers to a collection of different CL domain polypeptides or their amino acid sequences. The CL domains may be CLκ and / or CLλ. Furthermore, the term "CH1-CL domain-encoding polynucleotide set library" refers to a different set of polynucleotides encoding (i) CH1 domain polypeptides (WT or variant) and (ii) CL (CLκ and / or CLλ) domain polypeptides (WT or variant), or a collection of their polynucleotide sequences, and the term "CH1-CL domain polypeptide set library" refers to a different set of (i) CH1 domain polypeptides (WT or variant) and (ii) CL (CLκ and / or CLλ) domain polypeptides (WT or variant), or a collection of their amino acid sequences.

[0327] As used herein, "pharmaceutical carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents. In one embodiment, the carrier is suitable for parenteral, intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier can be a liquid in which the active therapeutic agent is formulated. Excipients generally do not provide any pharmacological activity to the formulation, but may provide chemical and / or biological stability and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, Gennaro, A. editor, 19th edition, Philadelphia, PA: Williams and Wilkins (1995), incorporated by reference.

[0328] "Conservative amino acid substitutions" are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain for another 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 for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0329] Variant CH1 domain, heavy chain containing same, variant CLκ domain, light chain containing same, and variant CH1-CLκ set As described herein, certain positions within the CH1 domain and certain amino acid substitutions within the CH1 domain have been found to affect pairing of the CH1 domain with the CL domain, which may be a CLκ domain or a CLλ domain.

[0330] In some embodiments, the variant CH1 domains described herein may include an amino acid substitution at one or more of the following amino acid positions: 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and / or 187 according to EU numbering. In some embodiments, the variant CH1 domains described herein may include any of the following combinations of positions: 168, 185, and 187; 128 and 147; 145, 147, and 181; 147 and 185; 148; 139, 141, and 187; 166 and 187; 168 and 185; 124 and 147; 147 and 148; 145; 145 and 181; 124, 145, and 147; 166 and 187; 147 and 175; 147R, 175, and 181; 145 and 147; or 147 and 185.

[0331] In some embodiments, the variant CH1 domains described herein may include one or more of the following amino acid substitutions: 124R, 128R, 139R, 141Q, 145Q, 145S, 147E, 147H, 147N, 147Q, 147R, 147T, 148E, 148R, 166K, 168R, 168S, 175D, 175E, 181E, 181Q, 185E, 185Q, 185S, 185Y, 187D, 187K, and / or 187Q.

[0332] In some embodiments, the variant CH1 domains described herein may comprise any of the combinations of CH1 substitutions listed in Table 2.

[0333] In some embodiments, the variant CH1 domains described herein may include any of the following combinations of amino acid substitutions: 168S, 185S, and 187D; 128R and 147R; 145Q, 147E, and 181E; 147T and 185Q; 148R; 139R, 141Q, and 187Q; 166K and 187K; 168R and 185E; 124R and 147R; 147H and 148E; 145S; 145S and 181Q; 145S; 145Q and 181E; 124R, 145S, and 147Q; 166K and 187K; 147R and 175D; 147R, 175E, and 181Q; 145S and 147N; or 147N and 185Y.

[0334] The parent CH1 domain sequence into which such amino acid substitutions can be incorporated can include a wild-type or naturally occurring CH1 domain sequence, or a variant or engineered version thereof. Exemplary sequences of such parent polypeptides include, but are not limited to, the reference CH1 sequence SEQ ID NO: 1.

[0335] In certain embodiments, the amino acid sequence of a variant CH1 domain described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 201.

[0336] In certain embodiments, the amino acid sequence of a variant CH1 domain described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 11, 21, 31, or 41.

[0337] As described herein, certain positions within the CL domain, and certain amino acid substitutions within the CL domain, have been found to affect pairing of the CL domain with the CH1 domain.

[0338] In some embodiments, a variant CL domain (variant CLκ or CLλ domain) described herein may comprise an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 114, 120, 124, 127, 129, 133, 135, 137, 138, 178, and 180. In some embodiments, a variant CLκ domain described herein may comprise any of the following combinations of positions: 135; 124, 133, and 178; 129, 178, and 180; 135 and 178; 124 and 129; 114, 135, and 138; 137 and 138; 127 and 129; 133; 124 and 133; 120, 178, and 180; 127, 129, and 178; 114, 137, and 138; 129, 178, and 180; 133 and 180; or 129 and 180. In some embodiments, a variant CLλ domain described herein may include any of the following combinations of positions: 135; 133 and 178; 129, 178, and 180; 135 and 178; 124 and 129; 114, 135, and 138; 138; 127 and 129; 133; 120, 178, and 180; 127, 129, and 178; 114, 137, and 138; 129, 178, and 180; 133 and 180; or 129.

[0339] In some embodiments, the variant CLκ domains described herein may comprise one or more of the following amino acid substitutions: 114D, 114Q, 120S, 124E, 124S, 127D, 127R, 127T, 129D, 129E, 129R, 133Q, 133Y, 135R, 135S, 137S, 137T, 138E, 138R, 178E, 178H, 178R, and 180H, 180Q, 180R, and / or 180S. In some embodiments, the variant CLκ domains described herein may comprise any of the combinations of CLκ substitutions listed in Table 2 or Appendix Table B. In some embodiments, variant CLλ domains described herein may comprise one or more of the following amino acid substitutions: 114D, 114Q, 120S, 124S, 127D, 127R, 127T, 129D, 129E, 129R, 133Q, 133Y, 135R, 135S, 137T, 138E, 138R, 178E, 178H, 178R, and 180H, 180Q, and / or 180R. In some embodiments, variant CLκ domains described herein may comprise any of the combinations of CLλ substitutions listed in Table 28 or Appendix Table C.

[0340] In some embodiments, the variant CLκ domains described herein may comprise any of the following combinations of amino acid substitutions: 135R; 124E, 133Q, and 178E; 129R, 178R, and 180Q; 135S and 178R; 124S and 129E; 114D, 135S, and 138R; 137S and 138E; 135S; 127D and 129E; 127R and 129R; 133Y; 133Y; 124E and 133Y; 120S, 178H, and 180Q; 127T, 129D, and 178R; 114Q, 137T, and 138E; 129D, 178R, and 180H; 129D and 180Q; 133Y and 180R; or 129R and 180S. In some embodiments, the variant CLλ domains described herein may include any of the following combinations of amino acid substitutions: 135R; 133Q and 178E; 129R, 178R, and 180Q; 135S and 178R; 124S and 129E; 114D, 135S, and 138R; 138E; 135S; 127D and 129E; 127R and 129R; 133Y; 133Y; 120S, 178H, and 180Q; 127T, 129D, and 178R; 114Q, 137T, and 138E; 129D, 178R, and 180H; 129D and 180Q; 133Y and 180R; or 129R.

[0341] Parent CL domain sequences into which such amino acid substitutions may be incorporated may include wild-type or naturally occurring CL domain sequences, or variant or engineered versions thereof. Exemplary sequences of such parent CLκ polypeptides include, but are not limited to, the reference CLκ sequence SEQ ID NO: 2. Exemplary sequences of such parent CLλ polypeptides include, but are not limited to, the reference CLλ sequence SEQ ID NO: 9.

[0342] In certain embodiments, the amino acid sequence of a variant CLκ domain described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202. In certain embodiments, the amino acid sequence of a variant CLλ domain described herein may comprise or consist of SEQ ID NO: 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 199, or 209.

[0343] In certain embodiments, the amino acid sequence of a variant CLκ domain described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 12, 22, 32, or 42. In certain embodiments, the amino acid sequence of a variant CLκ domain described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 59, 99, 39, 199, 49, or 29.

[0344] As described herein, combinations of certain amino acid positions within the CH1 and CL domains have been found to affect pairing between the CH1 and CL domains. In some embodiments, the CH1-CLκ sets described herein include the following amino acid positions within the CH1 and CLκ domains: CH1 positions 168, 185, and 187 together with CLκ position 135 (e.g., network 1039); CH1 positions 128 and 147 together with CLκ positions 124, 133, and 178 (e.g., network 1993); CH1 positions 145, 147, and 181 together with CLκ positions 129, 178, and 180 (e.g., network 1443); CH1 positions 147 and 185 together with positions 135 and 178 (e.g., network 2529); CH1 position 148 together with CLκ positions 124 and 129 (e.g., network 367); CH1 positions 139, 141, and 187 together with CLκ positions 114, 135, and 138 (e.g., network 1888); CH1 positions 166 and 187 together with CLκ positions 137 and 138 (e.g., network 1328); CH1 positions 168 and 185 together with CLκ position 135 (e.g., network 1329). , network 2366); CH1 positions 124 and 147 together with CLκ positions 127 and 129 (e.g., network 964); CH1 positions 147 and 148 together with CLκ positions 127 and 129 (e.g., network 767); CH1 position 145 together with CLκ position 133 (e.g., network 1148); CH1 positions 145 and 181 together with CLκ position 133 (e.g., network 384); CH1 position 145 together with CLκ positions 124 and 133 (e.g., network network 454); CH1 positions 145 and 181 together with CLκ positions 120, 178, and 180 (e.g., network 1048); CH1 positions 124, 145, and 147 together with CLκ positions 127, 129, and 178 (e.g., network 534); CH1 positions 166 and 187 together with CLκ positions 114, 137, and 138 (e.g., network 838); CH1 positions 147 and 175 together with CLκ positions 129, 178, and 180 (e.g., network 919);The amino acid substitutions may be included at one or more of: CH1 positions 147, 175, and 181, along with CLκ positions 129 and 180 (e.g., network 394); CH1 positions 145 and 147, along with CLκ positions 133 and 180 (e.g., network 1621); CH1 positions 147 and 185, along with CLκ positions 129 and 180 (e.g., network 742);

[0345] In some embodiments, the CH1-CLλ sets described herein include the following amino acid positions within the CH1 and CLλ domains: CH1 positions 168, 185, and 187 together with CLλ position 135 (e.g., network 1039); CH1 positions 128 and 147 together with CLλ positions 133 and 178 (e.g., network 1993); CH1 positions 145, 147, and 181 together with CLλ positions 129, 178, and 180 (e.g., network 1443); CH1 positions 147 and 181 together with CLλ positions 135 and 178 (e.g., network 1444). 5 (e.g., network 2529); CH1 position 148 together with CLλ positions 124 and 129 (e.g., network 367); CH1 positions 139, 141, and 187 together with CLλ positions 114, 135, and 138 (e.g., network 1888); CH1 positions 166 and 187 together with CLλ position 138 (e.g., network 1328); CH1 positions 168 and 185 together with CLλ position 135 (e.g., network 2366); CH1 positions 124 and 147 together with CLλ positions 127 and 129 (e.g., network 1328). network 964); CH1 positions 147 and 148 together with CLλ positions 127 and 129 (e.g., network 767); CH1 position 145 together with CLλ position 133 (e.g., network 1148); CH1 positions 145 and 181 together with CLλ position 133 (e.g., network 384); CH1 position 145 together with CLλ position 133 (e.g., network 454); CH1 positions 145 and 181 together with CLλ positions 120, 178, and 180 (e.g., network 1048); CLλ positions 127, 129, and 178 together with CH1 positions 124, 145, and 147 (e.g., network 534); together with CLλ positions 114, 137, and 138 with CH1 positions 166 and 187 (e.g., network 838); together with CLλ positions 129, 178, and 180 with CH1 positions 147 and 175 (e.g., network 919); together with CLλ positions 129 and 180 with CH1 positions 147, 175, and 181 (e.g., network 394); together with CLλ positions 133 and 180 with CH1 positions 145 and 147 (e.g., network 1621);It may include an amino acid substitution at one or more of CH1 positions 147 and 185 (e.g., network 742), along with CLλ position 129. In some embodiments, a CH1-CLκ set according to the present invention may include any of the combinations of CH1 and CLκ substitutions in the CH1-CLκ set listed in Table 2;

[0346] In some embodiments, a CH1-CLλ set according to the present invention can include any of the combinations of CH1 and CLλ substitutions in the CH1-CLλ set listed in Table 28.

[0347] In some embodiments, such CH1-CL sets according to the present invention are the following CH1-CL sets: H_168S_185S_187D-L_135R (e.g., network 1039), H_128R_147R-L_124E_133Q_178E (e.g., network 1993), H_145Q_147E_181E-L_129R_178R_180Q (e.g., network 1443), H_147T_185Q-L_135S_178R (e.g., network 2 529), H_148R-L_124S_129E (e.g., network 367), H_139R_141Q_187Q-L_114D_135S_138R (e.g., network 1888), H_166K_187K-L_137S_138E (e.g., network 1328), H_168R_185E-L_135S (e.g., network 2366), H_124R_147R-L_127D_129E (e.g., network 964), H_147H_148E-L_127 R_129R (e.g., network 767), H_145S-L_133Y (e.g., network 1148), H_145S_181Q-L_133Y (e.g., network 384), H_145S-L_124E_133Y (e.g., network 454), H_145Q_181E-L_120S_178H_180Q (e.g., network 1048), H_124R_145S_147Q-L_127T_129D_178R (e.g., network 534), H_166K It may be any of H_187K-L_114Q_137T_138E (e.g., network 838), H_147R_175D-L_129D_178R_180H (e.g., network 919), H_147R_175E_181Q-L_129D_180Q (e.g., network 394), H_145S_147N-L_133Y_180R (e.g., network 1621), or H_147N_185Y-L_129R_180S (e.g., network 742).In some embodiments, such CH1-CL sets according to the present invention are the following CH1-CL sets: H_168S_185S_187D-L_135R (e.g., network 1039), H_128R_147R-L_133Q_178E (e.g., network 1993), H_145Q_147E_181E-L_129R_178R_180Q (e.g., network 1443), H_147T_185Q-L_135S_178R (e.g., network 2 529), H_148R-L_124S_129E (e.g., network 367), H_139R_141Q_187Q-L_114D_135S_138R (e.g., network 1888), H_166K_187K-L_138E (e.g., network 1328), H_168R_185E-L_135S (e.g., network 2366), H_124R_147R-L_127D_129E (e.g., network 964), H_147H_148E-L_127 R_129R (e.g., network 767), H_145S-L_133Y (e.g., network 1148), H_145S_181Q-L_133Y (e.g., network 384), H_145S-L_133Y (e.g., network 454), H_145Q_181E-L_120S_178H_180Q (e.g., network 1048), H_124R_145S_147Q-L_127T_129D_178R (e.g., network 534), H_166K It may be any of H_187K-L_114Q_137T_138E (e.g., network 838), H_147R_175D-L_129D_178R_180H (e.g., network 919), H_147R_175E_181Q-L_129D_180Q (e.g., network 394), H_145S_147N-L_133Y_180R (e.g., network 1621), or H_147N_185Y-L_129R (e.g., network 742).

[0348] In certain embodiments, the amino acid sequences of the variant CH1 domains and variant CLκ domains of such CH1-CLκ sets may comprise the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, SEQ ID NOs: 21 and 22, respectively, SEQ ID NOs: 31 and 32, respectively, SEQ ID NOs: 41 and 42, respectively, SEQ ID NOs: 51 and 52, respectively, SEQ ID NOs: 61 and 62, respectively, SEQ ID NOs: 71 and 72, respectively, SEQ ID NOs: 81 and 82, respectively, SEQ ID NOs: 91 and 92, respectively, SEQ ID NOs: 101 and 102, respectively, SEQ ID NOs: 111 and 112, respectively, SEQ ID NOs: 121 and 122, respectively, SEQ ID NOs: 131 and 132, respectively, SEQ ID NOs: 141 and 142, respectively, SEQ ID NOs: 151 and 152, respectively, SEQ ID NOs: 161 and 162, respectively, SEQ ID NOs: 171 and 172, respectively, SEQ ID NOs: 181 and 182, respectively, SEQ ID NOs: 191 and 192, respectively, or SEQ ID NOs: 201 and 202, respectively.

[0349] In certain embodiments, the amino acid sequences of the variant CH1 domains and variant CLλ domains of such CH1-CLλ sets may comprise the amino acid sequences of SEQ ID NOs: 11 and 19, respectively, SEQ ID NOs: 21 and 29, respectively, SEQ ID NOs: 31 and 39, respectively, SEQ ID NOs: 41 and 49, respectively, SEQ ID NOs: 51 and 59, respectively, SEQ ID NOs: 61 and 69, respectively, SEQ ID NOs: 71 and 79, respectively, SEQ ID NOs: 81 and 89, respectively, SEQ ID NOs: 91 and 99, respectively, SEQ ID NOs: 101 and 109, respectively, SEQ ID NOs: 111 and 119, respectively, SEQ ID NOs: 121 and 129, respectively, SEQ ID NOs: 131 and 139, respectively, SEQ ID NOs: 141 and 149, respectively, SEQ ID NOs: 151 and 159, respectively, SEQ ID NOs: 161 and 169, respectively, SEQ ID NOs: 171 and 179, respectively, SEQ ID NOs: 181 and 189, respectively, SEQ ID NOs: 191 and 199, respectively, or SEQ ID NOs: 201 and 209, respectively.

[0350] In some preferred embodiments, the CH1-CLκ set according to the present invention may be H_168S_185S_187D-L_135R (e.g., network 1039), H_128R_147R-L_124E_133Q_178E (e.g., network 1993), H_145Q_147E_181E-L_129R_178R_180Q (e.g., network 1443), or H_147T_185Q-L_135S_178R (e.g., network 2529).

[0351] In some preferred embodiments, the CH1-CLλ sets according to the present invention may be H_148R-L_124S_129E (network 367), H_124R_147R-L_127D_129E (network 964), H_145Q_147E_181E-L_129R_178R_180Q (network 1443), H_145S_147N-L_133Y_180R (network 1621), H_168R_185E-L_135S (network 2366), H_147T_185Q-L_135S_178R (network 2529), H_128R_147R-L_133Q_178E (network 1993).

[0352] In certain embodiments, the amino acid sequences of the variant CH1 domain and the variant CLκ domain of such a CH1-CLκ set may comprise the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, SEQ ID NOs: 21 and 22, respectively, SEQ ID NOs: 31 and 32, respectively, or SEQ ID NOs: 41 and 42, respectively.

[0353] In certain embodiments, the amino acid sequences of the variant CH1 domains and variant CLλ domains of such CH1-CLλ sets may comprise the amino acid sequences of SEQ ID NOs: 19 and 592, respectively, SEQ ID NOs: 91 and 99, respectively, SEQ ID NOs: 31 and 39, respectively, SEQ ID NOs: 191 and 199, respectively, SEQ ID NOs: 81 and 89, respectively, SEQ ID NOs: 41 and 49, respectively, or SEQ ID NOs: 21 and 29, respectively.

[0354] Exemplary amino acid sequences of the CH1 and CLκ domains in some of the CH1-CLκ sets according to the present invention are shown in Appendix Tables AB.

[0355] Exemplary amino acid sequences of the CH1 and CLλ domains in some of the CH1-CLλ sets according to the present invention are shown in Appendix Tables A and C.

[0356] Rather than the variant CH1 domain pairing with another CL domain (e.g., a wild-type CLκ domain, another CLκ domain, a wild-type CLλ domain, or a variant CLλ domain), or the variant CL domain pairing with another CH1 domain (e.g., a wild-type CH1 domain or another variant CH1 domain), the resulting variant CH1 and CL domains preferentially pair with each other.

[0357] Such CH1 variant domains, variant CL domains, and / or CH1-CL sets may be useful for producing heterodimeric (or multimeric) polypeptides and molecules, such as multispecific antibodies and antibody fragments, by improving the fidelity of heavy-light chain pairing while maintaining the native IgG structure of the bispecific antibody, which is beneficial due to their well-established properties as therapeutic molecules, including long in vivo half-lives and the ability to trigger effector functions. Such variant CH1 domains, variant CL domains, and / or CH1-CL sets disclosed herein may also facilitate the creation of bispecific antibodies based on two existing and desired mAbs. These variant CH1 domains, variant CL domains, and / or CH1-CL sets may be used to completely or partially resolve heavy-light chain mispairing when generating multispecific, e.g., bispecific, antibodies, by promoting proper heavy-light chain pairing. More specifically, multispecific antibodies comprising the variant CH1 domains, variant CL domains, and / or CH1-CL sets disclosed herein will form fewer undesired product-related contaminants, i.e., molecules containing mismatched domains or chains, which can be difficult to eliminate during manufacturing.

[0358] A variant CH1-CLκ set according to the present disclosure that preferentially forms a CH1-CLκ pair is not identical to one identified as a pre-existing CH1-CLκ set, such as the pre-existing CH1-CLκ sets listed in Table 1. A variant CH1-CLλ set according to the present disclosure that preferentially forms a CH1-CLλ pair is not identical to one identified as a pre-existing CH1-CLλ set, such as the pre-existing CH1-CLλ set "CTL3l" shown in Table 1. However, any of the variant CH1 domains, variant CL domains, and / or variant CH1-CL sets of the present invention described herein may be combined with one or more pre-existing CH1-CL sets, such as those in Table 1. For example, one or more of the substitutions in Table 1 may be added to a variant CH1 and / or variant CL domain and / or CH1-CL set of the present invention. In some cases, a molecule, such as a multispecific antibody having a structure shown in Figures 2-7, comprising one or more CH1-CL sets according to the present invention may comprise one or more CH1-CLκ sets of Table 1. [Table 1-1] [Table 1-2]

[0359] In further embodiments, any of the CH1-CL design sets according to the present invention may be combined with one or more other CH1-CL design sets, i.e., multiple different CH1-CL design sets may be incorporated into a single molecule, such as a single polypeptide or multispecific antibody or antibody fragment, as described in detail below.

[0360] In some embodiments, one or more other CH1-CL design sets can comprise a CH1-CL design set according to the present invention, i.e., at least two different CH1-CL sets according to the present invention can be incorporated into one molecule, such as a single polypeptide or multispecific antibody or antibody fragment.

[0361] An antibody or antibody fragment comprising a CH1-CL set in one Fab arm and a WT CH1-CL set in the other Fab arm (i.e., both the CH1 and CL domains are WT) may be referred to as having a single-interface design (SID) or SIG format. A monospecific SID antibody ("monospecific SID") is an SID antibody in which one Fab arm and the other Fab arm have the same specificity. A bispecific SID antibody ("bispecific SID") is an SID antibody in which one Fab and the other Fab have different specificities. An antibody or antibody fragment comprising two different CH1-CL sets may be referred to as having a dual-interface design (DID) or DID format. A monospecific DID antibody ("monospecific DID") is an DID antibody in which one Fab arm and the other Fab arm have the same specificity. A bispecific DID antibody ("bispecific DID") is an DID antibody in which one Fab and the other Fab have different specificities. Furthermore, for each of the specific amino acid substitutions in the CH1 and / or CL domains disclosed herein as providing preferential pairing with one another, the resulting amino acids may be further substituted via conservative amino acid substitution to obtain another variant CH1 and / or CL domain that provides the same (or even higher) pairing preference. Alternatively, for each of the variant CH1 and / or CL domains of the CH1-CL set of invention, one or more amino acid positions that were unaffected (i.e., have the same amino acid relative to the wild-type sequence of CH1 or CL) may be altered via conservative substitution to obtain another variant CH1 and / or CL domain that provides the same or even higher CH1-CL pairing preference.

[0362] Below is provided a brief summary of some CH1-CL sets, among many identified as shown in the Examples, that provide at least one superior property, such as higher correct heavy chain-light chain pairing, compared to the WT CH1-CL set. For example, all sets in (1) through (7) show improved binding energies between the variant CH1 domain and the variant CLκ relative to the binding energies between the WT CH1 domain and the WT CLκ domain, based on a Rosetta score-based comparison, as shown in Examples 1 and 2. Some additional superior properties (non-exhaustive) for each of (1) through (7) are also provided below.

[0363] (1) Network 1039 The CH1-CL set of network 1039 comprises a CH1 domain comprising amino acids S at position 168, S at position 185, and D at position 187 (168S, 185S, and 187D), and a CL domain comprising amino acid R at position 135 (135R). The CH1-CLκ set of network 1039 comprises a CH1 domain comprising amino acid substitutions (relative to the WT CH1 sequence) at positions 168, 185, and 187 providing 168S, 185S, and 187D, and a CLκ domain comprising an amino acid substitution (relative to the WT CLκ sequence) at position 135 providing 135R, and has the set name "H_168S_185S_187D-L_135R." The CH1-CLλ set of network 1039 contains a CH1 domain with amino acid substitutions (relative to the WT CH1 sequence) at positions 168, 185, and 187 providing 168S, 185S, and 187D, and a CLλ domain with an amino acid substitution (relative to the WT CLλ sequence) at position 135 providing R at 135R, and has the set name "H_168S_185S_187D-L_135R".

[0364] For example, the "H_168S_185S_187D-L_135R" (network 1039) set exhibits a higher % of correct CH1-CLκ pairing values ​​when used in SID in an exemplary BsAb, i.e., the variant CH1-CLκ set is used in one Fab arm of a full-size IgG-like bispecific antibody as measured by LC-MS compared to the WT CH1-CLκ set (see Tables 6 and 10). The "H_168S_185S_187D-L_135R" set (network 1039) further improves the % correct CH1-CLκ pairing value when used in addition to another CH1-CLκ set such as the "H_145Q_147E_181E-L_129R_178R_180Q" set (network 1443) in an exemplary DID (to achieve 95% correct pairing), i.e., network 1039 is used in one Fab arm while network 1443 is used in the other Fab arm of a full-size IgG-like bispecific antibody (see Table 10). Additionally, combination of network 1039 with the "H_128R_147R-L_124E_133Q_178E" set (network 1993) in the exemplary DID achieved 97% correct CH1-CLκ pairing in the exemplary BsAb (see Table 10).

[0365] In this example, when Network 1039 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 11 and 12, respectively. When Network 1039 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 11 and 19, respectively. Network 1039 substitutions can be engineered into any reference CH1 and CL domain sequences to provide preferential pairing between heavy and light chains comprising the engineered variant domains.

[0366] (2) Network 1993 The CH1-CL set of network 1993 comprises a CH1 domain comprising an amino acid R at position 128 and an R at position 147 (128R and 147R), and a CL domain comprising an amino acid E at position 124, a Q at position 133, and an E at position 178 (124E, 133Q, and 178E). The CH1-CLκ set of network 1993 comprises a CH1 domain comprising amino acid substitutions (relative to the WT CH1 sequence) at positions 128 and 147 providing 128R and 147R, and a CLκ domain comprising amino acid substitutions (relative to the WT CLκ sequence) at 124, 133, and 178 providing 124E, 133Q, and 178E, and has the set name "H_128R_147R-L_124E_133Q_178E". The CH1-CLλ set of network 1993 contains a CH1 domain containing amino acid substitutions (relative to the WT CH1 sequence) at positions 128 and 147 providing 128R and 147R, and a CLλ domain containing amino acid substitutions (relative to the WT CLλ sequence) at positions 133 and 178 providing 133Q and 178E (note that position 124 is E in WT CLλ), and has the set name "H_128R_147R-L_133Q_178E".

[0367] For example, the "H_128R_147R-L_124E_133Q_178E" set (Network 1993) shows a higher % correct CH1-CLκ pairing value when used in the exemplary SID as measured by LC-MS compared to WT CH1-CLκ (see Table 6). Furthermore, the "H_128R_147R-L_124E_133Q_178E" set (Network 1993) dramatically improves the % correct CH1-CLκ pairing value when used in addition to another CH1-CLκ set, such as the "H_145Q_147E_181E-L_129R_178R_180Q" set (Network 1443) (to achieve 100% correct matching) or the "H_168S_185S_187D-L_135R" set (Network 1039) (to achieve 95% correct matching) in an exemplary DID (see Table 10). The extremely high % correct CH1-CLκ pairing when Network 1993 and Network 1443 are used together in exemplary DIDs with various specificity combinations is further confirmed, for example, in Table 16.

[0368] Furthermore, when the CH1-CLλ set of H_128R_147R-L_133Q_178E (network 1993) was used in combination with the CH1-CLλ set of H_145Q_147E_181E-L_129R_178R_180Q (network 1443) in a bsAb, it was predicted to provide particularly preferential pairing between the CH1 and CLλ domains in both CH1-CLλ sets, as shown, for example, in Figure 19.

[0369] In this example, when Network 1993 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 21 and 22, respectively. When Network 1993 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 21 and 29, respectively. Network 1993 substitutions can be engineered into any reference CH1 and CL domain sequences to provide preferential pairing between heavy and light chains comprising the engineered variant domains.

[0370] (3) Network 1443 The CH1-CL set of network 1443 comprises a CH1 domain comprising amino acids Q at position 145, E at position 147, and E at position 181 (145Q, 147E, and 181E), and a CL domain comprising amino acids R at position 129, R at position 178, and Q at position 180 (129R, 178R, and 180Q). The CH1-CLκ set of network 1443 comprises a CH1 domain comprising amino acid substitutions (relative to the WT CH1 sequence) at positions 145, 147, and 181 providing 145Q, 147E, and 181E, and a CLκ domain comprising amino acid substitutions (relative to the WT CLκ sequence) at 129, 178, and 180 providing 129R, 178R, and 180Q, and has the set name "H_145Q_147E_181E-L_129R_178R_180Q." The CH1-CLλ set of network 1443 also includes a CH1 domain containing amino acid substitutions (relative to the WT CH1 sequence) at positions 145, 147, and 181 to provide 145Q, 147E, and 181E, and a CLλ domain containing amino acid substitutions (relative to the WT CLλ sequence) at positions 129, 178, and 180 to provide 129R, 178R, and 180Q, and has the set name "H_145Q_147E_181E-L_129R_178R_180Q."

[0371] For example, the "H_145Q_147E_181E-L_129R_178R_180Q" set (network 1443) exhibits a higher % correct CH1-CLκ pairing value when used in the exemplary SID as measured by LC-MS compared to WT CH1-CLκ (see Tables 6 and 10). Furthermore, the "H_145Q_147E_181E-L_129R_178R_180Q" set (network 1443) dramatically improves the % correct CH1-CLκ pairing value when used in addition to another CH1-CLκ set, such as the "H_168S_185S_187D-L_135R" set (network 1039) (to achieve 97% correct pairing) in the exemplary DID as measured by LC-MS (see Table 10). In addition, the combination with the "H_128R_147R-L_124E_133Q_178E" set (Network 1993) in the exemplary DID achieved 100% correct CH1-CLκ pairing, the combination with the "H_124R_147R-L_127D_129E" set (Network 964) in the exemplary DID achieved 95% correct CH1-CLκ pairing, the combination with the "H_148R-L_124S_129E" set (Network 367) in the exemplary DID achieved 94% correct CH1-CLκ pairing, and the combination with the "H_168R_185E-L_135S" set (Network 2366) in the exemplary DID achieved 91% correct CH1-CLκ pairing, all of which were consistent with the two WT A much higher percentage of correct CH1-CLκ pairs was achieved compared to when the CH1-CLκ set was used (see Table 10). The extremely high percentage of correct CH1-CLκ pairs when network 1993 and network 1443 are used together in exemplary DIDs with various specificity combinations is further confirmed, for example, in Table 16.

[0372] Furthermore, when the CH1-CLλ set of H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) was used in combination with the CH1-CLλ sets of H_128R_147R-L_133Q_178E (Network 1993), H_124R_147R-L_127D_129E (Network 964), or H_148R-L_124S_129E (Network 367) in a bsAb, it was predicted to provide particularly preferential pairing between the CH1 and CLλ domains in both CH1-CLλ sets, as shown, for example, in Figure 19.

[0373] In this example, when Network 1443 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 31 and 32, respectively. When Network 1443 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 31 and 39, respectively. Network 1443 substitutions can be engineered into any reference CH1 and CL domain sequences to provide preferential pairing between heavy and light chains comprising the engineered variant domains.

[0374] (4) Network 2529 The CH1-CL set of network 2529 comprises a CH1 domain comprising an amino acid T at position 147 and a Q at position 185 (147T and 185Q), and a CL domain comprising an amino acid S at position 135 and an R at position 178 (135S and 178R). The CH1-CLκ set of network 2529 comprises a CH1 domain comprising amino acid substitutions (relative to the WT CH1 sequence) at positions 147 and 185 providing 147T and 185Q, and a CLκ domain comprising amino acid substitutions (relative to the WT CLκ sequence) at positions 135 and 178 providing 135S and 178R, and has the set name "H_147T_185Q-L_135S_178R". The CH1-CLλ set of network 2529 also includes a CH1 domain containing amino acid substitutions (relative to the WT CH1 sequence) at positions 147 and 185 providing 147T and 185Q, and a CLκ domain containing amino acid substitutions (relative to the WT CLκ sequence) at positions 135 and 178 providing 135S and 178R, and has the set name "H_147T_185Q-L_135S_178R".

[0375] For example, the "H_147T_185Q-L_135S_178R" set (network 2529) shows a higher % correct CH1-CLκ pairing value when used in the exemplary SID as measured by LC-MS compared to WT CH1-CLκ (see Table 6).

[0376] Furthermore, when the CH1-CLλ set of H_147T_185Q-L_135S_178R (network 2529) was used in combination with the CH1-CLλ sets of H_148R-L_124S_129E (network 367) or H_124R_147R-L_127D_129E (network 964) in a bsAb, it was predicted to provide particularly preferential pairing between the CH1 and CLλ domains in both CH1-CLλ sets, as shown, for example, in Figure 19.

[0377] In this example, when Network 2529 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 41 and 42, respectively. When Network 2529 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 41 and 49, respectively.

[0378] Network 2529 substitutions can be engineered into any reference CH1 and CL domain sequence to provide preferential pairing between heavy and light chains containing the engineered variant domains.

[0379] (5) Network 367 The CH1-CL set of network 367 comprises a CH1 domain comprising an amino acid R at position 148 (148R) and a CL domain comprising an amino acid S at position 124 and an E at position 129 (124S and 129E). The CH1-CLκ set of network 367 comprises a CH1 domain comprising an amino acid substitution at position 148 (relative to the WT CH1 sequence) providing 148R and a CLκ domain comprising amino acid substitutions at 124 and 129 (relative to the WT CLκ sequence) providing 124S and 129E, and has the set name "H_148R-L_124S_129E". The CH1-CLλ set of network 367 also includes a CH1 domain containing an amino acid substitution at position 148 (relative to the WT CH1 sequence) providing 148R, and a CLλ domain containing amino acid substitutions at positions 124 and 129 (relative to the WT CLλ sequence) providing 124S and 129E, and has the set name "H_148R-L_124S_129E."

[0380] For example, the "H_148R-L_124S_129E" set (network 367) improves the % correct CH1-CLκ pairing values ​​when used in addition to another CH1-CLκ set such as the "H_145Q_147E_181E-L_129R_178R_180Q" set (network 1443) or the "H_168S_185S_187D-L_135R" set (network 1039) in an exemplary DID as measured by LC-MS (see Table 10).

[0381] Furthermore, when the CH1-CLλ set of H_148R-L_124S_129E (Network 367) was used in combination with the CH1-CLλ sets of H_145S_147N-L_133Y_180R (Network 1621), H_147T_185Q-L_135S_178R (Network 2529), or H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) in a bsAb, it was predicted to provide particularly preferential pairing between the CH1 and CLλ domains in both CH1-CLλ sets, as shown, for example, in Figure 19.

[0382] In this example, when Network 367 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 51 and 52, respectively. When Network 367 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 51 and 59, respectively.

[0383] Network 367 substitutions can be engineered into any reference CH1 and CL domain sequence to provide preferential pairing between heavy and light chains containing the engineered variant domains.

[0384] (6) Network 964 The CH1-CL set of network 964 includes a CH1 domain that includes amino acids R at position 124 and R at position 147 (124R and 147R), and a CL domain that includes amino acids D at position 127 and E at position 129 (127D and 129E). The CH1-CLκ set of network 964 includes a CH1 domain that includes amino acid substitutions (relative to the WT CH1 sequence) at positions 124 and 147 that provide 124R and 147R, and a CLκ domain that includes amino acid substitutions (relative to the WT CLκ sequence) at 127 and 129 that provide 127D and 129E, and has the set name "H_124R_147R-L_127D_129E." The CH1-CLλ set of network 964 also includes a CH1 domain containing amino acid substitutions (relative to the WT CH1 sequence) at positions 124 and 147 providing 124R and 147R, and a CLλ domain containing amino acid substitutions (relative to the WT CLλ sequence) at positions 127 and 129 providing 127D and 129E, and has the set name "H_124R_147R-L_127D_129E."

[0385] Furthermore, the "H_124R_147R-L_127D_129E" set (network 964) improves the % correct CH1-CLκ pairing value when used in addition to another CH1-CLκ set, such as the "H_145Q_147E_181E-L_129R_178R_180Q" set (network 1443) (to achieve 95% correct CH1-CLκ pairing) in the exemplary DID as measured by LC-MS (see Table 10).

[0386] Furthermore, when the CH1-CLλ set of H_124R_147R-L_127D_129E (Network 964) was used in combination with the CH1-CLλ sets of H_145Q_147E_181E-L_129R_178R_180Q (Network 1443), H_145S_147N-L_133Y_180R (Network 1621), or H_147T_185Q-L_135S_178R (Network 2529) in a bsAb, it was predicted to provide particularly preferential pairing between the CH1 and CLλ domains in both CH1-CLλ sets, as shown, for example, in Figure 19.

[0387] In this example, when Network 964 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 91 and 92, respectively. When Network 964 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 91 and 99, respectively.

[0388] Network 964 substitutions can be engineered into any reference CH1 and CL domain sequence to provide preferential pairing between heavy and light chains containing the engineered variant domains.

[0389] (7) Network 742 The CH1-CL set of network 742 includes a CH1 domain that includes an amino acid N at position 147 and a Y at position 185 (147N and 185Y), and a CL domain that includes an amino acid R at position 129 and an S at position 180 (129R and 180S). The CH1-CLκ set of network 742 includes a CH1 domain that includes amino acid substitutions (relative to the WT CH1 sequence) at positions 147 and 185 that provide 147N and 185Y, and a CLκ domain that includes amino acid substitutions (relative to the WT CLκ sequence) at positions 129 and 180 that provide 129R and 180S, and has the set name "H_147N_185Y-L_129R_180S." The CH1-CLλ set of network 742 includes a CH1 domain containing amino acid substitutions (relative to the WT CH1 sequence) at positions 147 and 185 providing 147N and 185Y, and a CLλ domain containing an amino acid substitution (relative to the WT CLλ sequence) at position 129 providing 129R (note that position 180 is S in WT CLλ), and has the set name "H_147N_185Y-L_129R."

[0390] For example, the "H_147N_185Y-L_129R_180S" set (network 742) shows a higher % correct CH1-CLκ pairing value when used in the exemplary SID as measured by LC-MS compared to WT CH1-CLκ (see Table 6).

[0391] In this example, when network 742 substitutions are made into the reference CH1 and CLκ domain sequences of SEQ ID NOs: 1 and 2, the variant CH1 and CLκ domains comprise the amino acid sequences of SEQ ID NOs: 201 and 202, respectively. When network 742 substitutions are made into the reference CH1 and CLλ domain sequences of SEQ ID NOs: 1 and 9, the variant CH1 and CLλ domains comprise the amino acid sequences of SEQ ID NOs: 201 and 209, respectively.

[0392] Network 742 substitutions can be engineered into any reference CH1 and CL domain sequence to provide preferential pairing between heavy and light chains containing the engineered variant domains.

[0393] It should be noted that heavy chain polypeptides comprising any of the variant CH1 domain polypeptides described above, and light chain polypeptides comprising any of the variant CLκ or CLλ domain polypeptides described above, are also encompassed by the present invention.

[0394] Polypeptides, molecules, and multispecific antibodies A variant CH1 domain, a variant CL domain, and / or a variant CH1-CL domain set according to the present disclosure may be present in a polypeptide, molecule, and / or a multispecific antibody.

[0395] As used herein, an "immunoglobulin polypeptide" refers to a polypeptide comprising at least one immunoglobulin domain (or a variant thereof) (e.g., a CH1 domain, a CL domain, etc.). In certain cases, the CH1 domain may be present in a first polypeptide. The CH1 domain may be a variant CH1 domain according to the present disclosure. In certain cases, the CL domain may be present in a second polypeptide. The CL domain may be a variant CL domain according to the present disclosure (e.g., a variant CLκ domain or a variant CLλ domain). When the CH1 domain in the first polypeptide preferentially pairs with the CL domain in the second polypeptide (e.g., the CH1 and CL are a variant CH1-CL set according to the present disclosure), a heterodimeric molecule may be formed between the first and second polypeptides. Such molecules may be multispecific antibodies having structures such as, but not limited to, those disclosed in Figures 2-7.

[0396] In some embodiments, such CH1-CL sets are the following CH1-CLκ sets: H_168S_185S_187D-L_135R (e.g., network 1039), H_128R_147R-L_124E_133Q_178E (network 1993), H_145Q_147E_181E-L_129R_178R_180Q (network 1443), H_147T_185Q-L_135S_178R (network 2529), H_148R-L_124S_129E (Network 367), H_139R_141Q_187Q-L_114D_135S_138R (Network 1888), H_166K_187K-L_137S_138E (Network 1328), H_168R_185E-L_135S (Network 2366), H_124R_147R-L_127D_129E (Network 964), H_147H_148E-L_1 27R_129R (Network 767), H_145S-L_133Y (Network 1148), H_145S_181Q-L_133Y (Network 384), H_145S-L_124E_133Y (Network 454), H_145Q_181E-L_120S_178H_180Q (Network 1048), H_124R_145S_147Q-L_127T_129D_178R (Network 534), H_166K_ 187K-L_114Q_137T_138E (Network 838), H_147R_175D-L_129D_178R_180H (Network 919), H_147R_175E_181Q-L_129D_180Q (Network 394), H_145S_147N-L_133Y_180R (Network 1621), or H_147N_185Y-L_129R_180S (Network 742). Exemplary CH1 and CLκ sequences in these CH1-CLκ sets are provided in Appendix Tables A-B and the Sequence Listing.

[0397] In certain embodiments, such a CH1-CLκ set may be any of the following CH1-CLκ sets: H_168S_185S_187D-L_135R (network 1039), H_128R_147R-L_124E_133Q_178E (network 1993), H_145Q_147E_181E-L_129R_178R_180Q (network 1443), or H_147T_185Q-L_135S_178R (network 2529).

[0398] In some embodiments, such CH1-CL sets include the following CH1-CL lambda sets: H_168S_185S_187D-L_135R (network 1039), H_128R_147R-L_133Q_178E (network 1993), H_145Q_147E_181E-L_129R_178R_180Q (network 1443), H_147T_185Q-L_135S_178R (network 252 9), H_148R-L_124S_129E (Network 367), H_139R_141Q_187Q-L_114D_135S_138R (Network 1888), H_166K_187K-L_138E (Network 1328), H_168R_185E-L_135S (Network 2366), H_124R_147R-L_127D_129E (Network 964), H_147H_148E-L_127 R_129R (Network 767), H_145S-L_133Y (Network 1148), H_145S_181Q-L_133Y (Network 384), H_145S-L_133Y (Network 454), H_145Q_181E-L_120S_178H_180Q (Network 1048), H_124R_145S_147Q-L_127T_129D_178R (Network 534), H_166K_1 87K-L_114Q_137T_138E (Network 838), H_147R_175D-L_129D_178R_180H (Network 919), H_147R_175E_181Q-L_129D_180Q (Network 394), H_145S_147N-L_133Y_180R (Network 1621), or H_147N_185Y-L_129R (Network 742). Exemplary CH1 and CLλ sequences in these CH1-CLλ sets are provided in Appendix Tables A and C and in the Sequence Listing.

[0399] In certain embodiments, such a CH1-CL λ set may be any of the following CH1-CL λ sets: H_148R-L_124S_129E (network 367), H_124R_147R-L_127D_129E (network 964), H_145Q_147E_181E-L_129R_178R_180Q (network 1443), H_145S_147N-L_133Y_180R (network 1621), H_168R_185E-L_135S (network 2366), H_147T_185Q-L_135S_178R (network 2529), or H_128R_147R-L_133Q_178E (network 1993).

[0400] Such immunoglobulin polypeptides may further comprise one or more antigen-binding domains (such as VH, VL, scFv, or nanobody), CH1, CH2, CH3, and / or CL domains. Such polypeptides may be part of a multispecific antibody molecule.

[0401] In some embodiments, a polypeptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody), a variant CH1 domain, and optionally a CH2, CH3, and / or CL domain. In some embodiments, a polypeptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody), a variant CL domain, and optionally a CH1, CH2, and / or CH3 domain. In some embodiments, two such polypeptides may pair with each other. In such cases, when the antigen-binding domains of the two polypeptides are a cognate VH and VL pair or a cognate VL and VH pair, the VH and VL may form an antigen-binding site for the cognate epitope.

[0402] Alternatively, an immunoglobulin polypeptide may not comprise a VH, VL, CH1, or CH2 domain. For example, a first polypeptide may comprise a first domain in addition to a variant CH1 domain. If a second polypeptide further comprises a second domain in addition to a variant CL domain that preferentially pairs with the variant CH1 domain, and if heterodimer formation between the first and second domains is desired, the preferential pairing between the variant CH1 domain and the variant CL domain will promote heterodimerization of the first and second domains.

[0403] In one embodiment, such a polypeptide may be comprised in a molecule such as a multispecific antibody or fragment thereof. When the molecule is a multispecific antibody or fragment thereof, a variety of structures are possible, including but not limited to those shown in Figures 2-7.

[0404] In some embodiments, such molecules can include a first polypeptide comprising a variant CH1 domain and a second polypeptide comprising a variant CL domain, wherein the variant CH1 domain and the variant CL domain preferentially form a pair. For example, the variant CH1 domain and the variant CL domain can be a first CH1-CL set, which can be, for example, any of the CH1-CL sets described herein. The CL isotype can be kappa or lambda.

[0405] In some embodiments, such molecules can include a third polypeptide comprising a variant CH1 domain and a fourth polypeptide comprising a variant CL domain, wherein the variant CH1 domain and the variant CL domain preferentially form a pair. For example, the variant CH1 domain and the variant CL domain can be, for example, a second CH1-CL set, which can be any of the CH1-CL sets according to the present invention and can be different from the first CH1-CL set. The CL isotype in the second CH1-CL set can be kappa or lambda and can be the same as or different from the CL isotype in the first CH1-CL set.

[0406] In some cases, a CH1 in the first set does not preferentially pair with a CL in the second set, a CL in the first set does not preferentially pair with a CH1 in the second set, a CH1 in the second set does not preferentially pair with a CL in the first set, and a CL in the second set does not preferentially pair with a CH1 in the first set.

[0407] In certain cases, such molecules are distinct from one another (all sets may be CH1-CLκ sets, all sets may be CH1-CLλ sets, or two or more CH1-CL sets may be a mixture of CH1-CLκ sets and CH1-CLλ sets), but both comprise two or more CH1-CL sets according to the present invention. Each of the two or more CH1-CL sets may be a CH1-CL set of a network selected from Network 1039, Network 1993, Network 1443, Network 2529, Network 367, HNetwork 1888, Network 1328, Network 2366, Network 964, Network 767, Network 1148, Network 384, Network 454, Network 1048, Network 534, Network 838, Network 919, Network 394, Network 1621, or Network 742.

[0408] In certain cases, when such molecules comprise two CH1-CLκ sets that are different from each other but both are according to the invention, the combinations of CH1-CLκ sets can be, for example, (i) H_145Q_147E_181E-L_129R_178R_180Q (network 1443) and H_128R_147R-L_124E_133Q_178E (network 1993), (ii) H_168S_185S_187D-L_135R (network 1039) and H_128R_147R-L_124E_133Q_178E (network 1040). 124E_133Q_178E (Network 1993), (iii) H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) and H_124R_147R-L_127D_129E (Network 964), (iv) H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) and H_168S_185S_187D-L_135R (Network 1039), (v) H_145Q_147E_181E-L_ 129R_178R_180Q (Network 1443) and H_148R-L_124S_129E (Network 367), (vi) H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) and H_168R_185E-L_135S (Network 2366), (vii) H_168S_185S_187D-L_135R (Network 1039) and H_148R-L_124S_129E (Network 367), (viii) H_16 8S_185S_187D-L_135R (network 1039) and H_147T_185Q-L_135S_178R (network 2529), (ix) H_168S_185S_187D-L_135R (network 1039) and H_147N_185Y-L_129R_180S (network 742), or (x) H_168S_185S_187D-L_135R (network 1039) and H_168R_185E-L_135S (network 2366).

[0409] In a further example, when such a molecule comprises two CH1-CLκ sets that are different from each other but both are according to the invention, the combinations of CH1-CLκ sets can be, for example: (i) H_145Q_147E_181E-L_129R_178R_180Q (Network 1443) and H_128R_147R-L_124E_133Q_178E (Network 1993), (ii) H_168S_185S_187D-L_135R (Network 1039) and H_128R_147R-L_124E_133Q_178E (Network 1993), (iii) H_14 5Q 147E 181E-L 129R 178R 180Q (network 1443) and H 124R 147R-L 127D 129E (network 964), or (iv) H 145Q 147E 181E 129R 178R 180Q (network 1443) and H 168S 185S 187D-L 135R (network 1039), and preferably (i) H 145Q 147E 181E 129R 178R 180Q (network 1443) and H 128R 147R-L 124E 133Q 178E. In some cases, the network combination provides at least 95% correct matches.In certain cases, when such molecules comprise two CH1-CL sets that are different from each other but both are according to the invention, the combinations of CH1-CL sets can be, for example, (i) H_148R-L_124S_129E (Network 367) and H_145S_147N-L_133Y_180R (Network 1621), (ii) H_124R_147R-L_127D_129E (Network 964) and H_145Q_147E_181E-L_129R_178R_180Q (Network 1443), (iii) H_148R-L_124S_129E (Network 367) and H_147T_185Q-L_135S_178R (Network 2529), (iv) H_124R _147R-L_127D_129E (Network 964) and H_145S_147N-L_133Y_180R (Network 1621), (v) H_148R-L_124S_129E (Network 367) and H_145Q_147E_181E-L_129R_178R_180Q (Network 1443), (vi) H_124R_ 147R-L_127D_129E (network 964) and H_147T_185Q-L_135S_178R (network 2529), (vii) H_145Q_147E_181E-L_129R_178R_180Q (network 1443) and H_128R_147R-L_133Q_178E (network 1993).

[0410] In further instances, when such molecules comprise two CH1-CLλ sets that are distinct from one another but both are in accordance with the present invention, the combination of CH1-CLλ sets can be, for example, (i) H_148R-L_124S_129E (Network 367) and H_145S_147N-L_133Y_180R (Network 1621), or (ii) H_124R_147R-L_127D_129E (Network 964) and H_145Q_147E_181E-L_129R_178R_180Q (Network 1443). In some instances, the combination of networks provides at least 95% correct matching.

[0411] In some embodiments, such molecules include, in addition to a first polypeptide and a second polypeptide, a third polypeptide comprising a CH1 domain and a fourth polypeptide comprising a CL domain that is distinct from the CL isotype of the second polypeptide, wherein the CH1 domain of the third polypeptide and the CL domain of the fourth polypeptide may preferentially pair. Such variant CH1 domains and variant CL domains may be referred to as a second CH1-CL set.

[0412] In some cases, a CH1 in the first set does not preferentially pair with a CL in the second set, a CL in the first set does not preferentially pair with a CH1 in the second set, a CH1 in the second set does not preferentially pair with a CL in the first set, and / or a CL in the second set does not preferentially pair with a CH1 in the first set.

[0413] In some embodiments, such molecules may utilize, in addition to the first variant CH1 and CL domains, optionally other variants outside of the CH1 and CL domains, such as variants in the antigen binding domain and / or hinge, to further promote preferential heteropairing between the two polypeptides.

[0414] In some cases, the first and second polypeptides may be further linked, e.g., via one or more disulfide bonds, linkers, etc. In some cases, the third and fourth polypeptides may be further linked, e.g., via one or more disulfide bonds, linkers, etc.

[0415] Such molecules may be multispecific antibodies having structures such as, but not limited to, those disclosed in Figures 2-7. Multispecific antibodies according to the present disclosure may be bispecific, trispecific, tetraspecific, or specific for five, six, or more epitopes. Multispecific antibodies according to the present disclosure may be bivalent, trivalent, or tetravalent, or may have five, six, or more valencies.

[0416] In some embodiments, a multispecific antibody or antibody fragment according to the present disclosure may comprise multiple CH1-CL design sets. In certain embodiments, all of the multiple CH1-CL design sets may be CH1-CLκ sets. In certain embodiments, all of the multiple CH1-CL design sets may be CH1-CLλ sets. In certain embodiments, the multiple CH1-CL design sets may be a mixture of one or more CH1-CLκ sets and one or more CLλ sets.

[0417] In such multispecific antibodies or antibody fragments, each CH1-CL set can be directly or indirectly bound to an antigen-binding site (e.g., formed by a VH and a VL, or in the case of nanobodies, by a VH). Because such multispecific antibodies or antibody fragments comprise multiple CH1-CL design sets (e.g., set A, set B, set C, etc.) and multiple antigen-binding sites (e.g., site A, site B, site C, etc.), multiple combinations of CH1-CL design sets with antigen-binding sites may be possible. For example, in one case, set A may be bound to site A, set B may be bound to site B, and set C may be bound to site C, while in another case, set A may be bound to site B, set B may be bound to site C, etc.

[0418] In some cases, specific combinations (of CH1-CL design sets with antigen binding sites) may result in multispecific antibodies or fragments thereof with improved developability properties.Such properties include (i) production yield and / or suitability for a particular purification method (e.g., protein A affinity purification), which may be assessed in one or more cell types (e.g., mammalian cells such as CHO and HEK cells, yeast cells, insect cells, plant cells, etc.) using any suitable method or as described herein; (ii) the degree of aggregation (e.g., the presence of multimers of the complete antibody) (also referred to herein as purity), which may be quantified by, for example, chromatography, such as size exclusion chromatography (SEC), or electrophoresis, such as SDS-PAGE, using any suitable method or as described herein; (iii) the degree of aggregation (e.g., the presence of multimers of the complete antibody), which may be assessed by, for example, LC-MS using any suitable method or as described herein; (iv) the melting temperature (Tm) and / or aggregation temperature (Tagg) (e.g., Tagg266), which may be measured using any suitable method or as described herein, for example, by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC), or using an instrument such as Uncle®; (v) the "pI", isoelectric point ("pI"), which may be measured using any suitable method; (vi) the level of interaction with a multispecific reagent ("PSR"), which may be measured using any suitable method or as described herein, for example, as in WO2014 / 179363; (vii) the level of interaction with a multispecific reagent ("PSR"), which may be measured using any suitable method or as described herein, for example, as in WO2014 / 179363; These may include, but are not limited to, antibody hydrophobic interactions, which may be measured by hydrophobic interaction chromatography ("HIC") as described in p, et al. MAbs. 2015 May-Jun;7(3):553-561, (viii) self-interactions, (ix) stability to high or low pH stress, (x) solubility, (xi) production cost and / or time, (xii) other stability parameters, (xiii) shelf life, (xiv) in vivo half-life, and / or (xv) immunogenicity, which may be assessed using any suitable method.

[0419] Reduced self-interaction can be predicted in silico or measured by in vitro assays. Such in vitro assays can include, but are not limited to, affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) and dynamic light scattering (DLS) analysis. In some embodiments, various combinations of CH1-CL design sets with antigen-binding sites, each with equivalent multispecific antigen-binding functionality, can be screened to select combinations with improved developability properties (e.g., reduced self-interaction).

[0420] In certain cases, self-interactions can be measured in vitro by AC-SINS using a previously described protocol (Liu et al., MAbs. Mar-Apr 2014;6(2):483-92). For example, a polyclonal goat anti-human IgG Fc antibody (capture; Jackson ImmunoResearch Laboratories) and a polyclonal goat nonspecific antibody (non-capture; Jackson ImmunoResearch Laboratories) can be buffer-exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to 0.4 mg / ml. A 4:1 volume ratio of capture:non-capture can be prepared and further incubated with 20 nm gold nanoparticles (AuNP, Ted Pella Inc.) in a 1:9 volume ratio for 1 hour at room temperature. Thiolated PEG (Sigma-Aldrich) can then be used to block vacant sites on the AuNP and filtered through a 0.22 μm PVDF membrane (Millipore). The coated particles are then added to the test antibody solution and allowed to incubate for 2 hours at room temperature before measuring the absorbance at 510-570 nm on a plate reader. Data points can be fitted with a second-order polynomial in Excel to obtain the wavelength at maximum absorbance. Values ​​can be reported as the difference between the sample and background plasmon wavelengths (Δλmax). The self-interaction level can be determined based on Δλmax. Self-interaction can be considered low when Δλmax<5 nm, moderate when Δλmax≥5 nm and <20 nM, and high when Δλmax≥20 nm.

[0421] In certain cases, self-interactions can be measured in vitro by DLS. The diffusion interaction parameter (kD) of a monoclonal antibody, typically measured at concentrations below 12 mg / mL, correlates strongly with its solution behavior at very high concentrations (>100 mg / mL). Positive kD values ​​indicate repulsive interactions between molecules and correlate positively with low viscosity at high concentrations in the same formulation buffer. kD values ​​can be obtained by measuring the mutual diffusion coefficient (D) at a series of different concentrations (C) by DLS. For example, DLS kD measurements can be performed at multiple concentrations from 0.5 to 12 mg / mL in a 10 mM histidine buffer at pH 6.0. The method can be easily modified for different formats of antibodies, including bsAbs, and in different formulation buffers.

[0422] Stability to high or low pH stress can be measured by placing an antibody or fragment thereof in a high or low pH environment for a certain period of time, followed by one or more biochemical analyses. For example, to test stability to high pH, ​​100 μL of a 2 mg / mL IgG sample can be buffer-exchanged into 20 mM Tris, 10 mM EDTA (pH 8.5) and incubated at 40° C. After 7 days, the stressed sample can be collected and subjected to tryptic peptide mapping and CZE analysis; to test stability to low pH, 100 μL of a 2 mg / mL IgG sample can be buffer-exchanged into 50 mM sodium acetate buffer (pH 5.5) and incubated at 40° C. After 14 days, the stressed sample can be collected and subjected to tryptic peptide mapping and reduced intact mass analysis.

[0423] Polynucleotides, Vectors, Cells, and Compositions Polypeptides, molecules, and / or multispecific antibodies comprising the variant CH1 domains and / or CL domains described herein may be encoded by one or more polynucleotides. Such one or more polynucleotides may be DNA or RNA, or a combination thereof.

[0424] Any of the polypeptides described herein can be present in a vector.

[0425] Any of the variant CH1 domains, variant CL domains, CH1-CL sets, polypeptides, molecules, multispecific antibodies, polynucleotides, and / or vectors may be present in a cell, e.g., a eukaryotic cell. In some embodiments, such polypeptides may be expressed in mammalian cells, such as HEK923 cells or Chinese hamster ovary (CHO) cells. In some embodiments, the variant CH1 and / or CL domains are expressed in yeast.

[0426] Any of the variant CH1 domain, variant CL domain, CH1-CL set, polypeptide, molecule, multispecific antibody, polynucleotide, vector, and / or cell may be present in the composition. If the composition is a therapeutic composition, the composition may further comprise a pharmaceutically acceptable carrier.

[0427] CH1 domain library, CL domain library, and CH1-CL set screening / selection Also contemplated by the present disclosure are methods for generating CH1 domain libraries, which may be used specifically to screen for CH1 sequences that preferentially pair with CL domains or variant CL domains (which may be of the kappa or lambda isotype).

[0428] In some embodiments, at least one nucleic acid position within a codon encoding any of the amino acid positions of CH1 that are within or adjacent to the CH1-CL interface can be varied, in certain embodiments, adjacent can mean 1, 2, 3, 4, or 5 amino acids upstream or downstream of an amino acid that is present at the CH1-CL interface.

[0429] In some embodiments, at least one nucleic acid position within a codon encoding any of the CH1 amino acid positions at which an amino acid substitution occurs in any of the variant CH1 domains of the invention can be varied. For example, such predetermined amino acid positions can be 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and / or 187 according to EU numbering.

[0430] In some embodiments, any of the following combinations of amino acid positions may be varied: 168, 185, and 187; 128 and 147; 145, 147, and 181; 147 and 185; 148; 139, 141, and 187; 166 and 187; 168 and 185; 124 and 147; 147 and 148; 145; 145 and 181; 124, 145, and 147; 166 and 187; 147 and 175; 147R, 175, and 181; 145 and 147; or 147 and 185.

[0431] In some embodiments, 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, may be used to induce variability at a given position.

[0432] Also contemplated by the present disclosure are CH1 domain libraries. In some embodiments, the CH1 domain library can be a library generated by any method for generating a CH1 domain library described herein.

[0433] Also contemplated by the present disclosure are methods for generating CL domain libraries. The libraries can be particularly used to screen for CL sequences that preferentially pair with variant CH1 domains. The libraries can be CLκ domain libraries, CLλ domain libraries, or libraries containing both CLκ and CLλ domains.

[0434] In some embodiments, at least one nucleic acid position within a codon encoding any of the CL amino acid positions present within or adjacent to the CH1-CL interface can be varied, in certain embodiments, adjacent can mean 1, 2, 3, 4, or 5 amino acids upstream or downstream of an amino acid present at the CH1-CL interface.

[0435] In some embodiments, at least one nucleic acid position within a codon encoding any of the CL amino acid positions at which an amino acid substitution occurs in any of the variant CL domains of the invention may be varied, for example, such predetermined amino acid positions may be 114, 120, 124, 127, 129, 133, 135, 137, 138, 178, and 180 according to EU numbering.

[0436] In some embodiments, any of the combinations of amino acid positions selected from 135; 124, 133, and 178; 129, 178, and 180; 135 and 178; 124 and 129; 114, 135, and 138; 137 and 138; 127 and 129; 133; 124 and 133; 120, 178, and 180; 127, 129, and 178; 114, 137, and 138; 129, 178, and 180; 133 and 180; or 129 and 180 may be varied.

[0437] In some embodiments, any of the following combinations of amino acid positions may be varied in CLλ: 135; 133 and 178; 129, 178, and 180; 135 and 178; 124 and 129; 114, 135, and 138; 138; 127 and 129; 133; 120, 178, and 180; 127, 129, and 178; 114, 137, and 138; 129, 178, and 180; 133 and 180; or 129.

[0438] In some embodiments, 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, may be used to induce variability at a given position.

[0439] CL domain libraries are also contemplated by the present disclosure. In some embodiments, the CL domain library can be a library generated by any of the methods for generating a CL domain library described herein. The CL library can be a CLκ domain library, a CLλ domain library, or a library containing both CLκ and CLλ domains.

[0440] Also contemplated by the present disclosure are methods for generating CH1-CL domain set libraries. The libraries can be particularly used to screen for CH1-CL domain sets in which the CH1 and CL domains in the set preferentially pair with each other. The CL domains included in such libraries can be all CLκ domains, all CLλ domains, or a mixture of both CLκ domains and CLλ domains.

[0441] In some embodiments, the method may include selecting a combination of CH1 and CL domain positions predicted to influence CH1-CL domain interactions, such as interactions mediated by hydrogen bonds. In certain embodiments, the prediction may be made in silico. In certain embodiments, the prediction may be made in vitro. In certain embodiments, the in silico or in vitro prediction may be made based on a model antibody or antibody fragment, which may be, for example, a full-sized Ig molecule such as an IgG (IgG1, IgG2, IgG3, or IgG4), a Fab fragment, an scFv, or a bispecific antibody or antibody fragment such as one having any of the structures in Figures 2-7. In certain embodiments, CH1+CLκ domain coordinates from the Protein Data Bank (PDB), e.g., published CH1+CLκ domain coordinates such as ID 1fvd, may be used for the prediction using any suitable method (e.g., Maguire JB, et al., J Chem Theory Comput. 2018 May 8;14(5):2751-2760).

[0442] In some embodiments, the method may include preselecting combinations of CH1 and CL domain substitutions predicted to increase CH1-CL domain interactions, such as interactions mediated by hydrogen bonds. In certain embodiments, the predictions may be performed in silico. For example, the Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet) (see, e.g., Maguire JB, et al., J Chem Theory Comput. 2018 May 8;14(5):2751-2760), a computational protocol for in silico modeling of amino acid substitutions at protein-protein interfaces to design self-contained hydrogen bond networks, may be used. In certain embodiments, the predictions may be performed in vitro. In certain embodiments, the in silico or in vitro predictions may be based on a model antibody or antibody fragment, which may be, for example, a full-sized Ig molecule such as an IgG (IgG1, IgG2, IgG3, or IgG4), a Fab fragment, an scFv, or a bispecific antibody or antibody fragment such as one having the structure of any of Figures 2-7. In certain embodiments, published CH1+CLκ domain coordinates from the Protein Data Bank (PDB), such as ID 1fvd, can be used for prediction using any suitable method (e.g., Maguire JB, et al., J Chem Theory Comput. 2018 May 8;14(5):2751-2760).

[0443] In some embodiments, the number of CH1 substitution positions included in the CH1-CL domain set library can be predetermined, e.g., the number can be predetermined to be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5.

[0444] In some embodiments, the number of CL substitution positions included in the CH1-CL domain set library can be predetermined. For example, the number can be predetermined to be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0445] In some embodiments, certain known CH1-CL design sets or all known CH1-CL design sets can be removed from the CH1-CL domain set library.

[0446] In some embodiments, the method may include varying any combination of (i) a CH1 substitution position contained in any of the CH1 domain libraries described herein and (ii) a CL substitution position contained in any of the CL domain libraries described herein.

[0447] In certain embodiments, 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, may be used to induce variability at a given position.

[0448] In some embodiments, the method can include varying any combination of (i) a CH1 substitution contained in any of the CH1 domain libraries described herein and (ii) a CL substitution contained in any of the CL domain libraries described herein.

[0449] In certain embodiments, the method may include introducing any combination of (i) a CH1 substitution contained in any of the CH1 domain libraries described herein and (ii) a CLκ substitution contained in any of the CLκ domain libraries described herein, in a CH1-CLλ domain set library, where (i) a CH1 substitution contained in any of the CH1 domain libraries described herein and (ii) a CLλ substitution contained in any of the CLλ domain libraries described herein.

[0450] Also contemplated by the present disclosure are CH1-CL domain set libraries. In some embodiments, the CH1-CL domain set library can be a library generated by any method for generating a CH1-CL domain set library described herein. The CH1-CL domain set library can be a CH1-CLκ domain set library, a CH1-CLλ domain set library, or a library comprising both a CH1-CLκ domain set and a CH1-CLλ domain set. Also provided herein are methods for identifying one or more variant CH1 domains that preferentially pair with a CL domain or a variant CL domain, identifying one or more CL domains and / or variant CL domains that preferentially pair with a variant CH1 domain, and / or identifying one or more sets of CH1 domains and CL domains that preferentially pair with each other.

[0451] In some embodiments, the method is a method for identifying one or more sets of CH1 domains and CL domains that preferentially pair with each other. Such a method may include at least three steps.

[0452] The first step may include computationally or recombinantly co-expressing or combining (a-1) a first polypeptide or a first set of polypeptides, each comprising a wild-type CH1 domain or a variant CH1 domain, and (a-2) a second polypeptide or a second set of polypeptides, each comprising a wild-type CL domain or a variant CL domain. In certain embodiments, the variant CH1 domain may be expressed from the variant CH1 domain library described above. In certain embodiments, the variant CL domain may be expressed from the variant CL domain library described above. In certain embodiments, the variant CH1 domain and the variant CL domain may be expressed from the CH1-CL domain set library described above. In certain embodiments, the variant CH1 domain and the variant CL domain may be expressed from the CH1-CL domain set library comprising random mutations that can cause random amino acid modifications in the CH1 domain and / or CL domain.

[0453] The second step can involve quantifying binding or binding preference between a CH1 domain or variant CH1 domain and a CL domain or variant CL domain. In some embodiments, CH1-CL domain interactions, such as interactions mediated by hydrogen bonds, can be quantified. In certain embodiments, CH1-CL domain interactions can be quantified in silico. In certain embodiments, CH1-CL domain interactions can be quantified in vitro. In certain embodiments, in silico or in vitro quantification can be performed using Rosetta Monte Carlo (MC) hydrogen bond network (HBNet) (see, e.g., Maguire JB, et al., J Chem Theory Comput. 2018 May 8;14(5):2751-2760), a computational protocol for in silico modeling of amino acid substitutions at protein-protein interfaces to design built-in hydrogen bond networks. In certain embodiments, in silico or in vitro quantification may be performed based on a model antibody or antibody fragment, which may be, for example, a full-sized Ig molecule such as an IgG (IgG1, IgG2, IgG3, or IgG4), a Fab fragment, an scFv, a bispecific antibody or antibody fragment such as one having any of the structures in Figures 2-7. In certain embodiments, published CH1+CLκ domain coordinates from the Protein Data Bank (PDB), e.g., ID 1fvd, may be used for CH1-CL domain interaction quantification using any suitable method (e.g., Maguire JB, et al., J Chem Theory Comput. 2018 May 8;14(5):2751-2760).

[0454] The third step may include selecting one or more sets of CH1 domains or variant CH1 domains and CL domains or variant CL domains that provide preferential CH1-CL pairing. Such preferential CH1-CL pairing may optionally be equivalent to or higher than the pairing provided by a reference CH1-CL set. In certain cases, the reference CH1-CL set may optionally include a wild-type CH1 domain, a wild-type CL domain, a variant CH1 domain according to the present invention, and / or a variant CL domain according to the present invention. In certain cases, the reference CH1-CL set may optionally be a wild-type CH1-CL domain set and / or a CH1-CL domain set according to the present invention.

[0455] Mutabilization can be performed on any available CH1 and / or CL sequence, i.e., wild-type or modified sequences. In some embodiments, CH1 mutagenesis can be performed on the reference CH1 sequence of SEQ ID NO: 1. In some embodiments, CL mutagenesis can be performed on the reference CLκ sequence of SEQ ID NO: 2 and / or the reference CLλ sequence of SEQ ID NO: 9.

[0456] In some embodiments, a first polypeptide may contain or be ...

Claims

1. 1. A variant immunoglobulin heavy chain constant region 1 ("CH1") domain polypeptide, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide, the variant CH1 domain polypeptide comprises at least one amino acid substitution comprising, or consisting of, an amino acid substitution at one or more of the following CH1 amino acid positions according to EU numbering: 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187, and optionally (A) preferentially pairs with a variant immunoglobulin kappa light chain constant region (CLK) or variant lambda light chain constant region (CLλ) domain polypeptide, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide, wherein said variant CLK or CLλ domain polypeptide comprises at least one amino acid substitution, optionally comprising or consisting of an amino acid substitution at one or more of the following CLK or CLλ positions according to EU numbering: 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138; (B) the variant CH1 domain polypeptide is a variant of the CH1 domain of human IgG, and optionally of human IgG1, human IgG2, or IgG4; (C) the one or more amino acid substitutions in the variant CH1 domain polypeptide are (i) positions 145, 147, and 181, or (ii) positions 128 and 147, or (iii) positions 168, 185, and 187, or (iv) positions 147 and 185, or (v) position 148, or (vi) positions 139, 141, and 187, or (vii) positions 166 and 187, or (viii) positions 168 and 185, or (ix) positions 124 and 147, or (x) positions 147 and 148, or (xi) position 145, or (xii) positions 145 and 181, or (xiii) positions 124, 145, and 147, or (xiv) positions 166 and 187, or (xv) positions 147 and 175, or (xvi) positions 147, 175, and 181, or (xvii) positions 145 and 147, or (xviii) comprising, or consisting of, amino acid substitutions at positions 147 and 185; Optionally, the variant CH1 domain polypeptide preferentially pairs with a variant CLK or CLλ domain polypeptide, and further optionally, (i), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 129, 178, and 180; (ii), wherein the amino acid substitutions in the variant CLK domain polypeptide comprise or consist of amino acid substitutions at positions 124, 133, and 178, or wherein the amino acid substitutions in the variant CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 133 and 178, (iii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 135; (iv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 135 and 178; (v), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 124 and 129; (vi), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 114, 135, and 138; (vii), wherein the amino acid substitution in the variant CLK domain polypeptide comprises or consists of an amino acid substitution at positions 137 and 138, or wherein the amino acid substitution in the variant CLλ domain polypeptide comprises or consists of an amino acid substitution at position 138, (viii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 135; (ix), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 127 and 129; (x), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 127 and 129; (xi), wherein the amino acid substitution in the variant CLK domain polypeptide comprises or consists of an amino acid substitution at position 133 or amino acid substitutions at positions 124 and 133, or wherein the amino acid substitution in the variant CLλ domain polypeptide comprises or consists of an amino acid substitution at position 133; (xii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 133, or amino acid substitutions at positions 120, 178, and 180; (xiii), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 127, 129, and 178; (xiv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 114, 137, and 138; (xv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 129, 178, and 180; (xvi), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 129 and 180; (xvii), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of amino acid substitutions at positions 133 and 180; or (xviii), wherein the amino acid substitution in the variant CLK domain polypeptide comprises, or consists of, amino acid substitutions at positions 129 and 180, or wherein the amino acid substitution in the variant CLλ domain polypeptide comprises, or consists of, an amino acid substitution at position 129; In each of the foregoing, the substitution positions are, according to EU numbering: (D) the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of: 145Q, 147E, 181E, 128R, 147R, 124R, 139R, 141Q, 145S, 147H, 147N, 147Q, 147T, 148E, 148R, 166K, 168R, 168S, 175D, 175E, 181Q, 185E, 185Q, 185S, 185Y, 187D, 187K, and / or 187Q, according to EU numbering; (E) the amino acid substitutions in the variant CH1 domain are (i) 145Q, 147E, and 181E, (ii) 128R and 147R, (iii) 168S, 185S, and 187D, (iv) 147T and 185Q, (v) 148R, (vi) 139R, 141Q, and 187Q, (vii) 166K and 187K, (viii) 168R and 185E, (ix) 124R and 147R, (x) 147H and 148E, (xi) 145S, (xii) 145S and 181Q, (xiii) 145Q and 181E, (xiv) 124R, 145S, and 147Q, (xv) 166K and 187K, (xvi) 147R and 175D, (xvii) 147R, 175E, and 181Q, (xiii) 145S and 147N, or (xix) 147N and 185Y, and optionally wherein said variant CH1 domain polypeptide preferentially pairs with a variant CLK or CLλ domain polypeptide, and further optionally wherein (i), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 129R, 178R, and 180Q; (ii), wherein the amino acid substitutions in the variant CLK domain polypeptide comprise or consist of 124E, 133Q, and 178E, or wherein the amino acid substitutions in the variant CLλ domain polypeptide comprise or consist of 133Q and 178E; (iii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of 135R; (iv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 135S and 178R; (v), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 124S and 129E; (vi), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 114D, 135S, and 138R; (vii), wherein the amino acid substitutions in the variant CLK domain polypeptide comprise or consist of 137S and 138E, or wherein the amino acid substitutions in the variant CLλ domain polypeptide comprise or consist of 138E; (viii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of 135S; (ix), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 127D and 129E; (x), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 127R and 129R; (xi), wherein the amino acid substitutions in the variant CLK domain polypeptide comprise or consist of 133Y or 124E and 133Y, or wherein the amino acid substitutions in the variant CLλ domain polypeptide comprise or consist of 133Y; (xii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of 133Y; (xiii), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 120S, 178H, and 180Q; (xiv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 127T, 129D, and 178R; (xv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 114Q, 137T, and 138E; (xvi), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 129D, 178R, and 180H; (xvii), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 129D and 180Q; (xiii), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 133Y and 180R; or In (xix), the amino acid substitutions in the variant CLK domain polypeptide comprise, or consist of, 129R and 180S; or the amino acid substitutions in the variant CLλ domain polypeptide comprise, or consist of, 129R; In each of the foregoing, the substitution positions are, according to EU numbering: (F) the amino acid substitution in the variant CH1 domain polypeptide is (i) 145Q, 147E, and 181E; or (ii) 128R and 147R, (iii) 168S, 185S, and 187D, or (iv) 147T and 185Q, wherein optionally said variant CH1 domain polypeptide preferentially pairs with a variant CLK or CLλ domain polypeptide; In (i), the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 129R, 178R, and 180Q; or (ii), wherein the amino acid substitutions in the variant CLK domain polypeptide comprise or consist of 124E, 133Q, and 178E, or wherein the amino acid substitutions in the variant CLλ domain polypeptide comprise or consist of 133Q and 178E; (iii), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of 135R; (iv), wherein the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of 135S and 178R; In each of the foregoing, the substitution positions are, according to EU numbering: (G) the variant polypeptide comprises an amino acid sequence selected from one of SEQ ID NOs: 31, 21, 11, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, and 201; (H) the variant polypeptide comprises an amino acid sequence selected from one of SEQ ID NOs: 31, 21, 11, and 41; (I) One or more of the following: (i) the amino acid substitution in the variant CH1 domain polypeptide consists of T187E, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitution in the variant CLK domain polypeptide does not consist of N137K and S114A; (ii) the amino acid substitutions in the variant CH1 domain polypeptide consist of L145Q and S183V, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of V133T and S176V; (iii) the amino acid substitutions in the variant CH1 domain polypeptide consist of K147A and K213E, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide are free of S131R and E123K; (iv) the amino acid substitutions in the variant CH1 domain polypeptide consist of S183A and K147A, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S176I and S131R; (v) the amino acid substitutions in the variant CH1 domain polypeptide consist of S183G and K147A, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S176I and S131R; (vi) the amino acid substitutions in the variant CH1 domain polypeptide consist of K147A, K213E, and S183A, and the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S131R, E123K, and S176I; (vii) the amino acid substitutions in the variant CH1 domain polypeptide consist of K147A, K213E, and S183G, and the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S131R, E123K, and S176I; (viii) the amino acid substitutions in the variant CH1 domain polypeptide consist of A141I, F170S, S181M, S183A, and V185A, and the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of F116A, L135V, S174A, S176F, and T178V; (ix) the amino acid substitution in the variant CH1 domain polypeptide consists of A141L, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitution in the variant CLK domain polypeptide does not consist of F118S, F118A, or F118V; (x) the amino acid substitution in the variant CH1 domain polypeptide consists of K147D, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitution in the variant CLK domain polypeptide does not consist of T129R; (xi) the amino acid substitutions in the variant CH1 domain polypeptide consist of S181E and S183V, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S176 and T178; (xii) the amino acid substitutions in the variant CH1 domain polypeptide consist of S183L and V185Y, the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of V133S; (xiii) the amino acid substitutions in the variant CH1 domain polypeptide consist of S183K and K214R, the variant CH1 domain polypeptide preferentially pairs with a variant CL λ domain polypeptide, and the amino acid substitutions in the variant CL λ domain polypeptide do not consist of S176E, Y178E, and T212A; or (xiv) the amino acid substitutions in the variant CH1 domain polypeptide consist of L128E, K147T, Q175E, S183W, and K214R, and the variant CH1 domain polypeptide preferentially pairs with a variant CLK domain polypeptide, and the amino acid substitutions in the variant CLK domain polypeptide do not consist of S131R, V133G, S176R, and T178A; and / or (J) the amino acid substitution in the variant CH1 domain polypeptide is (I) positions 185 and / or 187, (II) positions 145, 147, and / or 148; (III) position 147 or 148, (IV) 145th place, (V) positions 166 and / or 187, (VI) positions 145 and / or 147, or (VII) comprising or consisting of an amino acid substitution at position 124 and / or 147; Optionally, the variant CH1 domain polypeptide preferentially pairs with a variant CLK or CLλ domain polypeptide, and further optionally, (I), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 135; (II), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 124; (III), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 129; (IV), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 133; (V), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 137 and / or 138; (VI), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises or consists of an amino acid substitution at position 178 and / or 180; or (VII), wherein the amino acid substitution in the variant CLK or CLλ domain polypeptide comprises, or consists of, an amino acid substitution at position 127; In each of the foregoing, the substitution positions are according to EU numbering: A variant CH1 domain polypeptide, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide.

2. 1. A variant CLK or CLλ domain polypeptide, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide, comprising, or consisting of, an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138, optionally (A) the variant CLK or CLλ domain polypeptide, or a light chain polypeptide comprising the variant CLK or CLλ domain polypeptide, preferentially pairs with a variant CH1 domain polypeptide comprising at least one amino acid substitution, wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of an amino acid substitution at one or more of the following positions according to EU numbering: 124, 128, 139, 141, 145, 147, 148, 166, 168, 175, 181, 185, and 187; (B) the amino acid substitution of the variant CLK or CLλ is: (i) positions 129, 178, and 180; (ii) positions 124, 133, and 178, or positions 133 and 178; (iii) 135th place, (iv) positions 135 and 178, (v) positions 124 and 129, (vi) positions 114, 135, and 138, (vii) positions 137 and 138, or position 138, (viii) positions 127 and 129, (ix) 133rd place, (x) positions 124 and 133; (xi) positions 120, 178, and 180; (xii) positions 127, 129, and 178; (xiii) positions 114, 137, and 138; (xiv) positions 129 and 180, (xv) positions 133 and 180, and (xvi) 129th place, and optionally said variant CLK or CLλ domain polypeptide preferentially pairs with a variant CH1 domain polypeptide, and further optionally (i), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 145, 147, and 181, or 147 and 175; (ii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 128 and 147; (iii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 168 and 185, or at positions 168, 185, and 187; (iv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 147 and 185; (v), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises, or consists of, an amino acid substitution at position 148; (vi), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 139, 141, and 187; (vii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 166 and 187; (viii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 124 and 147, or 147 and 148; (ix), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises, or consists of, amino acid substitutions at positions 145, or 145 and 181; (x), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises, or consists of, an amino acid substitution at position 145; (xi), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 145 and 181; (xii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 124, 145, and 147; (xiii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 166 and 187; (xiv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 147 and 185, or at positions 147, 175, and 181; (xv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 145 and 147; or (xvi), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, amino acid substitutions at positions 147 and 185; In each of the foregoing, the substitution positions are, according to EU numbering: (C) the amino acid substitutions in the variant CLK or CLλ domain polypeptide comprise or consist of: 129R, 178R, 180Q, 124E, 133Q, 178E, 114D, 114Q, 120S, 124S, 127D, 127R, 127T, 129D, 129E, 133Y, 135R, 135S, 137S, 137T, 138E, 138R, 178H, and 180H, 180R, and / or 180S, according to EU numbering; (D) the amino acid substitution in the variant CLK domain polypeptide is (i) 129R, 178R, and 180Q, (ii) 124E, 133Q, and 178E, or 133Q and 178E, (iii) 135R, (iv) 135S and 178R, (v) 124S and 129E, (vi) 114D, 135S, and 138R, (vii) 137S and 138E, or 138E, (viii) 135S, (ix) 127D and 129E, (x) 127R and 129R, (xi) 133Y, (xii) 133Y, (xiii) 124E and 133Y, or 133Y, (xiv) 120S, 178H, and 180Q, (xv) 127T, 129D, and 178R, (xvi) 114Q, 137T, and 138E, (xvii) 129D, 178R, and 180H, (xviii) 129D and 180Q, (xix) 133Y and 180R, or (xx) 129R and 180S, or 129R, and optionally wherein said variant CLK or CLλ domain polypeptide preferentially pairs with a variant CH1 domain polypeptide, and further optionally wherein (i), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 145Q, 147E, and 181E; (ii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 128R and 147R; (iii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 168S, 185S, and 187D; (iv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 147T and 185Q; (v), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of 148R; (vi), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 139R, 141Q, and 187Q; (vii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 166K and 187K; (viii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 168R and 185E; (ix), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 124R and 147R; (x), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, 147H and 148E; (xi), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of 145S; (xii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 145S and 181Q; (xiii), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of 145S; (xiv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 145Q and 181E; (xv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 124R, 145S, and 147Q; (xvi), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 166K and 187K; (xvii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 147R and 175D; (xviii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 147R, 175E, and 181Q; (xix), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 145S and 147N; or In (xx), the amino acid substitutions in the variant CH1 domain polypeptide comprise, or consist of, 147N and 185Y; In each of the foregoing, the substitution positions are, according to EU numbering: (E) the amino acid substitution in the variant CLK or CLλ domain polypeptide is (i) 129R, 178R, and 180Q, or (ii) 124E, 133Q, and 178E, or 133Q and 178E, (iii) 135R, (iv) 135S and 178R, wherein optionally said variant CLK or CLλ domain polypeptide preferentially pairs with a variant CH1 domain polypeptide; (i), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 145Q, 147E, and 181E; or (ii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 128R and 147R; (iii), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 168S, 185S, and 187D; (iv), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of 147T and 185Q; In each of the foregoing, the substitution positions are, according to EU numbering: (F) comprising an amino acid sequence selected from one of SEQ ID NOs: 32, 22, 12, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, or 202, or any one of SEQ ID NOs: 59, 99, 39, 199, 89, 49, 29, 19, 69, 79, 109, 119, 129, 139, 149, 159, 169, 179, 189, or 209; (G) comprising an amino acid sequence selected from one of SEQ ID NOs: 32, 12, 22, 42, or any one of SEQ ID NOs: 59, 99, 39, 199, 89, 49, or 29; (H) (i) the amino acid substitutions in the variant CLK domain polypeptide consist of N137K and S114A, and when preferentially paired with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of T187E; (ii) the amino acid substitutions in the variant CLK domain polypeptide consist of V133T, S176V, and when preferentially paired with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of L145Q and S183V; (iii) the amino acid substitution in the variant CLK domain polypeptide consists of V133E, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitution in the variant CH1 domain polypeptide does not consist of S183K; (iv) the amino acid substitutions in the variant CLK domain polypeptide consist of F116A, L135V, S174A, S176F, and T178V, and when preferentially paired with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of A141I, F170S, S181M, S183A, and V185A; (v) the amino acid substitution in the variant CLK domain polypeptide consists of T129R, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitution in the variant CH1 domain polypeptide does not consist of K147D; (vi) the amino acid substitutions in the variant CLK domain polypeptide consist of S176 and T178, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of S181E and S183V; or (vii) the amino acid substitution in the variant CLK domain polypeptide consists of V133S, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitution in the variant CH1 domain polypeptide does not consist of S183L and V185Y; or (viii) the amino acid substitutions in the variant CL λ domain polypeptide consist of S176E, Y178E, and T212A, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of S183K and K214R; or (ix) the amino acid substitutions in the variant CLK domain polypeptide consist of S131R, V133G, S176R, and T178A, and when preferentially pairing with a variant CH1 domain polypeptide, the amino acid substitutions in the variant CH1 domain polypeptide do not consist of L128E, K147T, Q175E, S183W, and K214R; In each of the foregoing, the substitution positions are according to EU numbering, and / or (I) the amino acid substitution in the variant CLK domain polypeptide is (I) 135th place, (II) 124th place, (III) 129th place, (IV) 133rd place, (V) positions 137 and / or 138, (VI) positions 178 and / or 180, or (VII) comprising or consisting of an amino acid substitution at position 127; Optionally, the variant CLK or CLλ domain polypeptide preferentially pairs with a variant CH1 domain polypeptide, and further optionally, (I), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of amino acid positions 185 and / or 187; (II), wherein the amino acid substitutions in the variant CH1 domain polypeptide comprise or consist of amino acid substitutions at positions 145, 147, and / or 148; (III), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of an amino acid substitution at position 147 or 148; (IV), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises, or consists of, an amino acid substitution at position 145; (V), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of an amino acid substitution at position 166 and / or 187; (VI), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of an amino acid substitution at position 145 and / or 147; or (VII), wherein the amino acid substitution in the variant CH1 domain polypeptide comprises or consists of an amino acid substitution at position 124 and / or 147; In each of the foregoing, the substitution positions are according to EU numbering in a variant CLK or CLλ domain polypeptide, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide.

3. 2. An immunoglobulin polypeptide comprising at least one variant immunoglobulin heavy chain constant region 1 ("CH1") domain polypeptide of claim 1, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide, optionally comprising: (A) one or more of the following: (i) an antigen-binding domain, (ii) a second CH1 domain or a variant CH1 domain; (iii) an immunoglobulin heavy chain constant region 2 ("CH2") domain or a variant CH2 domain; (iv) an immunoglobulin heavy chain constant region 3 ("CH3") domain or a variant CH3 domain, and / or (v) a light chain constant region (CL) domain or a variant CL domain, optionally a variant CLK or CLλ domain; and optionally In (i), the antigen-binding domain is an immunoglobulin heavy chain variable region ("VH") domain, an immunoglobulin light chain variable region ("VL") domain, a single chain fragment variable ("scFv"), an antigen-binding fragment (Fab), F(ab'), F(ab') ２ , F(ab')2, or a combination thereof; (ii), wherein the CH1 domain comprises a wild-type CH1 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH1 amino acid sequence; (iii), wherein the CH2 domain comprises a wild-type CH2 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH2 amino acid sequence; (iv), wherein the CH3 domain comprises a wild-type CH3 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH3 amino acid sequence; and / or (v), wherein the CL domain comprises a wild-type CL amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CL amino acid sequence; or (B) (I) comprises a VH domain and is bound to or paired with another polypeptide comprising a VL domain, the VH domain and the VL domain forming an antigen-binding site; or (II) An immunoglobulin polypeptide comprising a VL domain and bound to or paired with another polypeptide comprising a VH domain, wherein the VL domain and the VH domain form an antigen-binding site.

4. 3. An immunoglobulin polypeptide comprising at least one variant CLK or CLλ domain polypeptide according to claim 2, or a light chain polypeptide comprising a variant CLK or CLλ domain polypeptide, respectively, optionally comprising (A) one or more of the following: (i) an antigen-binding domain, (ii) a CH1 domain or a variant CH1 domain, (iii) a CH2 domain or a variant CH2 domain, (iv) a CH3 domain or a variant CH3 domain, and / or (v) a second CL domain or a variant CL domain; and optionally In (i), the antigen-binding domain is a VH domain, a VL domain, a scFv, a Fab, a F(ab'), a F(ab') ２ , F(ab')2, or a combination thereof; (ii), wherein the CH1 domain comprises a wild-type CH1 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH1 amino acid sequence; (iii), wherein the CH2 domain comprises a wild-type CH2 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH2 amino acid sequence; (iv), wherein the CH3 domain comprises a wild-type CH3 amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CH3 amino acid sequence; and / or (v), wherein the CL domain comprises a wild-type CL amino acid sequence or comprises one or more amino acid substitutions relative to the wild-type CL amino acid sequence; or (B) (I) comprises a VH domain and is bound to or paired with another polypeptide comprising a VL domain, the VH domain and the VL domain forming an antigen-binding site; or (II) It comprises a VL domain and is bound to or paired with another polypeptide comprising a VH domain, wherein the VL domain and the VH domain form an antigen-binding site; Immunoglobulin polypeptides.

5. A molecule comprising at least a first polypeptide and a second polypeptide, (A) the first polypeptide comprises a variant CH1 domain polypeptide of claim 1, or a heavy chain polypeptide comprising a variant CH1 domain polypeptide; and (B) the second polypeptide comprises a variant CLK domain polypeptide or CLλ domain polypeptide of claim 2, or a light chain polypeptide comprising a variant CLK domain polypeptide or CLλ domain polypeptide; further, the first polypeptide and the second polypeptide are optionally linked to each other or paired via a disulfide bond; Optionally, （１） (A) the first polypeptide is a polypeptide according to claim 3; and / or (B) the second polypeptide is a polypeptide according to claim 4; and / or （２） (A) the first polypeptide comprises an antigen-binding domain, and / or (B) the second polypeptide comprises an antigen-binding domain; Optionally, (I) the antigen-binding domain of the first polypeptide and the antigen-binding domain of the second polypeptide comprise a VH and a VL, or a VL and a VH, respectively, and optionally form an antigen-binding site specific for a first epitope; or (II) the antigen-binding domain of the first polypeptide comprises an scFv or nanobody specific for a first epitope, and / or the antigen-binding domain of the second polypeptide comprises an scFv or nanobody specific for a second epitope, and further optionally, the first epitope is the same as or different from the second epitope; （３） Furthermore, (C) a third polypeptide comprising at least one variant CH1 domain polypeptide of claim 1, or a heavy chain polypeptide comprising the variant CH1 domain polypeptide of claim 1; and (D) a fourth polypeptide comprising at least one variant CLK or CLλ domain polypeptide of claim 2, or a light chain polypeptide comprising a variant CLK or CLλ domain polypeptide of claim 2; the third polypeptide and the fourth polypeptide are optionally linked to each other or paired via a disulfide bond; Further optionally, (C) the variant CH1 domain polypeptide of the third polypeptide is the same as or different from the variant CH1 domain polypeptide of the first polypeptide; and / or (D) the variant CLK or CLλ domain polypeptide of the fourth polypeptide is the same as or different from the variant CLK or CLλ domain polypeptide of the second polypeptide; (3-i) (C) the third polypeptide is a polypeptide according to claim 3; and / or (D) the fourth polypeptide is a polypeptide according to claim 4; and / or (3-ii) (C) the third polypeptide comprises an antigen-binding domain; and / or (D) the fourth polypeptide comprises an antigen-binding domain; Optionally, (I) the antigen-binding domain of the third polypeptide and the antigen-binding domain of the fourth polypeptide comprise a VH and a VL, or a VL and a VH, respectively, and optionally form an antigen-binding site specific for a third epitope, and further optionally, the third epitope is the same as or different from the first epitope and / or the second epitope; or (II) the antigen-binding domain of the third polypeptide comprises an scFv or nanobody specific for a third epitope, and / or the antigen-binding domain of the fourth polypeptide comprises an scFv or nanobody specific for a fourth epitope, optionally the third epitope is the same as or different from the fourth epitope, and further optionally the third and / or fourth epitopes are the same as or different from the first and / or second epitopes, respectively; (4) One or more of the following: (i) it is a multispecific antibody or antigen-binding antibody fragment, optionally a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific antibody or antigen-binding antibody fragment, further optionally comprising a structure depicted in any one of Figures 2-7, further optionally an IgG, still further optionally an IgG1, IgG2, IgG3, or IgG4; (ii) the amino acid substitutions in the variant CLK Domain of the first polypeptide comprise or consist of 145Q, 147E, and 181E; the amino acid substitutions in the variant CLK Domain of the second polypeptide comprise or consist of 129R, 178R, and 180Q; the amino acid substitutions in the variant CLK Domain of the third polypeptide comprise or consist of 128R and 147R; and the amino acid substitutions in the variant CLK Domain of the fourth polypeptide comprise or consist of 124E, 133Q, and 178E; (iii) the amino acid substitutions in the variant CLK Domain of the first polypeptide comprise or consist of 128R and 147R; the amino acid substitutions in the variant CLK Domain of the second polypeptide comprise or consist of 124E, 133Q, and 178E; the amino acid substitutions in the variant CLK Domain of the third polypeptide comprise or consist of 145Q, 147E, and 181E; and the amino acid substitutions in the variant CLK Domain of the fourth polypeptide comprise or consist of 129R, 178R, and 180Q; (iv) the amino acid substitution in the variant CL λ domain of the first polypeptide comprises or consists of 148R, the amino acid substitution in the variant CL λ domain of the second polypeptide comprises or consists of 124S and 129E, the amino acid substitution in the variant CL λ domain of the third polypeptide comprises or consists of 145S and 147N, and the amino acid substitution in the variant CL λ domain of the fourth polypeptide comprises or consists of 133Y and 180R; (v) the amino acid substitutions in the variant CL λ domain of the first polypeptide comprise or consist of 145S and 147N, the amino acid substitutions in the variant CL λ domain of the second polypeptide comprise or consist of 133Y and 180R, the amino acid substitutions in the variant CL λ domain of the third polypeptide comprise or consist of 148R, and the amino acid substitutions in the variant CL λ domain of the fourth polypeptide comprise or consist of 124S and 129E; (vi) the amino acid substitutions in the variant CL domain of the first polypeptide comprise or consist of 124R and 147R, the amino acid substitutions in the variant CL domain of the second polypeptide comprise or consist of 127D and 129E, the amino acid substitutions in the variant CL domain of the third polypeptide comprise or consist of 145Q, 147E, and 181E, and the amino acid substitutions in the variant CL domain of the fourth polypeptide comprise or consist of 129R, 178R, and 180Q; (vii) the amino acid substitutions in the variant CL domain of the first polypeptide comprise or consist of 145Q, 147E, and 181E; the amino acid substitutions in the variant CL domain of the second polypeptide comprise or consist of 129R, 178R, and 180Q; the amino acid substitutions in the variant CL domain of the third polypeptide comprise or consist of 124R and 147R; and the amino acid substitutions in the variant CL domain of the fourth polypeptide comprise or consist of 127D and 129E; (viii) the variant CH1 domain of the first polypeptide, the variant CLK domain or CLλ domain of the second polypeptide, the variant CH1 domain of the third polypeptide, and the variant CLKCLλ domain of the fourth polypeptide are selected from the group consisting of: (A) SEQ ID NOs: 31, 32, 21, and 22, respectively; (B) SEQ ID NOs: 21, 22, 31, and 32, respectively; (C) SEQ ID NOs: 51, 59, 191, and 199, respectively; (D) SEQ ID NOs: 191, 199, 51, and 59, respectively; (E) SEQ ID NOs: 91, 99, 31, and 39, respectively; or (F) comprising an amino acid sequence selected from SEQ ID NOs: 31, 39, 91, and 99, respectively; In each of the foregoing, the substitution positions are according to EU numbering:

6. (i) a variant CH1 domain polypeptide of claim 1, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide; (ii) a variant CLK or CLλ domain polypeptide according to claim 2, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide; (iii) an immunoglobulin polypeptide according to claim 3 or 4, and / or (iv) a molecule according to claim 5, or one or more vectors comprising said one or more polynucleotides encoding said one or more polynucleotides.

7. A cell comprising one or more of the following: (i) a variant CH1 domain polypeptide of claim 1, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide; (ii) a variant CLK or CLλ domain polypeptide according to claim 2, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide; (iii) an immunoglobulin polypeptide according to claim 3 or 4, (iv) a molecule according to claim 5, and / or (v) one or more polynucleotides or one or more vectors according to claim 6, and optionally the cell is a mammalian or yeast cell.

8. 1. A composition comprising: (I) (i) a variant CH1 domain polypeptide of claim 1, or a heavy chain polypeptide comprising said variant CH1 domain polypeptide; (ii) a variant CLK or CLλ domain polypeptide according to claim 2, or a light chain polypeptide comprising said variant CLK or CLλ domain polypeptide; (iii) an immunoglobulin polypeptide according to claim 3 or 4, (iv) a molecule according to claim 5, (v) one or more polynucleotides or one or more vectors according to claim 6, and / or (vii) a cell according to claim 7, and (II) A composition comprising a pharma- ceutically or diagnostically acceptable carrier.

9. 1. A method for generating a CH1 domain encoding polynucleotide library, or a CH1 domain encoding polynucleotide library generated using said method, comprising incorporating in silico or in vitro mutations or randomizing nucleic acid at one or more predefined nucleotide positions in a plurality of CH1 domain encoding polynucleotides, wherein at least one of said one or more predefined nucleotide positions is within a codon encoding said amino acid at at least one of the predefined CH1 domain amino acid positions, and wherein said amino acid position is (i) is located within or adjacent to the interface of the CH1 domain and the CL domain; (ii) predicted to affect CH1-CL domain interactions, optionally hydrogen bond mediated interactions, optionally said prediction being performed in silico or in vitro, further optionally said prediction being performed in silico using Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet), and / or (iii) selected from positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to the EU numbering; Optionally, the one or more mutations are generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups; Further optionally, the method or library is for identifying one or more variant CH1 domain polypeptides that preferentially pair with a given or variant CL domain polypeptide rather than with a wild-type or another given variant CL domain polypeptide.

10. 1. A method for generating a CH1 domain polypeptide library, or a CH1 domain polypeptide library generated using said method, comprising: (I) obtaining, in silico or in vitro, a plurality of CH1 domain polypeptides corresponding to a plurality of CH1 domain-encoding polynucleotides contained in the CH1 domain-encoding polynucleotide library according to claim 38; or (II) incorporating substitutions in silico or in vitro at one or more predetermined CH1 domain amino acid positions in a plurality of CH1 domain polypeptides, wherein one or more of the one or more predetermined CH1 domain amino acid positions are (i) is located within or adjacent to the interface of the CH1 domain and the CL domain; (ii) predicted to affect CH1-CL domain interactions, optionally hydrogen bond mediated interactions, optionally said prediction being performed in silico or in vitro, further optionally said prediction being performed in silico using Rosetta MC HBNet; and / or (iii) selected from positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering; Optionally, the library is for identifying one or more variant CH1 domain polypeptides that preferentially pair with a given or variant CL domain polypeptide rather than with a wild-type or another given variant CL domain polypeptide; Further optionally, the CH1 domain polypeptides of the library comprise a predetermined number of CH1 substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and / or 1, 2, 3, 4, or 5.

11. 5. A method for generating a CLK and / or CLλ domain encoding polynucleotide library, or a CLK and / or CLλ domain encoding polynucleotide library generated using said method, comprising incorporating in silico or in vitro mutations or randomizing nucleic acids at one or more predefined nucleotide positions in a plurality of CLK and / or CLλ domain encoding polynucleotides, wherein at least one of said one or more predefined nucleotide positions is within a codon encoding said amino acid at at least one of the predefined CLK and / or CLλ domain amino acid positions, and wherein said amino acid position is (i) is located within or adjacent to the interface of the CH1 domain and the CLK and / or CLλ domain; (ii) is predicted to affect CH1-CL domain interactions, optionally hydrogen bond mediated interactions; optionally, said prediction is performed in silico or in vitro; further optionally, said prediction is performed in silico using a Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet); and / or (iii) selected from positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering; Optionally, the one or more mutations are generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups; Further optionally, the library is for identifying one or more variant CLK domain polypeptides that preferentially pair with a given or variant CH1 domain polypeptide rather than a wild-type CH1 or another given variant CH1 domain polypeptide; Further optionally, the variant CLK and / or CL λ domain library comprises only CL domains of a κ isotype, only CL domains of a λ isotype, or at least one CL domain of a κ isotype and at least one CL domain of a λ isotype.

12. 1. A method for generating a CLK and / or CLλ domain polypeptide library, or a CLK and / or CLλ domain polypeptide library generated using said method, comprising: (I) Obtaining in silico or in vitro a plurality of CLK and / or CL λ domain polypeptides corresponding to a plurality of CLK and / or CL λ domain encoding polynucleotides contained in the CLK and / or CL λ domain encoding polynucleotide library according to claim 11; or (II) incorporating substitutions in silico or in vitro at one or more predetermined CLK and / or CL λ domain amino acid positions in a plurality of CLK and / or CL λ domain polypeptides, wherein one or more of the one or more predetermined CLK and / or CL λ domain amino acid positions are (i) is located within or adjacent to the interface of the CH1 domain and the CL domain; (ii) predicted to affect CH1-CL domain interactions, optionally hydrogen bond mediated interactions, optionally said prediction being performed in silico or in vitro, further optionally said prediction being performed in silico using Rosetta MC HBNet; and / or (iii) selected from positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering; Optionally, the library is for identifying one or more variant CLK and / or CLλ domain polypeptides that preferentially pair with a given or variant CH1 domain polypeptide rather than a wild-type or another given variant CH1 domain polypeptide; Further optionally, the CLK and / or CLλ domain polypeptides of the library comprise a predetermined number of CLK and / or CLλ substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and / or 1, 2, 3, 4, or 5.

13. A method for generating a CH1-CL domain-encoding polynucleotide set library, or a CH1-CL domain-encoding polynucleotide set library generated using said method, comprising in silico or in vitro mutagenesis or randomization of nucleic acid at one or more predefined nucleotide positions in a plurality of CH1-CL domain-encoding polynucleotide sets, wherein at least one of said one or more predefined nucleotide positions is within a codon encoding said amino acid at at least one of predefined CH1 and / or CL domain amino acid positions, and wherein said amino acid position is (i) is located within or adjacent to the interface of the CH1 domain and the CL domain; (ii) predicted to affect CH1-CL interdomain interactions, optionally hydrogen bond mediated interactions, optionally wherein said prediction is performed in silico or in vitro, further optionally wherein said prediction is performed in silico using Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet); (iii) selected from CH1 domain amino acid positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering; and / or (iv) selected from CL domain amino acid positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering; Optionally, the one or more mutations are generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups; Further optionally, the library is for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CL domain rather than with a wild-type or another given variant CL domain polypeptide, and / or for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CL domain rather than with a wild-type or another given variant CL domain polypeptide, Further optionally, the CL domain encoded by the CH1-CL domain encoding polynucleotide set library comprises a CLK domain and / or a CLλ domain.

14. A method for generating a CH1-CL domain polypeptide set library, or a CH1-CL domain polypeptide set library generated using said method, comprising: （１） (I) Obtaining in silico or in vitro a plurality of sets of CH1-CL domain polypeptides corresponding to a plurality of sets of CH1-CL domain-encoding polynucleotides contained in the CH1-CL domain-encoding polynucleotide set library according to claim 13; or (II) incorporating substitutions in silico or in vitro at one or more predetermined CH1 and / or CL domain amino acid positions in a set of a plurality of CH1-CL domain polypeptides, wherein one or more of the one or more predetermined CH1 and / or CL domain amino acid positions are (i) is located within or adjacent to the interface of the CH1 domain and the CL domain; (ii) predicted to affect CH1-CL interdomain interactions, optionally hydrogen bond mediated interactions, optionally wherein said prediction is performed in silico or in vitro, further optionally wherein said prediction is performed in silico using Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet); (iii) selected from CH1 domain amino acid positions 145, 147, 181, 128, 124, 139, 141, 148, 166, 168, 175, 185, and 187 according to EU numbering; and / or (iv) selected from CL domain amino acid positions 129, 178, 180, 124, 133, 114, 120, 127, 135, 137, and 138 according to EU numbering; Optionally, the library is for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CL domain rather than a wild-type or another given variant CL domain polypeptide, and / or for identifying one or more variant CL domain polypeptides that preferentially pair with a given or variant CL domain rather than a wild-type or another given variant CL domain polypeptide, or （２） (i) providing a set of multiple CH1-CL domain polypeptides; (ii) calculating a CH1-CL domain interaction strength for one or more of said plurality of CH1-CL domain polypeptide sets, optionally comprising: (a) in silico or in vitro, optionally in silico using Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet), and / or (b) calculating based on the strength of the hydrogen bonds and / or the bond energy between the CH1-CL domains; (iii) (a) a reference set of CH1-CL domain polypeptides, optionally a set of WT CH1-CL domain polypeptides or a set of known CH1-CL domain polypeptides; or (b) optionally selecting one or more CH1-CL domain polypeptide sets that are calculated to have stronger CH1-CL inter-domain interactions compared to a reference CH1-CL inter-domain interaction strength, which is a CH1-CL inter-domain interaction strength of a WT CH1-CL domain polypeptide set or a known CH1-CL polypeptide domain set; Optionally, in (1) and / or (2), The CL domain polypeptides in the CH1-CL domain polypeptide set library include a CLK domain and / or a CLλ domain; and / or The CH1-CL domain polypeptide set library is a CH1-CLK domain polypeptide set library, a CH1-CL λ domain polypeptide set library, or a CH1-CL domain polypeptide set library, wherein the CL domain polypeptide of the library is a CH1-CL domain polypeptide set library comprising one or more CLK domain polypeptides and one or more CL λ domain polypeptides; Further optionally, in (1) and / or (2), (A) the CH1 domain polypeptides of the CH1-CL domain polypeptide set library comprise a predetermined number of CH1 substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and / or 1, 2, 3, 4, or 5; and / or (B) the CL domain polypeptides of the CH1-CL domain polypeptide set library comprise a predetermined number of CL substitution positions, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and / or 1, 2, 3, 4, or 5.

15. 1. A method of identifying one or more sets of variant CH1 domain polypeptides and variant CLK or CLλ domain polypeptides, wherein the variant CH1 domain polypeptides and the variant CLK or CLλ domain polypeptides preferentially pair with each other, the method comprising: (a) providing a plurality of sets of (a-1) a first polypeptide comprising a wild-type or variant CH1 domain polypeptide and (a-2) a second polypeptide comprising a wild-type or variant CLK or CLλ domain polypeptide, optionally wherein the plurality of sets of (a-1) and (a-2) are provided in silico or in vitro; (b) quantifying a binding preference between said variant CH1 domain polypeptide and said variant CLK or CLλ domain polypeptide, optionally wherein said binding preference is based on the strength of CH1-CL inter-domain hydrogen bonds and / or CH1-CL inter-domain binding energy, further optionally wherein said quantifying is performed in silico or in vitro; (c) selecting one or more sets of variant CH1 domain polypeptides and variant CLK or CLλ domain polypeptides that provide preferential CH1-CL pairing, optionally an equivalent or higher preferential pairing relative to a reference set of CH1-CL domain polypeptides; (i) the reference CH1-CL domain polypeptide set comprises a wild-type CH1 domain polypeptide, a wild-type CLK or CLλ domain polypeptide, a variant CH1 domain polypeptide according to claim 1, and / or a variant CLK or CLλ domain polypeptide according to claim 2; or (ii) the reference CH1-CL domain polypeptide set is a CH1-CL domain polypeptide set shown in Table 1; (I) (a-1) the first polypeptide comprises or is bound to a first label, and / or (a-2) the second polypeptide comprises or is bound to a second label; Optionally, the quantifying step (b) comprises detecting the first label and / or the second label; (II) (II-1) In step (a), the providing is carried out in silico; and In step (b), the quantifying optionally includes: ΔΔG, ΔΔG ｃｏｇｎａｔｅ ｔｏｔａｌ ｓｃｏｒｅ , ΔΔG ｃｏｇｎａｔｅ ｈｂｏｎｄ＿ａｌｌ , RBPP, RBPP ｔｏｔａｌ ｓｃｏｒｅ , R.B.P.P. ｈｂｏｎｄ＿ａｌｌ , and / or RBPP ｂｏｎｄ ｅｌｅｃ ｂａｃｋｒｕｂ １８ｋ and / or said quantifying is performed in silico using Rosetta Multi-Carlo (MC) Hydrogen Bond Network (HBNet), or (II-2) In step (a), the providing is carried out in vitro, optionally recombinantly; and In step (b), said quantifying comprises measuring the amount of CH1-CL pairs via liquid chromatography-mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, and / or flow cytometry; (III) The method further comprises the step of selecting one or more sets of CH1-CL domain polypeptides based on one or more properties of the antibodies comprising the first and second sets of polypeptides selected in step (c), wherein the one or more properties are selected from the following: (i) (i-1) optionally, production yield assessed in one or more cell types, optionally mammalian cells such as CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) optionally suitability for one or more antibody purification methods including Protein A affinity purification; (ii) the extent of aggregation, optionally quantified using chromatography, optionally size exclusion chromatography (SEC) or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full size antibodies; (iii) the rate of correct pairing, optionally assessed using LC-MS, optionally correct pairing between CH1 domains and / or between CH1 domains and CL domains; (iv) optionally the melting temperature (Tm) and / or aggregation temperature (Tagg) measured using differential scanning fluorometry (DSF) and / or differential scanning calorimetry (DSC) and / or using an instrument, optionally an Uncle®, optionally Tagg266; (v) isoelectric point (“pI”); (vi) optionally, the level of interaction with a multispecific reagent ("PSR"), as measured by the method described in WO2014 / 179363; (vii) the hydrophobic interactions of the antibody, optionally measured using hydrophobic interaction chromatography ("HIC"), optionally as described in Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561; (viii) optionally, self-interaction as measured by (viii-1) affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92, or (viii-2) dynamic light scattering (DLS); (ix) stability against high or low pH stress; (x) solubility; (xi) production costs and / or times; (xii) other stability parameters; (xiii) shelf life; (xiv) in vivo half-life, and / or (xv) immunogenicity.

16. One or more of the following characteristics: (i) at least one of the first polypeptides in step (a) is derived from the CH1 domain polypeptide library of claim 10 or expressed from the CH1 domain encoding polynucleotide library of claim 9; (i) at least one of the second polypeptides in step (a) is derived from a CLK and / or CLλ domain polypeptide library according to claim 12 or expressed from a CLK and / or CLλ domain encoding polynucleotide library according to claim 11; (iii) at least one set of the first polypeptide and the second polypeptide in step (a) is derived from the CH1-CL domain polypeptide set library according to claim 14 or is expressed from the CH1-CL domain-encoding polynucleotide set library according to claim 13; and / or (iv) at least one of the sets of first and second polypeptides in step (a) is derived from a CH1-CL domain polypeptide set library, in which the CH1 and / or CL domain polypeptides comprise one or more random amino acid modifications, or is expressed from a CH1-CL domain polypeptide set library, in which the CH1 and / or CL domain polypeptides comprise one or more random mutations; Optionally, (i) the one or more predetermined CH1 domain amino acid positions comprise or consist of 145, 147, and / or 181, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 129, 178, and / or 180; (ii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 128 and / or 147, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 124, 133, and / or 178; (iii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 168, 185, and / or 187, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 135; (iv) the one or more predetermined CH1 domain amino acid positions comprise or consist of 147 and / or 185, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 135 and / or 178; (v) the one or more predetermined CH1 domain amino acid positions comprise or consist of 148, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 124 and / or 129; (vi) the one or more predetermined CH1 domain amino acid positions comprise or consist of 139, 141, and / or 187, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 114, 135, and / or 138; (vii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 166 and / or 187, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 137 and / or 138; (viii) the one or more predetermined CH1 domain amino acid positions comprise or consist of positions 168 and / or 185, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of position 135; (ix) the one or more predetermined CH1 domain amino acid positions comprise or consist of 124 and / or 147, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 127 and / or 129; (x) the one or more predetermined CH1 domain amino acid positions comprise or consist of 147 and / or 148, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 127 and / or 129; (xi) said one or more predetermined CH1 domain amino acid positions comprise or consist of position 145, and / or said one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of position 133; (xii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 145 and / or 181, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 133; (xiii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 145, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 124 and / or 133; (xiv) the one or more predetermined CH1 domain amino acid positions comprise or consist of 145 and / or 181, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 120, 178, and / or 180; (xv) the one or more predetermined CH1 domain amino acid positions include or consist of 124, 145, and / or 147, and / or the one or more predetermined CLK or CLλ domain amino acid positions include or consist of 127, 129, and / or 178; (xvi) the one or more predetermined CH1 domain amino acid positions comprise or consist of 166 and / or 187, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 114, 137, and / or 138; (xvii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 147 and / or 175, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 129, 178, and / or 180; (xviii) the one or more predetermined CH1 domain amino acid positions comprise or consist of 147, 175, and / or 181, and / or the one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 129 and / or 180; (xix) said one or more predetermined CH1 domain amino acid positions comprise or consist of 145 and / or 147, and / or said one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 133 and / or 180; or 16. The method of claim 15, wherein (xx) said one or more predetermined CH1 domain amino acid positions comprise or consist of 147 and / or 185, and / or said one or more predetermined CLK or CLλ domain amino acid positions comprise or consist of 129 and / or 180, each of the foregoing wherein said substitution positions are according to EU numbering.

17. 1. A method of screening a combination of (i) a first set of a first variant CH1 domain polypeptide and a first variant CL domain polypeptide (the "first CH1-CL domain polypeptide set"), and (ii) a second set of a second variant CH1 domain polypeptide and a second variant CL domain polypeptide (the "second CH1-CL domain polypeptide set"), which combination is suitable for a multispecific antibody or antigen-binding antibody fragment of interest, having an antibody or antibody fragment structure of interest, optionally having a variable region sequence of interest, said method comprising: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments comprising different combinations of (i) a first candidate set of CH1-CL domain polypeptides and (ii) a second candidate set of CH1-CL domain polypeptides; (b) selecting one or more combinations of (i) a first set of CH1-CL domain polypeptides and (ii) a second set of CH1-CL domain polypeptides based on one or more properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a), optionally wherein at least one of the one or more properties is selected from the following: (i) (i-1) optionally, production yield assessed in one or more cell types, optionally mammalian cells such as CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) optionally suitability for one or more antibody purification methods including Protein A affinity purification; (ii) the extent of aggregation, optionally quantified using chromatography, optionally size exclusion chromatography (SEC) or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full size antibodies; (iii) the rate of correct pairing, optionally assessed using LC-MS, optionally correct pairing between CH1 domains and / or between CH1 domains and CL domains; (iv) optionally the melting temperature (Tm) and / or aggregation temperature (Tagg) measured using differential scanning fluorometry (DSF) and / or differential scanning calorimetry (DSC) and / or using an instrument, optionally an Uncle®, optionally Tagg266; (v) isoelectric point (pI), (vi) optionally, the level of interaction with a multispecific reagent ("PSR"), as measured by the method described in WO2014 / 179363; (vii) the hydrophobic interactions of the antibody, optionally measured using hydrophobic interaction chromatography ("HIC"), optionally as described in Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561; (viii) optionally, self-interaction as measured by (viii-1) affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92, or (viii-2) dynamic light scattering (DLS); (ix) stability against high or low pH stress; (x) solubility; (xi) production costs and / or times; (xii) other stability parameters; (xiii) shelf life; (xiv) in vivo half-life, and / or (xv) is selected from: Optionally, the plurality of multispecific antibodies and / or antigen-binding antibody fragments comprises: (I) a first polypeptide comprising a first variant CH1 domain polypeptide and a first antigen-binding domain polypeptide; (II) a second polypeptide comprising a second variant CH1 domain polypeptide and a second antigen-binding domain polypeptide; and (III) a third polypeptide comprising a first variant CL domain polypeptide and a third antigen-binding domain polypeptide; and (IV) a fourth polypeptide comprising a second variant CL domain polypeptide and a fourth antigen-binding domain polypeptide; Optionally, the first and third polypeptides preferentially pair with each other, and the second and fourth polypeptides preferentially pair with each other; Optionally, the plurality of multispecific antibodies and / or antigen-binding antibody fragments comprises a structure depicted in any of Figures 2-7, further optionally, an IgG, still further optionally an IgG1, IgG2, IgG3, or IgG4; Optionally, (i) the first variant CH1 domain polypeptide is the variant CH1 domain polypeptide of claim 1; (ii) the second variant CH1 domain polypeptide is the variant CH1 domain polypeptide of claim 1; (iii) the first CLK or CLλ domain polypeptide is a variant CLK or CLλ domain polypeptide according to claim 2; and / or (iv) the second CLK or CLλ domain polypeptide is a variant CLK or CLλ domain polypeptide according to claim 2; Optionally, (A) the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope of interest, and the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope of interest, and optionally the first epitope of interest and the second epitope of interest are distinct from each other, or (B) the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope of interest, the second antigen-binding domain forms a second antigen-binding site specific for a second epitope of interest, and the fourth antigen-binding domain forms a third antigen-binding site specific for a third epitope of interest, optionally wherein the first epitope of interest is different from the second and / or third epitope of interest, (C) the first antigen-binding domain forms a first antigen-binding site specific for a first epitope of interest, the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope of interest, and the third antigen-binding domain forms a third antigen-binding site specific for a third epitope of interest, optionally wherein the second epitope of interest is different from the first and / or third epitope of interest; or (D) The method, wherein the first antigen binding domain forms a first antigen binding site specific for a first epitope of interest, the second antigen binding domain forms a second antigen binding site specific for a second epitope of interest, the third antigen binding domain forms a third antigen binding site specific for a third epitope of interest, and the fourth antigen binding domain forms a fourth antigen binding site specific for a fourth epitope of interest, optionally wherein the first and / or third epitope of interest are different from the second and / or fourth epitope of interest.

18. 1. A method for generating a library of a set of first and second candidate polypeptide-encoding polynucleotides, or a library of a set of first and second candidate polypeptide-encoding polynucleotides generated using said method, comprising: (i) the first candidate polypeptide is the same as or a variant of the first parent polypeptide; and (ii) the second candidate polypeptide is the same as or a variant of the second parent polypeptide; The method further comprising: (a) providing a set of a polynucleotide encoding the first parent polypeptide and a polynucleotide encoding the second parent polypeptide; (b) in silico or in vitro mutagenizing or randomizing nucleic acid at one or more predefined nucleotide positions in the set of polynucleotides of step (a), wherein at least one of the one or more predefined nucleotide positions is within a codon encoding an amino acid at one or more of the predefined amino acid positions of the first and / or second parent polypeptides, and wherein the amino acid position is (i) the amino acid positions located within or adjacent to the interface between the first parent polypeptide and the second parent polypeptide, optionally located within or adjacent to the interface, are predicted in silico or in vitro; and / or (ii) predicting interactions between said first parent polypeptide and said second parent polypeptide, optionally affecting inter-polypeptide hydrogen bond mediated interactions and / or inter-polypeptide binding energy, optionally wherein said prediction is performed in silico or in vitro, further optionally wherein said prediction is performed in silico using a Rosetta Monte Carlo (MC) Hydrogen Bond Network (HBNet); Optionally, the one or more mutations are generated via degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid groups; Optionally, the library is for identifying first and second polypeptides that preferentially pair with each other for a set of the first and second parent polypeptides.

19. 1. A method for generating a library of a set of a first candidate polypeptide and a second candidate polypeptide, or a library of a set of a first candidate polypeptide and a second candidate polypeptide generated using said method, comprising: (i) the first candidate polypeptide is the same as or a variant of the first parent polypeptide; and (ii) the second candidate polypeptide is the same as or a variant of the second parent polypeptide; The method further comprising: (I) obtaining in silico or in vitro a plurality of sets of first and second candidate polypeptides corresponding to the first and second candidate polypeptide-encoding polynucleotides contained in the polynucleotide library according to claim 18; or (II) incorporating, in silico or in vitro, substitutions at one or more predetermined amino acid positions in the first and / or second parent polypeptide, wherein one or more of the one or more predetermined amino acid positions are: (i) the amino acid positions located within or adjacent to the interface between the first parent polypeptide and the second parent polypeptide, optionally located within or adjacent to the interface, are predicted in silico or in vitro; and / or (ii) predicting interactions between said first parent polypeptide and said second parent polypeptide, optionally affecting inter-polypeptide hydrogen bond mediated interactions and / or inter-polypeptide binding energy, optionally wherein said prediction is performed in silico or in vitro, further optionally wherein said prediction is performed in silico using Rosetta MC HBNet; Optionally, the library is for identifying first and second polypeptides that preferentially pair with each other for the set of first and second parent polypeptides; Further optionally, (A) the first candidate polypeptide in the library comprises a predetermined number of substitutions relative to the first parent polypeptide, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and / or 1, 2, 3, 4, or 5; and / or (B) the second candidate polypeptide in the library comprises a predetermined number of substitutions relative to the second parent polypeptide, optionally wherein the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, and / or 1, 2, 3, 4, or 5.

20. 1. A method for identifying one or more sets of a first polypeptide and a second polypeptide, comprising: (i) the first polypeptide is the same as or a variant of a first parent polypeptide; (ii) the second polypeptide is the same as or a variant of a second parent polypeptide; (iii) the first polypeptide is a variant of the first parent polypeptide, and / or the second polypeptide is a variant of the second parent polypeptide; and (iv) the first and second polypeptides preferentially pair with each other, optionally more preferentially than the first and second parent polypeptides; The method further comprising: (a) providing a plurality of sets of first and second candidate polypeptides, optionally wherein said providing is performed in silico or in vitro; (b) quantifying binding preference between said first candidate domain polypeptide and said second candidate domain polypeptide, optionally wherein said binding preference is based on the strength of inter-polypeptide hydrogen bonds and / or inter-polypeptide binding energy, further optionally wherein said quantifying is performed in silico or in vitro; (c) selecting one or more sets of a first polypeptide and a second polypeptide that provide preferential inter-polypeptide associations, optionally equal or higher preferential associations relative to a reference set of polypeptides, further optionally wherein the reference set of polypeptides is a set of (I) a first parent polypeptide or variant thereof and (II) a second parent polypeptide or variant thereof; Optionally, (1) the at least one set of the first candidate polypeptide and the second candidate polypeptide in step (a) (i) derived from a library according to claim 19 or expressed from a library according to claim 18; and / or (ii) the first and / or second candidate polypeptides are derived from a library of a set of first and second candidate polypeptides, wherein the first and / or second candidate polypeptides comprise one or more random amino acid modifications, or are expressed from a library of a set of first and / or second candidate polypeptide-encoding polynucleotides, wherein the first and / or second candidate polypeptide-encoding polynucleotides comprise one or more random mutations; （２） (a-1) the first polypeptide comprises or is bound to a first label, and / or (a-2) the second polypeptide comprises or is bound to a second label; Optionally, the quantifying step (b) comprises detecting the first label and / or the second label; and / or （３） (3-i) In step (a), the providing is performed in silico; and In step (b), the quantifying optionally includes: ΔΔG, ΔΔG ｃｏｇｎａｔｅ ｔｏｔａｌ ｓｃｏｒｅ , ΔΔG ｃｏｇｎａｔｅ ｈｂｏｎｄ＿ａｌｌ , RBPP, RBPP ｔｏｔａｌ ｓｃｏｒｅ , R.B.P.P. ｈｂｏｎｄ＿ａｌｌ , and / or RBPP ｂｏｎｄ ｅｌｅｃ ｂａｃｋｒｕｂ １８ｋ and / or said quantifying is performed in silico using Rosetta Multi-Carlo (MC) Hydrogen Bond Network (HBNet), or (3-ii) In step (a), the providing is carried out in vitro, optionally recombinantly; and In step (b), the quantifying comprises measuring the amount of CH1-CL pairs via liquid chromatography-mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, and / or flow cytometry.

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