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

Abstract

Provided are CH1 domain variants engineered to preferentially bind to a kappa CL domain or a lambda CL domain, and polypeptides, such as antibody heavy chains or antibodies, comprising such engineered CH1 domain variants, and pharmaceutical compositions comprising such CH1 domain variants and / or such polypeptides, and methods for preparing and using such CH1 domain variants. The CH1 domain variants minimize heavy chain-light chain mispairing and promote homologous heavy chain-light chain pairing, thereby improving the generation of multispecific, such as bispecific, antibodies. Also provided are methods for preparing a CH1 domain variant library and methods for identifying one or more CH1 domain variants.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 908,367, filed on September 30, 2019, entitled “CH1 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL LIGHT CHAIN ​​PAIRING AND MULTISPECIFIC ANTIBODIES COMPRISING THE SAME,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to CH1 domain variants, antibody heavy chains, and antibodies, particularly multispecific antibodies, comprising such CH1 domain variants, wherein the CH1 domain variants contain at least one amino acid substitution that promotes proper heavy chain-light chain pairing. The present invention further relates to compositions comprising such antibodies and uses thereof, for example, as therapeutic or diagnostic agents. The present invention further relates to methods for preparing libraries of CH1 domain variants and methods for identifying one or more CH1 domain variants. Background Art

[0004] Efforts are underway to develop antibody therapeutic agents with more than one antigen binding specificity, such as bispecific antibodies. Bispecific antibodies can be used to interfere with multiple surface receptors associated with cancer, inflammatory processes or other disease states. Bispecific antibodies can also be used to place targets in close proximity and regulate protein complex formation or drive contact between cells. The generation of bispecific antibodies was first reported in the early 1960s (Nisonoff et al., " Archives of Biochemistry and Biophysics (Arch Biochem Biophys)" 1961 93 (2): 460-462), and the first monoclonal bispecific antibody was produced using hybridoma technology in the 1980s (Milstein et al., " Nature (Nature)" 1983 305 (5934): 537-540). Over the past decade, interest in bispecific antibodies has increased significantly due to their therapeutic potential, and bispecific antibodies are now used in the clinic, for example, blinatumomab and emicizumab have been approved for the treatment of specific cancers (see Sedykh et al., Drug Des Devel Ther 12:195-208 (2018) and Labrijn et al., Nature Reviews Drug Discovery 18:585-608 (2019) for recent reviews of methods for generating bispecific antibodies and the characteristics of bispecific antibodies approved for medical use).

[0005] Although bispecific antibodies have shown significant advantages over monospecific antibodies, their widespread commercial application has been hampered by the lack of efficient / low-cost production methods, the lack of stability of bispecific antibodies, and the lack of a long half-life in humans. Over the past few decades, various methods have been developed to improve the production of bispecific antibodies. These include recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)); "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168); immunoglobulin crossover technology (also known as Fab domain exchange or CrossMab format) (see, e.g., WO 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)); engineered electrostatic steering effects for making antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)); "diabody" technology (see, e.g., Hollinger et al., PNAS 90:6444-6448 (1993)); single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); and trispecific antibodies as described, e.g., in Tutt et al., J. Immunol, 147:60 (1991).

[0006] Despite these improvements, it remains a challenge to produce bispecific antibodies with the correct heavy chain-light chain pairing. Bispecific antibodies can be formed by co-expression of two different heavy chains and two different light chains. Properly forming bispecific antibodies in the desired form remains a challenge because the heavy chain has evolved to bind to the light chain in a relatively non-proximal manner. Therefore, the co-expression of two heavy chains and two light chains may result in a disorder of the heavy chain-light chain pairing - a complex mixture of sixteen possible combinations, which only represents one of the ten different antibodies corresponding to the desired bispecific antibody (if there is complete confusion, the maximum yield in the mixture is 12.5%). This mispairing (also known as the chain association problem) remains a major challenge in producing bispecifics because uniform pairing is essential for manufacturability and efficacy.

[0007] One strategy for alleviating mispairing is to generate bispecific antibodies with a common light chain (see, e.g., Merchant et al., Nat. Biotech. 16: 677-681 (1998)). Alternatively, a single common heavy chain and two different light chains (one kappa and one lambda) can be used (see, e.g., Fischer et al., Nature Commun. 6: 6113 (2015)). However, this strategy requires identification of antibodies with a common chain, which is difficult and tends to compromise the specificity of each binding arm and substantially reduce diversity (see, e.g., Wang et al., MABS 10(8): 1226-1235 (2018)).

[0008] Other approaches to improve correct heavy chain-light chain pairing include CrossMab technology (Roche), in which the light chain of a fragment antigen-binding (Fab) arm, or one of its subdomains, is exchanged with the corresponding region of the heavy chain Fd region, and DuetMab technology (MedImmune), in which the native disulfide bonds in one Fab arm are replaced by engineered disulfide bonds. However, these approaches require significant changes to the native IgG format, which may result in compounds that do not fully resemble native antibodies.

[0009] Another strategy is to reduce or eliminate heavy chain-light chain mispairing by amino acid substitutions in the constant and / or variable regions of the heavy and light chains in an IgG format. To the best of the inventors' knowledge, modifications of the CH1 domain alone have not previously been shown to resolve the chain association or mispairing issues often observed during multispecific antibody expression. Instead, multispecific antibodies engineered to include CH1 domain variants further require modifications outside of the CH1 domain to resolve chain association issues, such as the CL domain, and in some cases the VH, CH2, CH3, and / or VL domains. Examples include Lewis et al., Nature Biotechnology 32(2):191-198 (2014) who generated mutated CH1 and CL domains, CRD1 (heavy chain substituted with D148K, F170T, V185F, and light chain substituted with K129D, L135F; EU numbering) and CRD2 (heavy chain substituted with H168A and F170T, and light chain substituted with L135Y, S176W), in an attempt to drive preferential pairing of the altered heavy and light chains and to disfavor pairing of the heavy and light chain domains with wild-type constant domains. However, they report that any pairing specificity achieved with mutated CH1 and CL domains in the absence of variable domains would not translate to a full-length IgG format without additional engineering within the VH-VL interface, i.e., substitutions within the VH-VL interface as well as CL and CH1 domain substitutions were required to achieve preferential heavy-light chain pairing. Engineering the CH1 and CL domains to contain charged amino acid residues is also thought to promote preferential heavy chain-light chain pairing (see, for example, US10,047,163). Bispecific antibodies having at least two Fab fragments with different CH1 and CL domains are also known, wherein one of the Fab fragments has substitutions within the CH1 domain and the Cκ domain to drive preferential pairing (see US20180022829 and US9,631,031, which disclose CH1: T187E and Cκ: N137K + S114A; CH1: L145Q + S183V and Cκ: V133T + S176V; CH1: L128A + L145E and Cκ: V133W; CH1: V185A and Cκ: L135W + N137A). Additional examples of specific CH1 domain substitutions that are said to promote preferential heavy chain-light chain pairing include: A141C / L, K147D, G166D, G166K, or substitutions with cysteine ​​at positions 128, 129, 162, or 171 (WO2019183406 (Invenra Inc.))); substitution of the cysteine ​​at position 126 or 220 with valine or alanine or substitution of the non-cysteine ​​at position 128, 141 or 168 with cysteine, L145F, K147A, F170V, S183F or V185W / F (US9,527,927 (Medical Immunology)); 172A and 174G (WO2020060924 (Dualogics); A172R and 174G or substitution of residue 190 to M or I (US10,047,167 (University of North Carolina Chapel Hill and Eli Lilly and Company) Lilly); L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and V185A / L (US20180177873 (Genentech); 131C / S, 133R / K, 137E / G, 138S / G178S / Y, 192N / S and / or 193F / L (US10,487,156 (Argenx BVBA (Argenx) BVBA); 145D / E / R / H / K (IMGT position 26) (WO2018141894 (Merck); 124K / E / R / D (US10,392,438 (Pfizer); 133V, 150A, 150D, 152D, 173D or 188W (US20190023810 (MIT); 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L and / or 190S / A / G / Y (US20180179296 and US9,914,785 (Zymeworks)); 143A / E / R / K / D and 145T / L (US10,077,298 (Zymeworks); 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T, 139W / G / C, 179E, or 186R (US20170204199 (Zymeworks)); substitution with cysteine ​​at position 126, 127, 128, 134, 141, 171, or 173 (Zenyaku Kogyo); L145Q, H168A, F170G, S183V, and T187E (WO2020127354 (Alligator Biosciences) Bioscience); 143D / E, 145T, 190E / D, and 124R (WO2017 / 059551 (Zymeworks)). Additionally, US Pat. No. 9,150,639 to Kyowa Hakko Kirin Co., Ltd. reports the production of heavy chains containing A140C, K147C, or S183C to introduce cysteines to allow for chemical regulation. Kirin Co., Ltd. indicates that antibody variants containing these heavy chain mutations can include wild-type light chains; however, there is no indication that this favors preferential heavy-light chain pairing.

[0010] Another strategy for minimizing heavy chain-light chain mispairing is to utilize different light chains, such as light chains with different constant domains.For example, Loew et al. produce multispecific antibodies with κ light chains and λ light chains, and observe minimum mispairing because some naturally occurring κ light chains have high fidelity and are not paired with the heavy chain from λ antibodies, and vice versa (WO2018057955). Unfortunately, the applicability of this method is limited to those light chains with high fidelity. Others have used κ and λ light chains to produce multispecific antibodies, wherein amino acid substitutions are used for both heavy and light chains to drive preferential pairing in an electrostatic or stereochemical manner (see, for example, WO2017059551 (Zymeworks), US20140154254 (Amgen) and US10,047,163 (AbbVie Stentrx companies (AbbVie Stemcentrx))). However, introducing numerous amino acid substitutions into both the heavy and light chains presents additional technical hurdles and may additionally have deleterious effects on antibody function and / or immunogenicity. Summary of the Invention

[0011] The present invention aims to provide engineered bispecific antibodies with appropriate heavy chain-light chain pairing. In one aspect, provided herein are CH1 domain variant polypeptides (also referred to herein as CH1 domain variants) that promote preferential pairing of heavy chains with specific light chains and polypeptides, such as antibodies, comprising the same. The CH1 domain variants contain at least one amino acid substitution (relative to the parent, e.g., wild-type sequence).

[0012] In some embodiments, the CH1 domain variant contains at least one amino acid substitution at a CH1 domain position that forms an interface with the CL domain of the light chain, including but not limited to positions 140 and / or 141 or 147 and / or 183 (EU numbering). The substitution promotes preferential pairing of the heavy chain containing the CH1 domain variant with a particular light chain, for example, CH1 domain variant 141 preferentially pairs with a lambda CL domain, such as compared to a kappa CL domain, while CH1 domain variants 147F and / or 183R, 183K, or 183Y preferentially pair with a kappa CL domain, such as compared to a lambda CL domain.

[0013] In some embodiments, the CH1 domain variant contains at least one amino acid substitution at a CH1 domain position that forms the interface between the CH1 domain and VH, such as CH1 position 151 (EU numbering).

[0014] This preferential pairing of constant domains is expected to drive the pairing of full-length light and heavy chains, including variable domains, thereby generating a solution to the chain pairing problem of bispecifics. Specifically, the CH1 domain variant polypeptides include amino acid substitutions at one or more of the following positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218. Optionally, such CH1 domain variant polypeptides preferentially pair with: (i) a kappa light chain constant region ("CL") domain, as compared to a lambda CL domain, and / or a kappa light chain polypeptide, as compared to a lambda light chain polypeptide; (ii) a lambda CL domain, as compared to a kappa CL domain, and / or a lambda light chain polypeptide, as compared to a kappa light chain polypeptide.

[0015] Optionally, in some embodiments, certain CH1 domain variants may be excluded and the CH1 domain variants according to the present invention may satisfy the following:

[0016] (a) if residue 141 on CH1 is substituted with C or L, residue 166 is substituted with D or K, residues 128, 129, 162, or 171 on CH1 are substituted with C, and / or residue 147 is substituted with D, then the CL domain with which the CH1 domain variant preferentially pairs does not include an amino acid substitution;

[0017] (b) if position 126 or 220 on CH1 is substituted with valine or alanine, the non-cysteine ​​at position 128, 141 or 168 is substituted with cysteine, or CH1 is substituted with L145F, K147A, F170V, S183F or V185W / F, the CL domain with which the CH1 domain variant is preferentially paired does not include an amino acid substitution;

[0018] (c) if residue 172 in CH1 is substituted to 172R, residue 174 is mutated to 174G, or residue 190 is substituted to 190M or 190I, these are not the only substitutions included in CH1;

[0019] (d) if the CH1 substitution consists of L128F, A141I / M / T / L, F170S / A / Y / M, S181M / I / T, S183A / E / K / V and / or V185A / L, the CL domain with which the CH1 domain variant is preferentially paired is unmodified;

[0020] (e) if the CH1 substitutions consist of 131C / S, 133R / K, 137E / G, 138S / G, 178S / Y, 192N / S, and / or 193F / L, these are not the only CH1 substitutions and / or in the bispecific antibody, the CH1 domains are of the same human immunoglobulin subtype or allotype;

[0021] (f) if the CH1 substitution consists of 145D / E / R / H / K (IMGT position 26), there is no corresponding LC substitution, 129D / E / R / H / K (IMGT position 18);

[0022] (g) if the CH1 substitution consists of 124K / E / R / D, there is no corresponding substitution at position 176 of the LC, and the CH1 domain variant preferentially pairs with it;

[0023] (h) if the CH1 substitution consists of 133V, 150A, 150D, 152D, 173D and / or 188W, there are no corresponding substitutions in LC with which the CH1 domain variant preferentially pairs;

[0024] (i) if the CH1 substitution consists of 133S / W / A, 139W / V / G / I, 143K / E / A, 145E / T / L / Y, 146G, 147T / E, 174V, 175D / R / S, 179K / D / R, 181R, 186R, 188F / L, and / or 190S / A / G / Y, there are no corresponding substitutions in LC with which the CH1 domain variant preferentially pairs;

[0025] (j) if the CH1 substitution consists of 143A / E / R / K / D and 145T / L, there are no corresponding substitutions in LC with which the CH1 domain variant preferentially pairs;

[0026] (k) if the CH1 substitution consists of 124A / R / E / W, 145M / T, 143E / R / D / F, 172R / T and 139W / G / C, 179E and / or 186R, there are no corresponding substitutions in LC with which the CH1 domain variant preferentially pairs;

[0027] (1) if the CH1 substitution consists of substitution by cysteine ​​at position 126, 127, 128, 134, 141, 171 or 173, the corresponding LC position is not modified to form a disulfide bond;

[0028] (m) if the CH1 substitution consists of L145Q, H168A, F170G, S183V and / or T187E, there are no corresponding substitutions in the κ or λ LC with which the CH1 domain variant preferentially pairs;

[0029] (n) if the CH1 substitution consists of 143D / E, 145T, 190E / D and / or 124R, there are no corresponding substitutions in LC with which the CH1 domain variant preferentially pairs; or

[0030] (o) If the CH1 substitution consists of A140C, K147C and / or S183C, there is a corresponding substitution in LC with which the CH1 domain variant preferentially pairs.

[0031] In some embodiments, the CH1 domain variant polypeptide comprises an amino acid substitution at one or more of the following positions according to EU numbering: 118, 124, 126-129, 131, 132, 134, 136, 139, 143, 145, 147-151, 153, 154, 170, 172, 175, 176, 181, 183, 185, 190, 191, 197, 201, 203-206, 210, 212-214, and 218. Optionally, the CH1 domain variant polypeptide is configured to preferentially pair with: (i) a kappa CL domain (or a kappa CL-containing polypeptide), as compared to a lambda CL domain (or a lambda CL-containing polypeptide); and / or (ii) a kappa light chain polypeptide, as compared to a lambda light chain polypeptide.

[0032] In certain embodiments, such CH1 domain variants comprise an amino acid substitution at position 147, position 183, or positions 147 and 183.

[0033] In certain embodiments, such CH1 domain variants comprise one or more of the following amino acid substitutions: position 118 is substituted with G; position 124 is substituted with H, R, E, L, or V; position 126 is substituted with A, T, or L; position 127 is substituted with V or L; position 128 is substituted with H; position 129 is substituted with P; position 131 is substituted with A; position 132 is substituted with P; position 134 is substituted with G; position 136 is substituted with E; position 139 is substituted with I; position 143 is substituted with V or S; position 145 is substituted with F, I, N, or T; position 147 is substituted with F, I, L, R, T, S, M, V, N, E, H, Y, Q, A, or G; position 148 is substituted with I, Q, Y, or G; position 149 is substituted with C, S, or H; position 150 is substituted with L or S; position 151 is substituted with L or S; position 152 is substituted with L or S; position 153 is substituted with L or S; position 154 is substituted with L or S; position 155 is substituted with L or S; position 156 is substituted with L or S; position 157 is substituted with L or S; position 158 is substituted with L or S; position 159 is substituted with L or S; position 160 is substituted with L or S; position 161 is substituted with L or S; position 162 is substituted with L or S; position 163 is substituted with L or S; position 164 is substituted with L or S; position 165 is substituted with L or S; position 166 is substituted with L or S; position 167 is substituted with L or S; position 168 is substituted with L or S; position 151 is substituted by A or L; position 153 is substituted by S; position 154 is substituted by M or G; position 170 is substituted by G or L; position 172 is substituted by V; position 175 is substituted by G, L, E, or A; position 176 is substituted by P; position 181 is substituted by Y, Q, or G; position 183 is substituted by I, W, F, E, Y, L, K, Q, N, R, or H; position 185 is substituted by W; position 190 is substituted by P; position 191 is substituted by I; position 197 is substituted by A; position 201 is substituted by S; position 203 is substituted by S; position 204 is substituted by Y; position 205 is substituted by Q; position 206 is substituted by S; position 210 is substituted by R; position 212 is substituted by G; position 213 is substituted by E or R; position 214 is substituted by R; and position 218 is substituted by Q.

[0034] In certain embodiments, the κ-preferring CH1 domain variant polypeptide may include: (i) amino acid residue F, I, L, R, T, S, M, V, N, E, H, Y, or Q at position 147; and / or (ii) amino acid residue I, W, F, E, Y, L, K, Q, N, or R at position 183.

[0035] In some preferred embodiments of the κ-preferring CH1 domain variant, the CH1 domain variant polypeptide may include: (i) amino acid residue R, K or Y at position 183; and / or (ii) amino acid residue F at position 147.

[0036] In further embodiments, a CH1 domain variant polypeptide comprises: (i) amino acid residue F at position 147 and amino acid residue R at position 183; (ii) amino acid residue F at position 147 and amino acid residue K at position 183; (iii) amino acid residue F at position 147 and amino acid residue Y at position 183; (iv) amino acid residue R at position 183; (v) amino acid residue K at position 183; or (vi) amino acid residue Y at position 183. Optionally, the CH1 domain variant may comprise the following amino acid sequence: (i) SEQ ID NO: 137; (ii) SEQ ID NO: 138; (iii) SEQ ID NO: 139; (iv) SEQ ID NO: 60; (v) SEQ ID NO: 41; or (vi) SEQ ID NO: 136.

[0037] In some embodiments, the CH1 domain variant polypeptide comprises an amino acid substitution at a CH1 amino acid position within the interface between CH1 and VH. Optionally, the CH1 amino acid position within such interface is position 151. Further optionally, such CH1 domain variants may comprise an amino acid residue A or L at position 151.

[0038] In some embodiments, the CH1 domain variant polypeptide further comprises one or more amino acid substitutions that increase pairing of the CH1 domain with: (i) a κ CL domain, as compared to a λ CL domain; and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide.

[0039] In some embodiments, the CH1 domain variant polypeptide of any one of claims 2 to 10 increases pairing with: (i) a kappa CL domain, as compared to a lambda CL domain; and / or (ii) a kappa light chain polypeptide, as compared to a lambda light chain polypeptide, by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The increase in kappa pairing can optionally be measured by liquid chromatography-mass spectrometry (LCMS).

[0040] In some embodiments, the CH1 domain variant polypeptide of any one of claims 2 to 10 increases pairing with: (i) a κ CL domain, as compared to a λ CL domain; and / or (ii) a κ light chain polypeptide, as compared to a λ light chain polypeptide, by at least 1.2-fold, at least 1.5-fold, at least 2-fold, 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold. The increase in kappa pairing can optionally be quantified by flow cytometry, for example by comparing the mean fluorescence intensity (MFI) ratio of kappa CL staining to lambda CL staining.

[0041] In some embodiments, a CH1 domain variant polypeptide according to the invention comprises an amino acid substitution at one or more of the following positions according to EU numbering: 119, 124, 126, 127, 130, 131, 133, 134, 138-142, 152, 163, 168, 170, 171, 175, 176, 181, 183-185, 187, 197, 203, 208, 210-214, 216, and 218. Optionally, the CH1 domain variant preferentially pairs with: (i) a lambda CL domain, as compared to a kappa CL domain; and / or (ii) a lambda light chain polypeptide, as compared to a kappa light chain polypeptide.

[0042] In certain embodiments, the lambda-preferring CH1 domain variant polypeptide comprises an amino acid substitution at one or more of positions 141, 170, 171, 175, 181, 184, 185, 187, and 218.

[0043] In certain embodiments, the lambda-preferring CH1 domain variant polypeptide comprises one or more of the following amino acid substitutions: position 119 is substituted with R; position 124 is substituted with V; position 126 is substituted with V; position 127 is substituted with G; position 130 is substituted with H or S; position 131 is substituted with Q, T, N, R, V or D; position 133 is substituted with D, T, L, E, S or P; position 134 is substituted with A, H, I, P, V, N or L; position 138 is substituted with R; position 139 is substituted with A; position 140 is substituted with I, V, D, Y, K, S, W, R, L or P; position 141 is substituted with D, K, E, T, R, Q, V or M; position 142 is substituted with M; position 152 is substituted with G; position 163 is substituted with M; position 168 is substituted with F, I or V; position 170 is substituted with F, I or V; position 171 is substituted with F, I or V; position 172 is substituted with F, I or V; position 173 is substituted with F, I or V; position 174 is substituted with F, I or V; position 175 is substituted with F, I or V; position 176 is substituted with F, I or V; position 177 is substituted with F, I or V; position 178 is substituted with F, I or V; position 179 is substituted with F, I or V; position 180 is substituted with F, I or V; position 181 is substituted with F, I or V; position 182 is substituted with F, I or V; position 183 is substituted with F, I or V; position 184 is substituted with F, I or V; position 185 is substituted with F, I position 170 is substituted by N, G, E, S or T; position 171 is substituted by N, E, G, S, A or D; position 175 is substituted by D or M; position 176 is substituted by R or M; position 181 is substituted by V, L, A, K or T; position 183 is substituted by L or V; position 184 is substituted by R; position 185 is substituted by M, L, S, R or T; position 187 is substituted by R, D, E, Y or S; position 197 is substituted by S; position 203 is substituted by D; position 208 is substituted by I; position 210 is substituted by T; position 211 is substituted by A; position 212 is substituted by N; position 213 is substituted by E; position 214 is substituted by R; position 216 is substituted by G; and position 218 is substituted by L, E, D, P, A, H, S, Q, N, T, I, M, G, C, K or W.

[0044] In certain further embodiments, the lambda-preferring CH1 domain variant polypeptide includes any one or more of (i)-(xvii): (i) amino acid residue V at position 126; (ii) amino acid residue G at position 127; (iii) amino acid residue V at position 131; (iv) amino acid residue S at position 133; (v) amino acid residue R at position 138; (vi) amino acid residue I or V at position 140; (vii) amino acid residue D, K, E, or T at position 141; (viii) amino acid residue M at position 142; (ix) amino acid residue I at position 168; (xvii) amino acid residue D, K, E, or T at position 141; (xvii) amino acid residue M at position 142; (ix) amino acid residue I at position 168; (xvii) amino acid residue G at position 127; (xvii) amino acid residue S at position 133; (xvii) amino acid residue I at position 168; (xvii) amino acid residue D, K, E, or T at position 161; (xvii) amino acid residue M at position 162; (ix) amino acid residue I at position 168 ... S at position 133; (xvii) amino acid residue D, K, E, or T at position 161; (ix) amino acid residue M at position 162; (ix) amino acid residue I at position 168; (ix) amino acid residue I at position 168; (ix) amino acid residue S at position 133; (ix) amino acid residue S at position 133; (v) amino acid residue R at position 138; (ix) amino acid residue I at position 1 (x) an amino acid residue E, G, or S at position 170; (xi) an amino acid residue E, D, G, S, or A at position 171; (xii) an amino acid residue M at position 175; (xiii) an amino acid residue R at position 176; (xiv) an amino acid residue K, V, A, or L at position 181; (xv) an amino acid residue R at position 184; (xvi) an amino acid residue R at position 185; (xvii) an amino acid residue R at position 187; and (xviii) an amino acid residue L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W at position 218.

[0045] In certain preferred embodiments, the lambda-preferring CH1 domain variant polypeptides according to the present invention comprise or consist of one or more of the following substitutions: 141D, 141E, 171E, 170E, 185R, and 187R.

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

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

[0048] In certain preferred embodiments, the lambda-preferring CH1 domain variant polypeptides according to the present invention comprise or consist of the following substitutions: (i) 141E and 185R; (ii) 141E and 187R; (iii) 141E, 170E or 171E and 185R; (iv) 141E, 170E or 171E and 187R; (v) 141D and 185R; (vi) 141D and 187R; (vii) 141D, 170E or 171E and 185R; (viii) 141D, 170E or 171E and 187R; (ix) 141E, 185R and 187R; or (x) 141D, 185R and 187R.

[0049] In further embodiments, the lambda-preferring CH1 domain variant polypeptides according to the present invention include one or more substitutions at position 141 to D, K or E, which are optionally paired with a substitution at position 181 to K, and further optionally paired with a substitution at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

[0050] In further embodiments, the lambda-preferring CH1 domain variant polypeptides according to the present invention include a substitution at position 141 to D, K or E, which is paired with a substitution at position 181 to K and / or with a substitution at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C or W.

[0051] In further embodiments, the λ-preferring CH1 domain variant polypeptide according to the present invention includes any one or more of (i)-(xvii): (i) amino acid residue D, E or K at position 141; (ii) amino acid residue E at position 170; (iii) amino acid residue E at position 171; (iv) amino acid residue M at position 175; (v) amino acid residue K at position 181; (vi) amino acid residue R at position 184; (vii) amino acid residue R at position 185; (viii) amino acid residue R at position 187; (ix) amino acid residue P, A or E at position 218.

[0052] In further embodiments, a lambda-preferring CH1 domain variant polypeptide according to the present invention comprises: (i) amino acid residue D at position 141; (ii) amino acid residue D at position 141 and amino acid residue K at position 181; (iii) amino acid residue D at position 141, amino acid residue K at position 181, and amino acid residue A at position 218; (iv) amino acid residue D at position 141, amino acid residue K at position 181, and amino acid residue P at position 218; (v) amino acid residue E at position 141; (vi) amino acid residue E at position 141 and amino acid residue K at position 181; (vii) amino acid residue K at position 141; (viii) amino acid residue P at position 218. (ix) amino acid residue K at position 141, amino acid residue K at position 181, and amino acid residue E at position 218; (x) amino acid residue K at position 141, amino acid residue K at position 181, and amino acid residue P at position 218; (xi) amino acid residue E at position 141, amino acid residue E at position 170, amino acid residue V at position 181, and amino acid residue R at position 187; (xii) amino acid residue E at position 141, amino acid residue D at position 171, and amino acid residue R at position 185; (xiii) amino acid residue E at position 141, amino acid residue D at position 171, and amino acid residue R at position 185; (xvi) amino acid residue E at position 141, amino acid residue S at position 171, and amino acid residue K at position 181; (xvii) amino acid residue E at position 141, amino acid residue G at position 170, amino acid residue M at position 175, amino acid residue V at position 181, amino acid residue V at position 184, and amino acid residue R at position 185; (xvii) amino acid residue E at position 141, amino acid residue G at position 170, amino acid residue M at position 175, amino acid residue V at position 181, amino acid residue V at position 184, and amino acid residue R at position 185; (xvii) amino acid residue E at position 141, amino acid residue S at position 171, and amino acid residue K at position 181; (xvii) amino acid residue E at position 141, amino acid residue G at position 170, amino acid residue M at position 175, amino acid residue V at position 181, amino acid residue V at position 184 (xviii) amino acid residue E at position 141 and amino acid residue R at position 185; (xix) amino acid residue E at position 141 and amino acid residue R at position 187; (xx) amino acid residue E at position 141, amino acid residue E at position 170 and amino acid residue R at position 185; (xxi) amino acid residue E at position 141, amino acid residue E at position 170 and amino acid residue R at position 187; (xxii) amino acid residue D at position 141 and amino acid residue R at position 185; (xxiii) amino acid residue D at position 141 and amino acid residue R at position 187;(xxiv) amino acid residue D at position 141, amino acid residue R at position 185, and amino acid residue R at position 187; (xxv) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 185; (xxvi) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187; (xxvii) amino acid residue E at position 141, amino acid residue E at position 171, and amino acid residue R at position 187; (xxiii) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (xxix) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 187.

[0053] Optionally, the CH1 domain variant comprises the following amino acid sequence: (i) SEQ ID NO: 140; (ii) SEQ ID NO: 141; (iii) SEQ ID NO: 142; (iv) SEQ ID NO: 143; (v) SEQ ID NO: 144; (vi) SEQ ID NO: 145; (vii) SEQ ID NO: 146; (viii) SEQ ID NO: 147; (ix) SEQ ID NO: 148; (x) SEQ ID NO: 149; (xi) SEQ ID NO: 155; (xii) SEQ ID NO: 157; (xiii) SEQ ID NO: 159; (xiv) SEQ ID NO: 162; (xv) SEQ ID NO: 163; (xvi) SEQ ID NO: 164; (xvii) SEQ ID NO: 165; (xviii) SEQ ID NO: 178; (xix) SEQ ID NO: 179; (xx) SEQ ID NO: 180; (xxi) SEQ ID NO: 181; (xxii) SEQ ID NO: 182; (xxiii) SEQ ID NO: 183; (xxiv) SEQ ID NO: 184; (xxv) SEQ ID NO: 185; (xxvi) SEQ ID NO: 186; (xxvii) SEQ ID NO: 187; (xxviii) SEQ ID NO:188; or (xxix) SEQ ID NO:189.

[0054] In some preferred embodiments, the λ-preferring CH1 domain variant comprises: (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0055] In another preferred embodiment, the lambda-preferring CH1 domain variant comprises amino acid substitutions consisting of: (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0056] In certain preferred embodiments, the lambda-preferring CH1 domain variant comprises an amino acid substitution consisting of: (i) SEQ ID NO: 188; or (ii) SEQ ID NO: 186.

[0057] In some embodiments, the λ-preferring CH1 domain variant polypeptide may further comprise one or more amino acid substitutions that increase pairing of the CH1 domain with: (i) a λ CL domain, as compared to a κ CL domain; and / or (ii) a λ light chain polypeptide, as compared to a κ light chain polypeptide.

[0058] In some embodiments, the CH1 domain variant polypeptide can increase pairing with: (i) a lambda CL domain, such as compared to a kappa CL domain; and / or (ii) a lambda light chain polypeptide, such as compared to a kappa light chain polypeptide, by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The increase in lambda pairing can optionally be measured by liquid chromatography-mass spectrometry (LCMS).

[0059] In some embodiments, the CH1 domain variant polypeptide can increase pairing with: (i) a lambda CL domain, such as compared to a kappa CL domain; and / or (ii) a lambda light chain polypeptide, such as compared to a kappa light chain polypeptide by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold. The increase in lambda pairing can optionally be measured by flow cytometry, optionally by comparing the ratio of the MFI values ​​of lambda CL staining to kappa CL staining.

[0060] In another aspect, further provided herein are antibody heavy chain polypeptides comprising a variable region and a constant region, wherein the constant region comprises a CH1 domain variant according to any of those described above.

[0061] In some embodiments, the CH1 domain variants of such antibody heavy chain polypeptides are according to comprising amino acid substitutions consisting of: (I) (i) amino acid residue F at position 147 and amino acid residue R at position 183; (ii) amino acid residue F at position 147 and amino acid residue K at position 183; (iii) amino acid residue F at position 147 and amino acid residue Y at position 183; (iv) amino acid residue R at position 183; (v) amino acid residue K at position 183; or (vi) amino acid residue Y at position 183; or (II) (i) amino acid residue D at position 141, amino acid residue E at position 171, and amino acid residue R at position 185; or (ii) amino acid residue D at position 141, amino acid residue E at position 170, and amino acid residue R at position 187.

[0062] In another aspect, the present invention further provides an antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, wherein (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; and (b) the first heavy chain polypeptide comprises a first CH1 domain variant, the first CH1 domain variant comprising an amino acid substitution at one or more of the following positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, such that the first CH1 domain variant preferentially binds to the first light chain. Optionally, the first light chain polypeptide comprises a first CL domain, the first CL domain being a wild-type CL domain. Further optionally, certain CH1 domain variants may be excluded as described above and the CH1 domain variant according to the present invention may satisfy one or more of items (a)-(o) as described above. Also provided herein are such antibodies or antibody fragments, which further comprise a second heavy chain polypeptide and a second light chain polypeptide, wherein: (a) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; and (b) the second heavy chain polypeptide comprises a second CH1 domain variant, which comprises an amino acid substitution at one or more of the following positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216 and 218, such that the second CH1 domain variant preferentially binds to the second light chain polypeptide comprising a second CL domain. Likewise, optionally, certain CH1 domain variants may be excluded as described above and the CH1 domain variants according to the present invention may satisfy one or more of items (a)-(o) as described above.Further optionally, such antibodies or antibody fragments include one or more of features (i)-(vii): (i) the first CL domain is a wild-type CL domain; (ii) the second CL domain is a wild-type CL domain; (iii) the first CL domain is a κCL domain; (iv) the first CL domain is a λCL domain; (v) the second CL domain is a κCL domain; (vi) the second CL domain is a λCL domain; (vii) the first CH1 domain variant is a CH1 domain variant according to any one of claims 1 to 20; (viii) the second CH1 domain variant is a CH1 domain variant according to any one of claims 1 to 20; and / or (ix) the amino acid substitution in the first CH1 domain variant is different from the amino acid substitution in the second CH1 domain variant.

[0063] Further provided herein are antibodies or antibody fragments comprising a first heavy chain polypeptide and a first light chain polypeptide, wherein: (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises a first CH1 domain variant according to any of the κ-preferring CH1 domain variants described above; and (c) the first light chain polypeptide comprises a κCL domain and is optionally a κ light chain polypeptide. Optionally, (i) the κCL domain is a wild-type CL domain; and / or (ii) the first light chain polypeptide is a wild-type light chain polypeptide. In certain embodiments, the first heavy chain polypeptide optionally comprises one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with: (i) a κCL domain, such as compared to a λCL domain, and / or (ii) a κ light chain polypeptide, such as compared to a λ light chain polypeptide. The one or more amino acid substitutions outside the CH1 domain can, for example, be in VH.

[0064] Also provided herein are antibodies or antibody fragments comprising a second heavy chain polypeptide and a second light chain polypeptide, wherein: (a) the second heavy chain polypeptide and the second light chain polypeptide form a first cognate pair; (b) the second heavy chain polypeptide comprises a second CH1 domain variant according to any of the λ-preferring CH1 domain variants described above; and (c) the second light chain polypeptide comprises a λ CL domain, and is optionally a λ light chain polypeptide. Optionally, (i) the λ CL domain is a wild-type CL domain; and / or (ii) the second light chain polypeptide is a wild-type light chain polypeptide. In certain embodiments, the second heavy chain polypeptide optionally comprises one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with: (i) a λ CL domain, such as compared to a κ CL domain, and / or (ii) a λ light chain polypeptide, such as compared to a κ light chain polypeptide.

[0065] Also provided herein are antibodies or antibody fragments comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, wherein: (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises a first CH1 domain, which comprises a CH1 domain variant according to any one of the κ-preferring CH1 domain variants described above; (c) the first light chain polypeptide comprises a κCL domain and is optionally a κ light chain polypeptide; (d) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; (e) the second heavy chain polypeptide comprises a second CH1 domain, which comprises a CH1 domain variant according to any one of the λ-preferring CH1 domain variants described above; and (f) the second light chain polypeptide comprises a λCL domain and is optionally a λ light chain polypeptide. In certain embodiments, the first heavy chain polypeptide optionally includes one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with: (i) a κ CL domain, such as compared to a λ CL domain, and / or (ii) a κ light chain polypeptide, such as compared to a λ light chain polypeptide. The one or more amino acid substitutions outside the CH1 domain can, for example, be in VH. In certain embodiments, the second heavy chain polypeptide optionally includes one or more amino acid substitutions outside the CH1 domain that further promote preferential pairing of the heavy chain with: (i) a λ CL domain, such as compared to a κ CL domain, and / or (ii) a λ light chain polypeptide, such as compared to a κ light chain polypeptide.

[0066] Any of the antibodies or antibody fragments may be multispecific, optionally bispecific. Optionally, the structure of such an antibody or antibody fragment is as depicted in any one of Figures 24 to 29 .

[0067] In some embodiments, in a multispecific antibody or antibody fragment as described above, the first CH1 domain variant and the second CH1 domain variant reduce the formation of non-cognate heavy chain-light chain pairs by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, in a multispecific antibody or antibody fragment as described above, the first CH1 domain variant and the second CH1 domain variant increase the formation of cognate heavy chain-light chain pairs by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0068] In some embodiments, the reduction of non-cognate heavy-light pairing and / or the increase of cognate heavy-light pairing can be achieved by transfecting cells with HC (or VH plus CH1) comprising CH1, κLC and λLC of interest at a predetermined ratio, such as HC:κLC:λLC=2:1:1, and measuring the light chain species by LCMS as described in Examples 7 and Figure 23 、 30 or 31 for quantification. In certain embodiments using such or similar quantification methods, an exemplary WT CHI can produce HC-LC pairs, 60% of which are cognate pairs and 40% of which are non-cognate pairs, and by using CHI variants according to the present disclosure, the percentage of cognate pairs can be increased to at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, and the percentage of non-cognate pairs can be reduced to at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, or 0%. In specific embodiments using such or similar quantification methods, the percentage of cognate pairs can be increased to at least 85%, at least 90%, at least 95%, or 100%, while the percentage of non-cognate pairs can be reduced to at least 15%, at least 10%, at least 5%, or 0%.

[0069] In some embodiments, in a multispecific antibody or antibody fragment as described above, the first CHI domain variant and the second CHI domain variant reduce the formation of non-cognate heavy chain-light chain pairs by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold. In some embodiments, in a multispecific antibody or antibody fragment as described above, the first CHI domain variant and the second CHI domain variant increase the formation of cognate heavy chain-light chain pairs by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold.

[0070] In some embodiments, the reduction of non-cognate heavy-light pairs and / or the increase of cognate heavy-light pairs can be quantified by simultaneously expressing HC (or VH+CH1) comprising the CH1 of interest, κ LC, and λ LC at a predetermined ratio to allow for presentation of heavy-light pairs on cells (e.g., yeast cells), staining the cells with anti-κ and anti-λ antibodies, and quantifying the presence of κ and λ by FACS, for example, by comparing MFI values ​​as shown in Figures 2-5, 8-13, and 19-22. To compare the κ preference of a particular CH1, the ratio of the MFI of cells stained with anti-κ to the MFI of cells stained with anti-λ can be calculated and divided by such ratio of WT CH1 to obtain a multiple of the parent (FOP) value. To compare the λ preference of a particular CH1, the ratio of the MFI of cells stained with anti-λ to the MFI of cells stained with anti-κ can be calculated and divided by such ratio of WT CH1 to obtain a multiple of the parent (FOP) value. To compare the λ preference of a particular CH1, the ratio of the MFI of cells stained with anti-λ to the MFI of cells stained with anti-κ can be calculated and divided by such ratio of WT CH1.

[0071] In certain embodiments using such or similar quantitative methods, the FOP value (calculated for κ bias, i.e., MFI of κ:λ) can be increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold using the κ-biased CH1 variants according to the present disclosure. In certain embodiments using such or similar quantitative methods, the FOP value (calculated for lambda bias, i.e., MFI of lambda:κ) can be increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, or at least 25-fold using the lambda-biased CHI variants according to the present disclosure.

[0072] In some embodiments, the second CH1 domain variant comprises a substitution at position 141 and reduces the formation of non-cognate heavy chain-light chain pairs by at least 50%. In some embodiments, the second CH1 domain variant comprises a substitution at position 141 and the first CH1 domain variant comprises a substitution at position 183 and optionally at position 147, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises 141D or 141E and the second CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises one or more of 141D or 141E, 170E, 171E, 181K, 185R, 187R, and 218P, and the first CH1 domain variant comprises 183R, 183K, or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by at least 50% to at least 75%. In some embodiments, the second CH1 domain variant comprises a combination of 141D, 171E, and 185R, a combination of 141D, 171E, and 187R, or a combination of 141D, 181K, and 218P, and the second CH1 domain variant comprises 183R, 183K, or 183Y and optionally 147F, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by at least 50% to at least 75%.

[0073] In some embodiments, the first CH1 domain variant and the second CH1 domain variant provide at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% formation of the desired first and second homologous pairs. In some embodiments, the first CH1 domain variant and the second CH1 domain variant provide about 85% to about 95% formation of the desired first and second homologous pairs. In some embodiments, the second CH1 domain variant comprises a substitution at position 141, and the first CH1 domain variant comprises a substitution at position 183 and optionally at position 147, and provides about 85% to at least about 95% formation of the desired first and second homologous pairs. In some embodiments, the second CH1 domain variant comprises 141D or 141E, and the first CH1 domain variant comprises 183R, 183K or 183Y and optionally 147F, or vice versa, and provides about 85% to at least about 95% formation of the desired first and second homologous pairs. In some embodiments, the first and second CH1 domain variants reduce the formation of non-cognate heavy chain-light chain pairs by less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the second CH1 domain variant comprises a substitution at position 141, 170, 171, 181, 185, 187, and / or 218, and the first CH1 domain variant comprises a substitution at position 183 and, optionally, position 147, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by less than about 15%, less than about 10%, or less than about 5%. In some embodiments, the second CH1 domain variant comprises one or more of 141D or 141E, 170E, 171E, 181K, 185R, 187R, and 218P, and the first CH1 domain variant comprises 183R, 183K, or 183Y and, optionally, 147F, or vice versa, and reduces the formation of non-cognate heavy chain-light chain pairs by less than about 15%, less than about 10%, or less than about 5%.

[0074] In yet another aspect, further provided herein are pharmaceutical and diagnostic compositions comprising: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above.

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

[0076] In yet another aspect, further provided herein are nucleic acids encoding: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above.

[0077] In yet another aspect, the present invention further provides a vector comprising a nucleic acid encoding the following or a cell transfected with a nucleic acid encoding the following: (i) a CH1 domain variant polypeptide as described above; (ii) an antibody heavy chain polypeptide as described above; and / or (iii) an antibody or antibody fragment as described above and its use for producing the above.

[0078] In another aspect, the present disclosure provides a method for generating a library of CH1 variant domains, the method comprising steps (a)-(c): (a) providing (i) one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a κ CL domain ("Cκ set"); (ii) one or more sets of polypeptides comprising a CH1 domain paired with a polypeptide comprising a λ CL domain ("Cλ set"); and / or (iii) generating a library of CH1 variant domains comprising a Cκ set; κ Set and / or C λ (b) selecting one or more amino acid positions of the CH1 domain that contact one or more amino acid positions in the κCL domain in the Cκ focus and / or the λCL domain in the Cλ focus; and (c) generating a library of CH1 domain variant polypeptides or a library of CH1 domain variant encoding constructs, wherein one or more amino acid positions of the one or more amino acid positions selected in step (b) are substituted with any non-wild-type amino acid. Optionally, the polypeptide comprising the CH1 domain further comprises a heavy chain variable region (VH), and further optionally, the polypeptide comprising the κ or λCL domain further comprises a light chain variable region (VL).

[0079] Optionally: (I) in step (a), the CH1 domain, the κCL domain and the λCL domain are wild-type and / or human; (II) in step (a), (i) the polypeptide comprising the CH1 domain paired with the polypeptide comprising the κCL domain and (ii) the polypeptide comprising the CH1 domain paired with the polypeptide comprising the λCL domain are both intact antibodies or antigen-binding fragments ("Fab"); (III) in step (b), one or more amino acid positions of the CH1 domain are selected under the following circumstances: the amino acid residue at the one or more amino acid positions of the CH1 domain is between the following: and / or (IV) the generation in step (c) is by degenerate codons, optionally degenerate RMW codons representing six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues.

[0080] In some embodiments, the one or more CH1 amino acid positions selected in step (b) are: (i) located at the interface with a κ CL domain in at least 10% of a representative set of the Cκ set and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the Cκ set; (ii) located at the interface with a λ CL domain in at least 10% of a representative set of the Cλ group and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the Cλ group and / or (iii) located at the interface with a C κ and / or C λ At least 10% of the representative set is at the interface of VH and in C κ and / or C λ The fractional solvent accessible surface area in at least 90% of a representative set of sets is greater than 10%.

[0081] In some embodiments, the amino acid positions selected in step (b) include one or more of positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 according to EU numbering. Optionally, certain CH1 domain variants may be excluded as described above and the CH1 domain variants according to the present invention may meet criteria (a) to (o) as described above.

[0082] In some embodiments, in step (c), the synthetic polypeptide encoding the CH1 variant domain or CH1 domain variant library is expressed in a yeast strain. In some embodiments, the yeast strain is Saccharomyces cerevisiae. In some embodiments, a cell system such as a yeast strain co-expresses (i) one or more polypeptides comprising a κCL domain, such as a κ light chain, and (ii) one or more polypeptides comprising a λCL domain, such as a λ light chain. Optionally, the κ and / or λCL domains are wild-type. Further optionally, the κ and / or λCL domains are human.

[0083] In some embodiments, the methods of the present disclosure further comprise verifying that the one or more substituted CH1 amino acid residues drive preferential pairing of κ light chains or λ light chains. In some embodiments, fluorescence-activated cell sorting is used to verify that the one or more substituted CH1 amino acid residues drive preferential pairing of κ light chains or λ light chains.

[0084] In some embodiments, one or more kappa constant (CK) domains, one or more lambda constant (Cλ) domains, and one or more CH1 domains are wild type. In some embodiments, one or more kappa constant (CK) domains, one or more lambda constant (Cλ) domains, and one or more CH1 domains are human.

[0085] In some embodiments, the method of generating a CH1 domain library comprises steps (a)-(c): (a) selecting one or more CH1 amino acid positions of the following CH1 amino acid positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218; (b) selecting one or more CH1 amino acid positions of interest that are different from the positions selected in step (a); and (c) generating a library of CH1 domain variant polypeptides or a library of CH1 domain variant encoding constructs, wherein one or more of the one or more amino acid positions selected in steps (a) and (b) are substituted with any non-wild-type amino acid. In certain embodiments, the amino acid position selected in (a) can include positions 141, 147, 151, 170, 171, 181, 183, 185, 187 or 218 or any combination thereof. In certain embodiments, the generation in step (c) is by degenerate codons, optionally degenerate RMW codons representing six naturally occurring amino acids (D, T, A, E, K and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues. In certain embodiments, in step (c), the amino acid position selected in step (a) can be substituted with a predetermined amino acid, and the amino acid position selected in (b) is substituted by a degenerate codon. Optionally, the substitution to the predetermined amino acid may include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R or K218P, or any combination thereof.

[0086] In yet another aspect, the present disclosure provides a method for identifying one or more CH1 domain variant polypeptides that preferentially pair with: (A) a polypeptide comprising a κCL domain, as compared to a polypeptide comprising a λCL domain; or (B) a polypeptide comprising a λCL domain, as compared to a polypeptide comprising a κCL domain. Such methods comprise steps (a)-(c): (a) co-expressing one or more candidate CH1 domain variant polypeptides with (i) one or more polypeptides comprising a κCL domain and (ii) one or more polypeptides comprising a λCL domain; (b) comparing the amount of (i) the candidate CH1 domain variant polypeptide paired with the polypeptide comprising a κCL domain to the amount of (ii) the candidate CH1 domain variant polypeptide paired with the polypeptide comprising a λCL domain; (c) based on the comparison in step (b), selecting one or more CH1 domain variants that provide preferential pairing with: (A) a polypeptide comprising a κCL domain, as compared to a polypeptide comprising a λCL domain; or (B) a polypeptide comprising a λCL domain, as compared to a polypeptide comprising a κCL domain. In step (a), the total amount of the candidate CH1 domain variant polypeptide expressed and the total amount of the polypeptide comprising (κ and λ) CL domains expressed may be approximately the same. Optionally, in step (a), the candidate CH1 domain variant polypeptide, the polypeptide comprising a κ CL domain, and the polypeptide comprising a λ CL domain are expressed in a ratio of approximately 2:1:1.

[0087] In some embodiments, in step (a), the (i) one or more polypeptides comprising a κ CL domain and (ii) one or more polypeptides comprising a λ CL domain are wild-type and / or human.

[0088] In some embodiments, in step (b), the amount is determined by fluorescence activated cell sorting or by liquid chromatography-mass spectrometry.

[0089] In some embodiments, the method further comprises step (d): (d) co-expressing one or more control CH1 domain variants with (i) one or more polypeptides comprising a κCL domain and (ii) one or more polypeptides comprising a λCL domain, optionally wherein one or more of the one or more control CH1 domain variants is a CH1 domain variant according to any of those described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1A -C is a schematic diagram of the CH1 domain variant binding to the Cλ domain or the Cκ domain. Figure 1A Heterodimerization of the wild-type CH1 domain with Cλ and Cκ is shown (the wild-type or unmodified CH1 domain is referred to as CH1 WT ). Figure 1B CH1 domain variants that preferentially pair with CK are shown (such CH1 domain variants that preferentially pair with CK are referred to as CH1κ). Figure 1C CH1 domain variants that preferentially pair with Cλ are shown (such CH1 domain variants that preferentially pair with Cλ are referred to as CH1λ).

[0091] Figure 2A and 2B Exemplary FACS plots are shown for multiple rounds of selection to identify proteins with lambda CL domain preference ( Figure 2A ) or κCL domain preference ( Figure 2B ). R1 ​​= first round of selection, R2 = second round of selection, R3 = third round of selection. The x-axis shows the lambda light chain labeled with PE, and the y-axis shows the kappa light chain labeled with FITC.

[0092] Figure 3 Individual unique clones expressing CH1 domain variants with either a λ CL domain preference or a κ CL domain preference are shown. Clones were scored based on the ratio of their anti-κ median fluorescence intensity (MFI) to their anti-λ MFI (κ:λ ratio). The κ:λ ratio of any individual clone was compared to a matched strain with a wild-type CH1 sequence ("parent"). FOP refers to fold increase relative to the parent.

[0093] Figure 4 Individual unique clones expressing CH1 domain variants with amino acid substitutions at positions 141, 147, or 183 are shown (EU numbering). The ratio of anti-κ to anti-λ MFI of the clones was scored and compared to the parent to determine FOP. CH1 positions 147 and 183 were identified as two positions that confer a kappa CL domain preference. CH1 position 141 was identified as a position that confer a lambda CL domain preference.

[0094] Figure 5Specific amino acid substitutions at positions 141, 147, and / or 183 (EU numbering S183K) in the CH1 domain with a λ CL domain preference (A141T, Q, D, or R) or a κ CL domain preference (K147V, A, F, Y, or M) are shown, as measured by the ratio of anti-κ MFI to anti-λ MFI. Amino acid substitutions shown as white dots (V134; T141, V147; A151, and K183) were identified after initial selection from a library with diversity at multiple positions, and amino acid substitutions shown as black dots were identified after additional rounds of selection from a diverse library with targeted positions 141, 147, and 183. Parental κ:λ ratios (wild-type signal): GAL1 CK; GAL10 CK: 3.58 and GAL1 CK; GAL10 CK: 0.3. Parental ratios are the average of experimental replicates. For CH1 variants with substitutions at positions 147 and 183, the first amino acid listed is the variant at position 147, and the second amino acid listed is the variant at 183 (eg, YxF means a CH1 variant with substitutions K147Y and S183F).

[0095] Figure 6A -E shows representative binding data demonstrating that the CH1 domain variants do not alter target binding of the multispecific antibodies (BsAb2-BsAb14) compared to the wild-type CH1 domain (BsAb1 and BsAb15). Figure 6A IL12B and EGFR binding data for BsAbs 1-3 are shown. Figure 6B IL12B and EGFR binding data for BsAbs 5, 7, and 4 are shown. Figure 6C IL12B and EGFR binding data for BsAbs 9, 10, and 6 are shown. Figure 6D IL12B and EGFR binding data for BsAbs 11, 12, and 8 are shown. Figure 6E IL12B and EGFR binding data are shown for BsAbs 13-15. Pani = Panitumumab; Uste = Ustekinumab.

[0096] Figure 7 Shown is an increase in correct heavy chain-light chain pairing (HC1-LC1 or HC2-LC2) and a concurrent decrease in heavy chain-light chain mispairing (HC1-LC2 and HC2-LC1) in bispecific antibodies containing CH1 domain variants (BsAb2-BsAb14) compared to a bispecific antibody containing a wild-type CH1 domain (BsAb1).

[0097] Figure 8Shown are the lambda-biased FOP values ​​for WT clones, A141D clones, and individual clones with different amino acid substitutions at positions 141, 181, and 218 of the CH1 domain obtained from the 141x181x218 library selection output in Example 5. The 13 data points marked in the rectangles correspond to the clones with the highest FOP values, and the amino acid residues at CH1 positions 141, 181, and 218 and the FOP value for each clone are provided in Table 8.

[0098] Figure 9 Shown are the lambda-biased FOP values ​​for WT clones, A141D clones, and individual clones with a D at position 141, a K at position 181, and different amino acid substitutions at position 218 of the CH1 domain in the 141x181x218 library selection output in Example 5. The open data points represent the FOP of individual clones with the same CH1 sequence, and the solid data points represent the average FOP value.

[0099] Figure 10 Shown are the lambda-biased FOP values ​​measured with the recloned clones as well as the WT and A141D clones, confirming that the lambda bias was maintained.

[0100] Figure 11 Shown are exemplary scatter plots of HEK293-produced IgG from CH1 harboring one of the nine 141x181x218 leader sequences selected in Example 5, along with WT and A14D stained for κ and λ CL. The scatter plots for individual clones are overlaid with the WT plot. The x-axis shows the λ light chain labeled with PE, and the y-axis shows the κ light chain labeled with FITC.

[0101] Figure 12 Shown are the lambda-biased FOP values ​​for nine leader sequences and WT and A141 D from Example 5. The three CH1 variants with the highest FOP values ​​(D_K_WT, D_K_P, and D_K_A) were selected for subsequent double-chain (κ or λ) transfection in HEK293.

[0102] Figure 13 The lambda-biased FOP values ​​were compared among CH1 variants having the same amino acid at position 141. When position 141 was D, additional amino acid substitutions at position 181 or at positions 181 and 218 further increased the FOP value.

[0103] Figure 14 Shown are the light chain species (comparing kappa and lambda) % of nine leader sequence full-length IgGs produced in HEK293, as measured by liquid chromatography-mass spectrometry ("LCMS"). Three CH1 variants with the highest FOP values ​​(D_K_WT, D_K_P, and D_K_A) were selected for subsequent transfection in HEK293.

[0104] Figure 15 Exemplary process yields are shown for the three leader sequences (D_K_WT, D_K_P, and D_K_A) and A141D relative to WT, shown as fold-over-parent ("FOP") values.

[0105] Figure 16 Shown are the Tm (°C) of kappa-paired Fabs and lambda-paired Fabs with one of the three lead CH1 variants (D_K_WT, D_K_P, and D_K_A), or A141D or WT.

[0106] Figure 17 Relative λTm (°C) is shown, as defined as: [change in Tm of λ-paired variant Fab relative to λ-paired WT Fab ("ΔλTm")] - [change in Tm of κ-paired variant Fab relative to κ-paired WT Fab ("ΔκTm")].

[0107] Figure 18 Sequencing results from the re-cloning output in Example 6 are provided, visualizing the frequent amino acid substitutions observed in the output clones.

[0108] Figure 19 Shown are the lambda-biased FOP values ​​(λMFI:κMFI) for the leader sequences from the recloning output in Example 6 and some of the 141x181x218 leader sequences (DKP, DKA, KKE, KKP, and EKK) from Example 5, expressed as IgG in yeast. At least seven of the leader sequences marked with arrows had FOP values ​​equal to or higher than the values ​​for the 141x181x218 leader sequences tested.

[0109] Figure 20 The lambda-biased FOP values ​​for 14 leader sequences from Example 7, as well as DKP, A141D, and wild-type identified in Example 5, are shown. Two leader sequences, "A414D_P171E_V185R" and "A141D_F170E_T187R," marked with arrows, showed higher FOP values ​​than DKP. All 14 leader sequences had higher FOP values ​​than the wild-type.

[0110] Figure 21 Shown are exemplary FACS plots comparing the lambda preference of the 14 CH1 domain variants from Example 7 and three controls (DKP, A141D, and wild-type identified in Example 5). The x-axis shows the lambda light chain labeled with PE, and the y-axis shows the kappa light chain labeled with FITC. The numbers in each figure are the ranking # shown in Table 14. For example, the first two figures numbered "1" and "2" are for "A414D_P171E_V185R" and "A141D_F170E_T187R," respectively.

[0111] Figure 22 Shown Figure 21 Exemplary FACS plots overlay of a single plot (labeled "a"), a wild type plot (labeled "b"), and a DKP plot (labeled "c").

[0112] Figure 23 The light chain species (comparing kappa and lambda)% of 14 leader sequences and three control full-length IgGs produced in HEK293 are shown, as measured by LCMS in Example 7. The three controls are shown with open arrows. Compared to the positive control "DKP", "A414D_P171E_V185R" and "A141D_F170E_T187R" (solid arrows) show higher lambda% and lower kappa chain%.

[0113] Figures 24-29 provide exemplary and non-limiting examples of various multispecific antibody structures that can be used with the CH1 domain variants disclosed herein. In Figures 24-29, unless otherwise indicated, the following apply: (1) Each domain is presented as a rectangle with the text indicating the domain name (e.g., CH1, VH1, etc.); (2) Solid rectangles and dotted rectangles are CH1 domain variants with a κ or λ preference, which can be CH1 domain variants disclosed herein; (3) "CH1κ" is a CH1 domain variant with a κCL preference, "CH1λ" is a CH1 domain variant with a λ preference, and "CH1" without a "κ" or "λ" designation is any CH1 domain, wild type or variant, with or without a light chain allotype preference; (4) "Cκ" is a κCL domain, "Cλ" is a λ CL domain, and "CL" without the "κ" or "λ" designation, when shown paired with a solid or dotted CH1 domain, indicates a CL domain of the allotype (κ or λ) preferred by the paired solid or dotted CH1 domain; (5) when more than one solid and / or dotted CH1 domain is present in a multispecific structure, at least one is a CH1 domain variant disclosed herein, and the others may or may not be CH1 domain variants disclosed herein; (6) when both solid and dotted CH1 domains are present in a multispecific structure, the solid and dotted designations indicate CH1 domains with different light chain allotype preferences (i.e., when the solid designation indicates a CH1 domain with a κ preference, the dotted designation indicates a CH1 domain with a κ preference). (7) VH1 and VL1 form an antigen binding site for the first epitope, VH2 and VL2 form an antigen binding site for the second epitope, VH3 and VL3 form an antigen binding site for the third epitope, VH4 and VL4 form an antigen binding site for the fourth epitope, VH5 and VL5 form an antigen binding site for the fifth epitope, and VH6 and VL6 form an antigen binding site for the sixth epitope; (8) All of the first to sixth epitopes may be different from each other, or not all of the first to sixth epitopes may be different from each other, as long as the specific combination as a whole makes the presented structure multispecific; (9) A group of multiple domains connected to each other represents polypeptides (e.g., heavy chain polypeptides, light chain polypeptides, etc.); (10) the orientation of domains within a polypeptide is according to the textual direction of the domain names, from N-terminus to C-terminus; (11) linkers or hinges may be used between domains as needed, and disulfide bonds may be present between polypeptides (and / or within domains), perhaps to allow for proper formation of antigen binding sites, even if the figure does not explicitly show linkers, hinges, or disulfide bonds; (12) CH2 and / or CH3 domains shown in the figures may be omitted where possible and, where appropriate, replaced with hinges; (13) CH1, CH2, and CH3 domains may individually be wild-type or variant and may individually have any (heavy chain) allotype;and (14) when more than one CH1 domain is present in the structure, the CH1 domains may or may not be the same allotype, when more than one CH2 domain is present in the structure, the CH2 domains may or may not be the same allotype, and when more than one CH3 domain is present in the structure, the CH3 domains may or may not be the same allotype.;

[0114] Figures 24A-24C Some exemplary and non-limiting examples of various multispecific antibody structures that can be used with the CH1 domain variants disclosed herein are provided. Figure 24A In one polypeptide, a kappa-preferred CH1 domain ("CH1κ") is used. The other CH1 domain may or may not prefer a lambda CL domain and may or may not be a CH1 domain variant disclosed herein. Figure 24B In one polypeptide, a lambda preference CH1 domain ("CH1λ") is used. The other CH1 domain may or may not prefer a kappa CL domain and may or may not be a CH1 domain variant disclosed herein. Figure 24C In the embodiment, CH1κ is used for one polypeptide and CH1λ is used for one polypeptide. This general structure allows the production of bispecific compounds with no or minimal or less effort to remove mismatched compounds. At least one of the CH1κ and CH1λ domains is a CH1 domain variant disclosed herein. As described above in (10), the domain orientation within the polypeptide is based on the text direction of the displayed domain name from N-terminus to C-terminus. Thus, in Figure 24A In the case of the upper left compound, in the direction from N-terminus to C-terminus, the first polypeptide includes VH1-CH1k-CH2-CH3, the second polypeptide includes VL1-Ck, the third polypeptide includes VH2-CH1-CH2-CH3, and the fourth polypeptide includes VL2-CL. Figures 24A-24C (and all other applicable figures) represent mechanisms that promote heterodimerization of two non-identical polypeptides, such as "knob-in-hole" engineering. Figures 24A-24C (and all other applicable figures) show a hinge structure connecting a CH1κ-containing polypeptide and a CH1λ-containing polypeptide. Although two bonds (e.g., disulfide bonds) are explicitly shown connecting the two polypeptides, the number of bonds and the exact positioning / position of the bonds can vary and be selected appropriately. Figure 24C At the bottom right, “+” indicates a mixture of two different Fab fragments.

[0115] Figures 25A-25B Additional exemplary and non-limiting examples of various multispecific antibody structures that can be used with the CH1 domain variants disclosed herein are provided. Figure 24A-23 C, but with a different order of fields. Figure 25A In , CH1κ and VL are located in the same polypeptide, and CH1λ and VL are located in the same polypeptide. Figure 25B In the figure, Cλ is located in the same polypeptide as CH2 (top three and bottom left), and Cλ is located in a heavy chain-like polypeptide (a polypeptide containing a hinge) (bottom right).

[0116] Figures 26A-26C Additional exemplary embodiments and non-limiting examples of various multispecific antibody structures are provided, which include two sets of two antigen-binding sites in series and are therefore tetravalent. The structure can be bispecific, trispecific, or tetraspecific, depending on what the first, second, third, and fourth epitopes are. For example, if the first, second, and fourth epitopes are different from each other, and if the fourth epitope is the same as the first, second, or third epitope, then the structure will be a tetravalent trispecific structure.

[0117] Figures 27A-27C Additional exemplary and non-limiting examples of various multispecific antibody structures are provided, which are similar to Figures 26A-26C As described above in (10), the domain orientation within a polypeptide is based on the textual orientation of the displayed domain names from N-terminus to C-terminus. Thus, in Figure 27A In the case of the upper left structure, in the direction from N-terminus to C-terminus, the first polypeptide includes VH3-VH1-CH1(solid)-CH2-CH3, the second polypeptide includes VL1-VL3-CL, the third polypeptide includes VH4-VH2-CH1(dot)-CH2-CH3, and the fourth polypeptide includes VL2-VL4-CL. Figures 27A-27C In any structure, appropriate alignments can be used between the domains to achieve appropriate formation of the antigen binding site.

[0118] Figures 28A-28D Additional exemplary and non-limiting examples of various multispecific antibody structures containing at least one scFv are provided. Any of the structures provided in Figures 24-29 can additionally include or be modified to include one or more scFv-containing portions, such as conjugated to any one of the heavy chain constant domain, light chain constant domain, and / or antigen binding domain. For example, Figures 28A-28C Provided Figure 24A The upper left structure in Figure 1 is conjugated to two scFvs, allowing specificity for up to four epitopes. Figure 28A In the scFv is conjugated to the CH3 domain. Figure 28B In the scFv, the CL domain is conjugated. Figure 28C In some cases, more than two scFvs can be conjugated. For example, Figures 28A-28C Provided Figure 24A The upper left structure in Figure 1 is conjugated to four scFvs, allowing specificity for up to six epitopes.

[0119] Figures 29A-29D Still further exemplary and non-limiting examples of various multispecific antibody structures containing two additional Fab fragments are provided. Although two Fab fragments are conjugated to the CH3 domain, it should be noted that the Fab fragments can be conjugated to any other part of the structure, and it should also be noted that one (or three or more than three) rather than two Fab fragments can be conjugated. Figure 29A In the embodiment, the two CH1 domains are in the same polypeptide as the CH2 and CH3 domains. In the intermediate configuration, the kappa-preferring CH1 domain and the lambda-preferring CH1 domain are present in the same polypeptide (for both of the two CH1-containing polypeptides). When the two CH1-containing polypeptides are identical, the configuration facilitates the generation of tetravalent bispecific compounds without the need for mechanisms that promote heterodimerization of two non-identical polypeptides (such as "knob-in-hole" engineering), for example by simply using the three-chain transfection system used in the Examples. Figure 29B In the intermediate structure, when the two polypeptides without CH1 are identical, the structure facilitates the generation of tetravalent bispecific compounds without the need for mechanisms that promote heterodimerization of two non-identical polypeptides (such as "knob-in-hole" engineering), for example by simply using the three-chain transfection system used in the examples. Figure 29C and 29D Each polypeptide contains a CH1 domain. Figure 29C and 29D In an intermediate structure, if the first and third epitopes are the same epitope and the second and fourth epitopes are the same epitope but different from the first and third epitopes, then the structure is bispecific. In such a structure, if the two CH2 / CH3-containing polypeptides are identical, then the structure facilitates the generation of tetravalent bispecific compounds without the need for mechanisms that promote heterodimerization of two non-identical polypeptides (such as "knob-in-hole" engineering), for example, by simply using the three-chain transfection system used in the Examples.

[0120] Figure 30Shown are exemplary process yields normalized to the process yield of WT for intact IgG containing one of the first two λ-preferring CH1 variants identified in Example 7 ("A141D P171E V185R" or "A141D F170E T187R"), the κ-preferring CH1 variant identified in Example 4 ("K147F S183R"), or WT CH1. Striped bars (paired with κ) and solid bars (paired with λ) represent process yields normalized to the corresponding yield of WT. Open diamonds (paired with κ) and solid triangles (paired with λ) represent raw process yields (mg / L).

[0121] Figure 31 Exemplary process yields normalized to the process yield of WT are shown for Fabs containing one of the first two lambda-preferring CH1 variants identified in Example 7 ("A141D P171E V185R" or "A141D F170E T187R"), a lambda-preferring CH1 variant identified in Examples 4 or 5 ("A141D" or "A141D S181KK218P"), a kappa-preferring CH1 variant identified in Example 4 ("K147F S183R"), or WT CH1. Yields are normalized to the corresponding yields of WT. Striped bars represent Fabs containing kappa LC, and solid bars represent Fabs containing lambda LC.

[0122] Figure 32 The wild-type CH1-Cλ interface is shown in its electron density. Representative electron density in the region of interest of the Fab crystal structure of panitumumab variable fragment (Fv) and wild-type IgG1-CH1 paired with the wild-type lambda constant domain (Cλ). Heavy chain (HC) carbon atoms are colored in light gray, lambda light chain (λLC) carbon atoms are colored in white, nitrogen atoms are colored in dark gray, and oxygen atoms are colored in black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown with a 1σ outline. The gray mesh of the carve. The data for this crystal structure extend to Atomic resolution.

[0123] Figure 33 The A141D CHI-Cλ interface is shown in its electron density. Representative electron density in the region of interest of the Fab crystal structure of panitumumab variable fragment (Fv) and A141D substituted IgG1-CH1 paired with the wild-type lambda constant domain (Cλ). Heavy chain (HC) carbon atoms are colored in light gray, lambda light chain (λLC) carbon atoms are colored in white, nitrogen atoms are colored in dark gray, and oxygen atoms are colored in black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown with a 1σ outline. The gray mesh of the carve. The data for this crystal structure extend to Atomic resolution.

[0124] Figure 34 The wild-type CH1-Cκ interface is shown in its electron density. Representative electron density in the region of interest of the Fab crystal structure of panitumumab variable fragment (Fv) and wild-type IgG1-CH1 paired with the wild-type kappa constant domain (Cκ). Heavy chain (HC) carbon atoms are colored in light gray, kappa light chain (κLC) carbon atoms are colored in white, nitrogen atoms are colored in dark gray, and oxygen atoms are colored in black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown with a contour of 0.9σ. The gray mesh of the carve. The data for this crystal structure extend to Near-atomic resolution.

[0125] Figure 35 The K147F-S183R CH1-Cκ interface is shown in its electron density. Representative electron density in the region of interest of the crystal structure of the panitumumab variable fragment (Fv) and the K147F-S183R substituted IgG1-CH1 paired with the wild-type kappa constant domain (Cκ). Heavy chain (HC) carbon atoms are colored in light gray, kappa light chain (κLC) carbon atoms are colored in white, nitrogen atoms are colored in dark gray, and oxygen atoms are colored in black. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown with a contour of 0.9σ. The gray mesh of the carve. The data for this crystal structure extend to Near-atomic resolution.

[0126] Figure 36 A-36D shows that the HC-A141D substitution allows hydrogen bonding with λLC while destabilizing κ pairing through steric hindrance with κLC. Figure 36 A-36D provides a Figure 36 A) Between WT CH1 and κLC ( Figure 36 B) Between A141D CH1 and λLC ( Figure 36 C) or between A141D CH1 and κLC ( Figure 36 D) View of the pairing interface around the HC-Ala141 position. The kappa light chain constant domain (κLC) interface contains the κ light chain constant domain (κLC) shown in Figure 4. Figure 36 The three hydrophobic residues Phe116, Phe118, and Leu135 in B. The presence of Thr116 in λLC at the structurally equivalent position of κLC-Phe116 allows hydrogen bonding with the carboxyl group of HC-Asp141, as indicated by the black dashed line ( Figure 36 C) In Figure 36 In D, the HC alignment of A141D CH1-constant λ (Cλ) and WT CH1-CK shows the steric hindrance of the HC-Asp141 side chain and the κLC-Phe116 side chain. Heavy chain (HC) carbon atoms are colored light gray, light chain (LC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are shown in stick representation with a transparent molecular surface, and main chain atoms are shown in sketch representation.

[0127] Figure 37 A and 37B show that the wild-type CH1 sequence sequesters HC-Gln175 in an intrachain hydrogen bond network, which is likely disrupted by the K147F substitution, allowing HC-Gln175 to freely interact with κLC-Gln160. Figure 37 A and 37B provide views of the ternary hydrogen bond network in the HC, involving the panitumumab wild-type CH1-constant kappa (Cκ) structure ( Figure 37 A) and panitumumab K147F-S183R-CH1-Cκ structure ( Figure 37 B) Lys147, Asp148, and Gln175 in the heavy chain (HC) are colored in light gray, kappa light chain (κLC) carbon atoms are colored in white, nitrogen atoms are colored in dark gray, and oxygen atoms are colored in black. Side chains are shown in stick representation, and main chain atoms are shown in sketch representation. Hydrogen bonds are shown as dashed lines.

[0128] Figure 38 A-38D shows that hydrogen bonding between HC-Arg183 and κ LC-Thr178 can drive κ pairing, while steric hindrance between HC-Arg183 and λ LC-Tyr178 reduces the preference for λ pairing. Figure 38 A-38D provides the region surrounding the S183R substitution in IgG1-CH1 and in the panitumumab wild-type CH1-constant lambda (Cλ) structure ( Figure 38 B) between HC-Ser183 and λLC-Thr178 and in the panitumumab K147F-S183R-CH1-constant kappa (Cκ) structure ( Figure 38 C) View of the hydrogen bond between HC-Arg183 and κLC-Thr178. Figure 38 A shows that HC-Ser183 and κLC-Thr178 are too far apart to hydrogen bond. Heavy chain (HC) carbon atoms are colored light gray, light chain (LC) carbon atoms are colored white, nitrogen atoms are colored dark gray, and oxygen atoms are colored black. Side chains are shown in stick representation. The side chain of λLC-Tyr178 is also shown as a transparent molecular surface. Hydrogen bonds are shown as black dashed lines. Figure 38D provides a model in which the HC of the panitumumab K147F-S183R-CH1-CK structure is superimposed on the HC of the panitumumab wild-type CH1-Cλ structure. The resulting model shows a clear steric hindrance between HC-Arg183 and λLC-Tyr178. DETAILED DESCRIPTION

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about" when used with reference to a particular recited value means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0130] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising" aspects and embodiments, "consisting of" and "consisting essentially of" aspects and embodiments.

[0131] Provided herein are engineered CH1 domains containing at least one amino acid substitution that prevents heavy chain-light chain mispairing by promoting preferential pairing of a heavy chain containing the CH1 domain with a κ CL domain (or κ light chain) or a λ CL domain (or λ light chain). The term "preferential pairing" refers to the pairing of a heavy chain (or CH1 domain) with a polypeptide, such as an antibody, such as a light chain (or CL domain) in a bispecific antibody. When a heavy chain (H1) is co-expressed with two different light chains (L1 and L2), H1 will pair with each of L1 and L2, thereby producing a mixture of H1:L1 and H1:L2. In some cases, H1 can pair well with both L1 and L2, producing a mixture of approximately 50:50 H1:L1 to H1:L2. For example, if the amount of H1:L1 heterodimer formed is greater than the amount of H1:L2 heterodimer formed when H1 is co-expressed with L1 and L2, "preferential pairing" will occur between H1 and L1. In this example, H1 pairs with L1 relative to L2. If H1 inherently favors pairing with L1 over L2 (such that the ratio of H1:L1 to H1:L2 is not 50:50, but rather, for example, 60:40 or 70:30, in which case the formation of H1:L2 is still undesirable), then preferential pairing between the desired pairing, i.e., H1:L1, will occur when the number of pairings between H1:L1 improves (increases) compared to H1:L2. As used herein, the term "preferential pairing" encompasses pairing of heavy and light chains (as described above) as well as pairing of the CHI domain with the CL domain. For example, if the amount of CHI:Cκ formed is greater than the amount of CHI:Cλ formed when CHI is co-expressed with Cκ and Cλ, then "preferential pairing" will occur between the CHI domain and the κ CL domain. Likewise, if the amount of CH1:Cλ formed is greater than the amount of CH1:Cκ formed when CH1 is co-expressed with Cλ and CK, "preferential pairing" will occur between the CH1 domain and the λ CL domain.

[0132] It was found that certain positions within the CH1 domain, identified as part of the CH1-CL interface (for both Cκ and Cλ), affect the binding of the heavy chain to the light chain. In addition, positions within the CH1 domain at the CH1:VH interface have also been shown to affect the binding of the heavy chain to the light chain. The heavy chain pairs with the light chain through two sets of domain interfaces: one between the VH and VL domains, and the other between the CH1 and CL domains, and the region where the chains pair or meet or contact is referred to as an "interface." In addition, within the heavy chain, the CH1 domain also contacts a portion of the VH, and this space where the CH1 domain and VH are in close proximity is also encompassed by the term "interface." The interface includes amino acid residues in the heavy chain and amino acid residues in the light chain, or alternatively, amino acid residues in the CH1 domain and amino acid residues in the VH, which contact each other in three-dimensional space. In some embodiments, the interface includes the CH1 domain of the heavy chain and the CL domain of the light chain. In other embodiments, the interface includes the CH1 domain and the VH domain of the heavy chain. The "interface" is preferably derived from an IgG antibody or Fab thereof.

[0133] The CH1 variant domains described herein contain amino acid substitutions at one or more CH1:CL interface (CH1:κCL or CH1:λCL) positions, such as positions 141, 147, 170, 171, 175, 181, 183, 184, 185, 187, and / or 218, or one or more CH1:VH interface positions, such as position 151, as compared to the parent. The term "parent" refers to a polypeptide (and an amino acid sequence encoding the polypeptide) that is subsequently modified to generate a variant. A parent polypeptide can be a wild-type or naturally occurring polypeptide or a variant or engineered form thereof. Thus, a "parent CH1 domain" refers to a CH1 domain polypeptide (and an amino acid sequence encoding a CH1 domain polypeptide) that is subsequently modified to generate a CH1 domain variant. Such a parent CH1 domain can be a wild-type or naturally occurring CH1 domain or a variant or engineered form thereof, such as a wild-type CH1 domain modified to conjugate a toxin or a small molecule drug. Such a parent CH1 domain can be isolated or part of a larger construct, such as a Fab, F(ab')2 or IgG, which can optionally contain additional modifications, such as CH3 modifications that promote heterodimerization, alter Fc receptor binding, extend half-life and / or link additional binding domains.

[0134] The resulting CH1 variant domain has a preferential pairing with a κ CL (Cκ) domain or a λ CL (Cλ) domain, and the Cκ and Cλ domains can be part of a light chain. Amino acid variations at one or both of CH1 domain positions 147 and 183 (EU numbering) promote binding to Cκ (and simultaneously prevent pairing with Cλ), while amino acid variations at CH1 domain position 141 promote binding to Cλ (and simultaneously prevent pairing with Cκ). The κ and λ CL domains can exist in any number of forms, including but not limited to wild-type or chimeric Fabs or IgGs, such as Fabs or IgGs containing Vκ and Cκ, Vκ and Cλ, Vλ and Cκ, or Vλ and Cλ. By improving the fidelity of heavy chain-light chain pairing while maintaining the native IgG structure of bispecific antibodies, such CH1 variant domains can be used to engineer multispecific antibodies, which are advantageous due to their recognized properties as therapeutic molecules, including long in vivo half-life and the ability to elicit effector functions.

[0135] The term "CH1 domain" refers to the first constant domain of the heavy chain of an antibody, the C-terminus of the variable domain of the heavy chain, and the N-terminus of the hinge region. According to IMGT, the CH1 domain is the amino acid sequence from positions 118-215 (EU numbering), and the hinge region is the amino acid sequence from positions 216-230 (EU numbering). As used herein, the term "CH1 domain variant" refers to an amino acid sequence comprising the entire CH1 domain (positions 118-215 according to EU numbering) or a fragment thereof comprising at least 7 of CH1 residues 118-215 (according to EU numbering), wherein such fragments comprise one or more of the modifications disclosed herein, and a portion of the hinge region (positions 216-218). Libraries screened to identify described CH1 domain variants contain variations in the hinge region, such as positions 216 and 218.

[0136] The CHI domain pairs with the CL domain of the light chain. In some embodiments, the light chain is a κ chain. In some embodiments, the light chain is a λ chain. The terms "κ constant domain," "κ CL domain," or "Cκ" refer to the constant domain of a κ light chain. The terms "λ constant domain," "λ CL domain," or "Cλ" refer to the constant domain of a λ light chain. A single disulfide bond covalently links the CHI domain with the CL domain. As used herein, the CHI domain refers to all antibody allotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE.

[0137] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably those that exhibit the desired antigen-binding activity, which fragments are also referred to as "antigen-binding antibody fragments").

[0138] "Monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations and / or substitutions or arising during the production of the monoclonal antibody preparation), such variants generally being present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0139] 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 or at least two different epitopes. In some embodiments, the multispecific antibody comprises (1) a first heavy chain and a first light chain that form a 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.

[0140] "Bispecific antibodies", which may also be referred to herein as "bispecific compounds", are a type of multispecific antibody and refer to antibodies that include 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 on the same antigen. Bispecific antibodies can target, for example, two different surface receptors, two different cytokines / chemokines, receptors, and ligands on the same or different (e.g., immune cells and cancer cells) cells. Combinations of antigens that can be targeted by bispecific antibodies may include, but are not limited to: CD3 and Her2; CD3 and Her3; CD3 and EGFR; CD3 and CD19; CD3 and CD20; CD3 and EpCAM; CD3 and CD33; CD3 and PSMA; CD3 and CEA; CD3 and gp100; CD3 and gpA33; CD3 and B7-H3; CD64 and EGFR; CEA and HSG; TRAIL-R2 and LTβR; EGFR and IGFR; VEGFR2 and VEGFR3; VEGFR2 and PDGFRα; PDGFRα and PDGFRβ; EGFR and MET; EGFR and EDV-miR16; EGFR and CD64; EGFR and Her2; EG FR and Her3; Her2 domain ECD2 and Her2 domain ECD4; Her2 and Her3; IGF-1R and HER3; CD19 and CD22; CD20 and CD22; CD30 and CD16A; FceRI and CD32B; CD32B and CD79B; MP65 and SAP-2; IL-17A and IL-23; IL-1α and IL-1β; IL-12 and IL-18; VEGF and osteopontin; VEGF and Ang-2; VEGF and PDGFRβ; VEGF and Her2; VEGF and DLL4; FAP and DR5; FcgRII and IgE; CEA and DTPA; CEA and IMP288; and LukS-PV and LukF-PV.

[0141] "Different antigens" can refer to different and / or unique proteins, polypeptides, or molecules; and different and / or unique epitopes that can be contained within a protein, polypeptide, or other molecule. Thus, a bispecific antibody can bind to two epitopes on the same polypeptide.

[0142] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of the antigen bound by an antibody. An epitope may be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and has those residues that directly contribute to the affinity of the interaction. An epitope may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may comprise a determinant grouped as a chemically active surface of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0143] In some cases, antibodies include four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a variable region, such as a heavy chain variable region ("VH") and a heavy chain constant region ("CH"). In the case of a complete antibody, CH includes domains CHI, CH2, and CH3. In the case of an antibody fragment, CH may include CHI, CH2, and / or CH3 domains, and in some preferred embodiments, CH includes at least a CHI domain. The CHI domain variants disclosed herein can be used in combination with wild-type CH2 and / or CH3 domains or CH2 and / or CH3 domains that include one or more amino acid substitutions, such as amino acid substitutions that alter or improve antibody stability and / or effector function. Each light chain includes a variable region, such as a light chain variable region ("VL") and a light chain constant region ("CL"). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Thus, the CDRs in the heavy chain are named "CDRH1," "CDRH2," and "CDRH3," respectively, and the CDRs in the light chain are named "CDRL1," "CDRL2," and "CDRL3." In other cases, the antibody may include a multimer thereof (e.g., IgM) or an antigen-binding fragment thereof.

[0144] In some cases, VH and CL can be present in one polypeptide. In some cases, VL and CH1, CH2 and / or CH3 domains can be present in one polypeptide. For example, in some antibodies or antibody fragments, although the first polypeptide includes VH1 and CH1, and the second polypeptide includes VL1 and CL (VH1 and VL form the antigen binding site of the first epitope), the third polypeptide includes VH2 and CL, and the fourth polypeptide includes VL2 and CH1 (VH2 and VL2 form the antigen binding site of the second epitope). In another certain antibody or antibody fragment, although the first polypeptide includes VH1 and CH1, and the second polypeptide includes VL1 and CL (VH1 and VL form the antigen binding site of the first epitope), the third polypeptide includes VL2, CL, and one or more of the CH2 and / or CH3 domains, and the fourth polypeptide includes VH and CH1. The present invention encompasses any antibody or antibody fragment comprising any of the CH1 variants disclosed herein that provides preferential pairing with kappa CL or preferential pairing with lambda CL, regardless of whether the CH1 domain is in the heavy chain or light chain.

[0145] As used herein, the term "cognate pair" or "cognate pairing" refers to a pair or pairing of two antibody chains (e.g., a heavy chain and a light chain), each containing a variable region (e.g., VH and VL), wherein the combination of the variable regions provides the intended binding specificity to an epitope or antigen. As used herein, the term "non-cognate pair" or "non-cognate pairing" refers to a pair or pairing of two antibody chains (e.g., a heavy chain and a light chain), each containing a variable region (e.g., VH and VL), wherein the combination of the variable regions does not provide the intended binding specificity to an epitope or antigen.

[0146] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (allotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0147] Unless otherwise expressly indicated, the term "antibody" as used herein encompasses molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (sometimes referred to as "full-length antibodies" or "intact antibodies" or "whole antibodies," etc., in all cases referring to antibodies having a structure substantially similar to that of natural antibodies), as well as antigen-binding antibody fragments thereof. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody or a combination of portions derived from an intact antibody or multiple intact antibodies, and binds to the antigen to which the intact antibody or multiple intact antibodies bind.

[0148] Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to: Fv; fragment antigen-binding ("Fab") fragments; Fab' fragments; Fab' containing free sulfhydryl groups ('Fab'-SH'); F(ab')2 fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., single-chain variable fragments ("scFv"), nanobodies, or VHH or only VH or VL domains); and monospecific or multispecific compounds formed from one or more of the aforementioned antibody fragments. In some embodiments, the antigen-binding fragment of the bispecific antibodies described herein is an scFv. In preferred embodiments, the antigen-binding fragment includes a CH1 domain that preferentially pairs with κCL or λCL.

[0149] As with 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 include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or to a different epitope of the same antigen.

[0150] The present disclosure provides CH1 domain variants that preferentially pair with (or bind to) a kappa light chain CL domain or a lambda light chain CL domain. In one embodiment, the CH1 domain variant does not exhibit reduced binding to a kappa class light chain or a lambda class light chain, and at the same time exhibits exclusivity or increased preference for binding to another class of light chain (in this example, lambda or kappa, respectively). These CH1 domain variants can be used to fully or partially resolve heavy chain and light chain mispairing when generating multispecific, such as bispecific antibodies, by promoting appropriate heavy chain and light chain pairing. In one embodiment, the CH1 domain variants can be optionally used in combination with other variants outside of the CH1 domain to further promote preferential pairing with the kappa light chain CL domain or the lambda light chain CL domain (e.g., VH:VL substitutions such as Q39E / K:Q38K / E (Dillon et al., MAbs 2017 9(2):213-230); or Q39K+R62E:Q38D+D1R or Q39Y+Q105R:Q38R+K42D (Brinkmann et al., MAbs 2017 9(2):182-212). More specifically, bispecific antibodies comprising these CH1 variant domains will form fewer unwanted product-related contaminants, i.e., molecules containing mismatched domains, which can be challenging to eliminate during downstream processing. For example, by engineering the heavy chain CH1 domain of antibody A to a κ-preferring CH1 domain variant (e.g., 147Phe and / or 183Arg, Lys, Tyr) and engineering the heavy chain CH1 domain of antibody B to a λ-preferring CH1 domain variant ( For example, 141Asp), bispecific antibodies comprising (i) the heavy chain and light chain from antibody A (wherein the light chain is a kappa light chain) and (ii) the heavy chain and light chain from antibody B (wherein the light chain is a lambda light chain) may be produced more efficiently, i.e., with fewer unwanted product-related contaminants. Thus, the heavy chain of antibody A will favor binding to the light chain of antibody A (and disfavor binding to the light chain of antibody B), while the heavy chain of antibody B will favor binding to the light chain of antibody B (and disfavor binding to the light chain of antibody A). See Figures 1 and 7 and Table 6.

[0151] In some embodiments, the CH1 domain variants reduce the formation of mispairing, i.e., non-cognate HCl-LC2 and / or HC2-LC1 pairs by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, CH1 domain variants containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 147F+183R, 147F+183K, 147F+183Y, reduce the formation of mispairing, i.e., non-cognate HCl-LC2 and / or HC2-LC1 pairs by at least 25% to at least 80%. In some embodiments, CH1 domain variants containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduce the formation of mismatches, i.e., non-cognate HCl-LC2 and / or HC2-LC1 pairs, by at least 50%. In some embodiments, CH1 domain variants containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduce the formation of mismatches, i.e., non-cognate HCl-LC2 and / or HC2-LC1 pairs, by at least 75%.

[0152] In some embodiments, the CH1 domain variants preferentially pair with (bind to) a cognate CL domain (CK or CL) or a cognate light chain containing a corresponding CL domain (CK or CL) resulting in at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% formation of the desired first and second cognate pairs, i.e., HC1-LC1 and / or HC2-LC2. In some embodiments, the CH1 domain variants preferentially pair with (bind to) a cognate CL domain (Cκ or Cλ) or a cognate light chain containing a corresponding CL domain (Cκ or Cλ), resulting in about 80% to about 99% or more specifically, at least about 85% to at least about 95% formation of the desired first and second cognate pairs, i.e., HCl-LC1 and / or HC2-LC2. In some embodiments, CH1 domain variants containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, provide about 85% to at least about 95% formation of the desired first and second cognate pairs, i.e., HCl-LC1 and / or HC2-LC2.

[0153] In some embodiments, the CH1 domain variants reduce the formation of mispaired heavy chain-light chain heterodimers, i.e., HC1-LC2 and / or HC2-LC1 pairs, to less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, CH1 domain variants containing a substitution at position 141, e.g., 141D, alone or in combination with other substitutions, e.g., 183R, 183K, 183Y, 147F+183R, 147F+183K, 147F+183Y, reduce the formation of mispaired heavy chain-light chain heterodimers to less than about 15%, less than about 10%, or less than about 5%.

[0154] Several CH1 domain positions were identified as influencing light chain binding preference, i.e., preferentially pairing with a kappa CL domain or a lambda CL domain, including positions 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218 (EU numbering). Replacing the wild-type amino acid residue at any one or more of these positions in the CH1 domain with a variant (non-wild-type) amino acid residue generates a heavy chain that has preferential pairing for a light chain containing a kappa CL domain or a lambda CL domain. For example, positions 147 and 183 were each identified as having a pairing preference for the kappa CL domain, and positions 141, 170, 171, 175, 181, 184, 185, 187, and 218 were identified as having a pairing preference for the lambda CL domain.

[0155] Substitution of the wild-type amino acid residue at position 141 of the CH1 domain (Ala) with Thr, Asp, Lys, Glu, Arg, Met, Val or Gln has been shown to increase the preference of the heavy chain for binding to a light chain containing a λCL domain. Substitution of the wild-type amino acid residue at position 170 of the CH1 domain (Phe) with Glu, Gly, Ser, Asn or Thr; substitution of the wild-type amino acid residue at position 171 of the CH1 domain (Pro) with Glu, Gly, Ser, Asn, Asp or Ala; substitution of the wild-type amino acid residue at position 175 of the CH1 domain (Met) with Asp or Met; substitution of the wild-type amino acid residue at position 181 of the CH1 domain (Ser) with Val, Leu, Ala, Lys or Thr; substitution of the wild-type amino acid residue at position 184 of the CH1 domain (Ser) with Arg. ); substitution of the wild-type amino acid residue at position 185 of the CH1 domain (Val) with Met, Leu, Ser, Arg, Thr; substitution of the wild-type amino acid residue at position 187 of the CH1 domain (Thr) with Arg, Asp, Glu, Tyr or Ser; and / or substitution of the wild-type amino acid residue at position 218 of the CH1 domain (Lys) with Leu, Glu, Asp, Pro, Ala, His, Ser, Gln, Asn, Thr, Ile, Met, Gly, Cys, Lys or Trp also helps to increase pairing of the heavy chain with the light chain containing the λCL domain.

[0156] Substitution of the wild-type amino acid residue (Lys) at position 147 of the CH1 domain with Val, Ala, Phe, Ile, Thr, Ser, Tyr, Leu, Arg, Asn, Glu, His, Met, or Gln showed increased heavy chain preference for binding to light chains containing a kappa CL domain. Substitution of the wild-type amino acid residue (Ser) at position 183 of the CH1 domain with Arg, Lys, Tyr, Trp, Glu, Phe, Ile, Leu, Asn, or Gln showed increased heavy chain preference for binding to light chains containing a kappa CL domain (see Figure 5 ). The effect of a given variant amino acid residue at a particular position may vary, but all variants show improved preferential pairing with either CK or Cλ based on the amino acid position comprising the variant residue. In addition, given the high similarity in the CH1 region of IgG1, IgG2, IgG3, and IgG4, it is expected that the CH1 domain variants described herein will display similar preferential pairing characteristics within each allotype.

[0157] The first round of selection identified Thr at position 141 as promoting preferential pairing with Cλ compared to the wild-type CH1 domain sequence (Ala at position 141), but additional rounds of selection identified Asp, Arg, and Gln as providing increased preferential pairing compared to Thr (see Figure 5 Additional screening strategies identified Lys and Glu as also providing increased λ preference (see Example 5, Figure 10-14). It was also found that Glu at position 170; Glu at position 171; Met at position 175; Lys at position 181; Arg at position 184; Arg at position 185; Arg at position 187; and / or Pro, Ala or Glu at position 218 increased lambda preference (see Examples 5-7). In addition, the applicant showed that specific substitution combinations that increase lambda preference include, but are not limited to: Asp at position 141 and Lys at position 181; Asp at position 141, Lys at position 181 and Ala at position 218; Asp at position 141, Lys at position 181 and Pro at position 218; Glu at position 141, Glu at position 170, Val at position 181 and Arg at position 187; Glu at position 141, Asp at position 171 and Arg at position 185; Gl at position 141 u, Glu at position 171 and Arg at position 185; Glu at position 141, Gly at position 171, Arg at position 185 and Arg at position 187; Glu at position 141, Arg at position 185 and Arg at position 187; Glu at position 141, Ser at position 171 and Lys at position 181; Glu at position 141, Gly at position 170, Met at position 175, Val at position 181, Arg at position 184 and Arg at position 187. In additional screening work, "Asp at position 141, Glu at position 171 and Arg at position 185" and "Asp at position 141, Glu at position 170 and Arg at position 187" were identified as particularly lambda-preferred CH1 domain substitution combinations (see Figure 20 、 23 , 30 and 31).

[0158] Similarly, compared to the wild-type CH1 domain sequence, the first round of selection identified Val or Ala at position 147 and Lys at position 183 as promoting preferential pairing with CK, but additional rounds of selection identified Phe, Ile, Thr, Tyr, Leu, Arg, Asn, Glu, His, Met, or Gln at position 147 and / or Arg, Tyr, Trp, Glu, Phe, or Gln at position 183 as providing increased preferential pairing compared to 147Val or Ala or 183Lys, respectively. These CH1 domain variants, alone or in combination with other amino acid substitutions, can improve the preferential pairing of heavy chains containing such CH1 domain variants with light chains containing CK or CK.

[0159] Provided herein are variant CH1 domains comprising amino acid substitutions at one or more of the following positions according to EU numbering, and thus, the CH1 domain variants exhibit preferential pairing with CK or Cλ (or light chains comprising such domains): 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218. As demonstrated herein, substitutions of different amino acid residues at one or more of these positions can produce CH1 domains that preferentially pair with CK or Cλ (see Tables 3 and 4). In some embodiments, the amino acid substitution at position 147 (EU numbering) is not cysteine. In some embodiments, the amino acid substitution at position 183 (EU numbering) is not cysteine ​​or threonine. In some embodiments, the amino acid substitution at position 147 (EU numbering) is not cysteine ​​and the amino acid substitution at position 183 (EU numbering) is not cysteine ​​or threonine.

[0160] In some embodiments, the CH1 domain variant comprises an amino acid substitution at one or more of the following positions to drive preferential pairing of the CH1 domain variant (or a heavy chain comprising such a domain) with a CK (or a light chain comprising such a domain): 118, 124, 126-129, 131-132, 134, 136, 139, 143, 145, 147-151, 153-154, 170, 172, 175-176, 181, 183, 185, 190-191, 197, 201, 203-206, 210, 212-214, and 218 (EU numbering). In some embodiments, the amino acid substitution is one or more of: position 118 is substituted with G; position 124 is substituted with H, R, E, L, or V; position 126 is substituted with A, T, or L; position 127 is substituted with V or L; position 128 is substituted with H; position 129 is substituted with P; position 131 is substituted with A; position 132 is substituted with P; position 134 is substituted with G; position 136 is substituted with E; position 139 is substituted with I; position 143 is substituted with V or S; position 145 is substituted with F, I, N, or T; position 147 is substituted with F, I, L, R, T, S, M, V, E, H, Y, or Q; position 148 is substituted with I, Q, Y, or G; position 149 is substituted with C, S, or H; position 150 is substituted with L or S; position 151 is substituted with A or L substituted with S; position 153 is substituted with S; position 154 is substituted with M or G; position 170 is substituted with G or L; position 172 is substituted with V; position 175 is substituted with G, L, E, or A; position 176 is substituted with P; position 181 is substituted with Y, Q, or G; position 183 is substituted with I, W, F, E, Y, L, K, Q, N, or R; position 185 is substituted with W; position 190 is substituted with P; position 191 is substituted with I; position 197 is substituted with A; position 201 is substituted with S; position 203 is substituted with S; position 204 is substituted with Y; position 205 is substituted with Q; position 206 is substituted with S; position 210 is substituted with R; position 212 is substituted with G; position 213 is substituted with E or R; position 214 is substituted with R; and position 218 is substituted with Q. In some embodiments, the CH1 domain variants include amino acid substitutions at positions 147 and 183 to drive preferential pairing (binding to) with a kappa light chain. In some embodiments, the amino acid substituted at position 147 is selected from the group consisting of: F, I, L, R, T, S, M, V, E, H, Y, and Q, and wherein the amino acid substituted at position 183 is selected from the group consisting of: I, W, F, E, Y, L, K, Q, N, and R. In specific embodiments, the CH1 domain variant comprises R or K or Y at position 183 alone or in combination with F at position 147. Non-limiting examples of kappa-preferring CH1 domain variants can include the amino acid sequence of SEQ ID NO: 137, 138, 139, 60, 41, or 136.

[0161] In some embodiments, the CH1 domain variant comprises amino acid substitutions at one or more of the following positions to drive preferential pairing of the CH1 domain variant (or a heavy chain comprising such a domain) with Cλ (or a light chain comprising such a domain): 119, 124, 126-127, 130-131, 133-134, 138-142, 152, 163, 170-171, 175, 181, 183-185, 187, 197, 203, 208, 210-214, 216, and 218 (EU numbering). In some embodiments, the amino acid substitution is one or more of the following: position 119 is substituted by R; position 124 is substituted by V; position 126 is substituted by V; position 127 is substituted by G; position 130 is substituted by H or S; position 131 is substituted by Q, T, N, R, V or D; position 133 is substituted by D, T, L, E, S or P; position 134 is substituted by A, H, I, P, V, N or L; position 138 is substituted by R; position 139 is substituted by A; position 140 is substituted by I, V, D, Y, K, S, W, R, L or P; position 141 is substituted by D, T, R, E, K, Q, V or M, preferably D, E or K; position 142 is substituted by M; position 152 is substituted by G; position 163 is substituted by M; position 168 is substituted by F, I or V; position 170 is substituted by N, G, E, S or T, preferably position 171 is substituted by N, E, G, S, A, D, preferably D, E, G or S; position 175 is substituted by D or M, preferably M; position 181 is substituted by V, L, A, K or T, preferably K or V; position 183 is substituted by L or V; position 184 is substituted by R; position 185 is substituted by M, L, S, R or T, preferably R; position 187 is substituted by R, D, E, Y or S; position 197 is substituted by S; position 203 is substituted by D; position 208 is substituted by I; position 210 is substituted by T; position 211 is substituted by A; position 212 is substituted by N; position 213 is substituted by E; position 214 is substituted by R; position 216 is substituted by G; and position 218 is substituted by P, A, L, E, D, H, S, Q, N, T, I, M, G, C, K or W, preferably P or A. In some embodiments, the CH1 domain comprises an amino acid substitution at residue 141 to drive preferential pairing with a lambda light chain. In some embodiments, the amino acid substituted at residue 141 is selected from the group consisting of: T, R, E, K, V, D, and M. In specific embodiments, the CH1 domain variant comprises Asp or Glu at position 141. In some embodiments, the amino acid substitution at position 141 can be combined with one or more substitutions within CH1, such as Lys at position 181 or Lys at position 181 and Ala or Pro at position 218.Asp or Glu at position 141 can be combined with one or more substitutions at positions 170, 171, 175, 181, 184, 185 and / or 187, such as Glu or Gly at position 170, Asp, Glu, Gly or Ser at position 171, met at position 175, Val or Lys at position 181, Arg at position 184, Arg at position 185 and / or Arg at position 187. Non-limiting examples of lambda-preferring CH1 domain variants can include the amino acid sequence of SEQ ID NO: 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 155, 157, 159, 162, 163, 164, 165, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or 189.

[0162] In specific embodiments, the CH1 domain variant comprises a combination of 141D, 181K and 218P, a combination of 141D, 171E and 185R, or a combination of 141D, 170E and 187R. In further embodiments, the CH1 domain variant comprises the amino acid sequence of SEQ ID NO: 188, 186 or 143.

[0163] The present disclosure also contemplates polypeptides, such as antibodies, comprising CH1 domain variants. Such polypeptides may be multispecific antibodies comprising a first heavy chain comprising a first CH1 domain variant and a second heavy chain comprising a second CH1 domain variant. The first heavy chain and the second heavy chain may bind to different epitopes. In some embodiments, the antibody comprises a first heavy chain comprising a first CH1 domain. In some embodiments, the antibody further comprises a second heavy chain comprising a second CH1 domain, wherein the second CH1 domain comprises an amino acid sequence different from that of the first heavy chain CH1 domain.

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

[0165] In some embodiments, the first CH1 domain variant may preferentially pair with (bind to) Cλ, and the second CH1 domain may preferentially pair with (bind to) Cκ. In this case, the first light chain includes a Cλ domain, and the second light chain includes a Cκ domain. In some embodiments, the first light chain is a lambda light chain (Cλ and Vλ) or a chimeric light chain (Cλ and Vκ), and the second light chain is a kappa light chain (Cκ and Vκ) or a chimeric light chain (Cκ and Vλ).

[0166] The first light chain and the second light chain may include (or may not include) amino acid substitutions that drive preferential pairing with the CH1 domain. In some embodiments, the CL domain of the light chain is not modified to change binding to the heavy chain, such as the CH1 domain. In some embodiments, the first light chain contains a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain. In some embodiments, the second light chain contains a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain. The wild-type κ light chain or Cκ domain can be encoded by IGKC. The wild-type λ light chain or Cλ domain can be encoded by IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7.

[0167] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. Such multispecific and bispecific antibodies may include any form containing a CH1 domain, such as, but not limited to, the structures depicted in Figures 24-29. See also, for example, Brinkmann and Kontermann, MAbs 9(2):182-212 (2017), Table 2, which is incorporated herein by reference in its entirety.

[0168] The multispecific antibodies may include one or more of the CH1 domain variants having the amino acid sequences listed in Tables 3, 4, 7, 9, 12, or 13. In some embodiments, the antibodies include a first heavy chain and a first light chain comprising a first CH1 domain variant, wherein the first heavy chain and the first light chain form a first cognate pair. The first CH1 domain variant may include amino acid substitutions at one or more of the following positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218. Such first CH1 domain variants preferentially bind to the first light chain. The CL domain of the first light chain may or may not be modified to alter binding to the first heavy chain.

[0169] In some embodiments, the antibody further includes a second heavy chain and a second light chain containing a second CH1 domain variant, wherein the second heavy chain and the second light chain form a second homologous pair. The second CH1 domain variant may include amino acid substitutions at one or more of the following positions according to EU numbering: 118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, 218. Such second CH1 domain variants preferentially bind to the second light chain. The CL domain of the second light chain may or may not be modified to alter its binding to the second heavy chain.

[0170] In certain embodiments of a multispecific antibody or antibody fragment, the antibody or antibody fragment may include a κ-preferring CH1 domain variant and a λ-preferring CH1 domain variant. In some cases, the κ-preferring CH1 domain variant may be a κ-preferring CH1 domain variant as disclosed herein, and the λ-preferring CH1 domain may be a λ-preferring CH1 domain that may or may not be described herein. In some cases, the λ-preferring CH1 domain variant may be a λ-preferring CH1 domain variant as disclosed herein, and the κ-preferring CH1 domain may or may not be a κ-preferring CH1 domain that may or may not be described herein. In some cases, both the κ-preferring CH1 domain variant and the λ-preferring CH1 domain variant are variants as disclosed herein.

[0171] Any of the CH1 domain variants disclosed herein can be used to provide a pairing preference for a kappa CL domain or a lambda CL domain, and the CL domain can be wild type or non-wild type. In addition, any of the CH1 domain variants disclosed herein can be used to provide a kappa / lambda pairing preference in an antibody or antibody fragment structure with or without introducing further amino acid changes to the rest of the antibody structure, such as CH2, CH3, VH, VL or CL domains. For example, the CH1 domain variants disclosed herein can be used with VH substitutions that can further enhance light chain pairing preferences (e.g., VH:VL substitutions such as Q39E / K:Q38K / E (Dillon et al., MAbs 2017 9(2):213-230); or Q39K+R62E:Q38D+D1R or Q39Y+Q105R:Q38R+K42D (Brinkmann et al., MAbs 2017 9(2):182-212).

[0172] Without wishing to affect the scope of the present invention, it is emphasized that the CH1 domain variants provided herein provide a kappa / lambda pairing preference in the context of a wild-type light chain (or a polypeptide comprising a wild-type CL domain) without requiring another modification in the CH2, CH3 or variable domain, but such non-CH1 modifications can optionally be used in combination with the novel CH1 domain variants discovered by the inventors herein. This is particularly surprising given the many reported failures in generating antibodies, particularly multispecific antibodies, in which modification of the CH1 domain alone provides a meaningful kappa or lambda preference.

[0173] In some embodiments, the antibody is part of a pharmaceutical composition. Such a composition may contain multiple polypeptides, such as antibodies, that include the CH1 domain variants described herein.

[0174] The present disclosure also contemplates methods for obtaining such CH1 domain variants. The variant CH1 domains described herein can be identified by rational design (in silico) or can be identified randomly, for example using ePCR or other mutagenesis techniques known in the art. In one embodiment, a rational design approach is employed to design variant CH1 domains. For such methods, a set of structures, such as experimentally derived protein structures, such as Fab crystal structures, can be assembled and analyzed to identify solvent-exposed positions that involve contacts across the CH1-CL domain interface (also referred to as CH1-CL domain interface positions). The group can be curated by selecting structures with certain properties, such as a high percentage identity to a reference (wild type) CH1, Cκ, and Cλ. In some embodiments, if a pair of side chain atoms in A position is described or defined as contacting another residue (or being "in contact") if it is within a cutoff distance of . A "CH1 interface residue" can be defined as a residue in a CH1 domain that contacts a residue in a Cκ domain or a Cλ domain. In this context, the terms "residue" and "position" can be used interchangeably. The inventors have also unexpectedly discovered that amino acid substitutions at CH1 positions in the CH1-VH interface (e.g., CH1 position 151) alter light chain allotype preference. Thus, in some embodiments, CH1 positions that contact VH residues (e.g., a pair of side chain atoms in The cutoff distance of 1 was used for reasonable CH1 domain variant identification.

[0175] The selection of amino acid positions to be varied, either individually or in combination (e.g., singlets, doublets, triplets, etc.), can depend on various parameters, such as the consistent role of the position in forming the interface between CH1 and CL or between CH1 and VH in different structures, the accessibility of the position in the overall structure, the relationship of the position to positions that affect antigen binding, or the likelihood that the residue affects the formation of the CH1:CL or CH1:VH interface in an allosteric manner without directly participating in intermolecular contacts across the interface. In some embodiments, an amino acid residue in the CH1 domain is selected for variation if: 1) the residue is located at the interface with the light chain constant domain in at least 10% of the structures in the Cκ set and has a fractional solvent accessible surface area (SASA) greater than 10% in at least 90% of the structures in the Cκ set (see Example 1), or 2) the residue is located at the interface with the light chain constant domain in at least 10% of the structures in the Cλ set and has a fractional SASA greater than 10% in at least 90% of the structures in the Cλ set, or 3) the residue is located at the interface with the C κ and / or C λ At least 10% of the representative sets are at the interface of VH and in C κ and / or C λ The fractional solvent accessible surface area in at least 90% of a representative set of sets is greater than 10%.

[0176] In addition, for each of the specific amino acid substitutions provided herein to confer a κ or λ preference in the CH1 domain, the amino acids included as a result of the substitution can be further substituted by conservative amino acid substitutions to obtain another CH1 domain variant that provides an equivalent κ or λ preference. Alternatively, for each CH1 domain variant, one or more amino acid positions that are not affected in the CH1 domain variant relative to the wild-type sequence can be changed by conservative substitutions to obtain another CH1 domain variant that provides an equivalent κ or λ preference.

[0177] "Conservative amino acid substitutions" are known in the art and encompass amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of 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.), substitutions of an amino acid with a beta-branched side chain for another amino acid with a beta-branched side chain (e.g., Ile, Thr, and Val), substitutions of 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.

[0178] Next, a library in which the CH1 domain residues vary can be generated. One or more CH1 domain residues can vary in the library. In certain embodiments, about one to six CH1 domain residues vary in the library. The amino acid diversity at each residue position can be generated by a degenerate codon such as NNK, to allow at least all 20 naturally occurring amino acids to be represented at a given CH1 domain position. The selected CH1 domain position can be individually varied to generate a point substitution (also referred to as a single peak), or a subset of position combinations can be combined to vary, for example, to generate double substitutions and triple substitutions (also referred to as doublets and triplets). In certain embodiments, a variant combination is generated, the variant combination being included in adjacent CH1 domain positions in 3D space, for example, position 147x [124, 126, 145, 148, 175, and 181].

[0179] In some embodiments, a method of preparing a CH1 domain variant library comprises: a) providing a set of structures comprising one or more kappa constant (CK) domains, one or more lambda constant (Cλ) domains, and one or more CH1 domains; b) selecting to replace one or more solvent-exposed CH1 domain positions that are in contact with one or more CK domain positions and / or one or more Cλ domain positions; c) substituting the one or more CH1 domain positions identified in step b) with any amino acid other than the parent amino acid; and d) synthesizing polypeptides encoding the CH1 variant domains of step c) to assemble the CH1 variant domain library.

[0180] In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are wild-type. In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are human (including allele functional variants). In some embodiments, the Cκ amino acid sequence in step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In specific embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions across the CH1-CL interface or the CH1-VH interface.

[0181] In some embodiments, the one or more CH1 amino acid residues selected for substitution (i) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cκ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of CH1:Cκ structures; (ii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of CH1:Cλ structures; or (iii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures. κ and / or C λ At least 10% of the representative set of structures are at the interface of VH and in C κ and / or C λ At least 90% of a representative set of structures have a fractional solvent accessible surface area greater than 10%.

[0182] In some embodiments, a library is generated by altering one or more CH1 positions disclosed herein as altering light chain allotype preference (e.g., positions 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof), and optionally one or more additional CH1 positions of interest. In certain embodiments, a library can be generated by combining predetermined substitutions at one or more CH1 positions disclosed herein as altering light chain allotype preference (e.g., positions 141, 147, 151, 170, 171, 181, 183, 185, 187, or 218, or any combination thereof), with one or more additional CH1 positions of interest. In particular examples, the predetermined substitutions can include A141D, A141E, K147F, P151A, P151L, F170E, P171E, S181K, S183R, V185R, T187R, or K218P, or any combination thereof.

[0183] In some embodiments, the library is screened to identify CH1 domain variants that exhibit preferential binding to a kappa light chain or a lambda light chain. Such screening can begin by expressing the library in a suitable host cell, e.g., a eukaryotic cell, e.g., a yeast cell, e.g., a Saccharomyces cerevisiae cell. Following expression of the CH1 variant domains contained in the library in the host cell, the library of variants can be screened, e.g., by FACS or MACS, to identify those variants with the desired binding properties.

[0184] In some embodiments, methods for identifying CH1 domain variants with preferential CK or Cλ domain binding comprise: a) providing a set of structures comprising one or more κ constant (CK) domains, one or more λ constant (Cλ) domains, and one or more CH1 domains; b) selecting to replace one or more solvent-exposed CH1 domain positions that are in contact with one or more CK domain positions and / or one or more Cλ domain positions; c) substituting the one or more CH1 domain positions identified in step b) with any amino acid other than the parent amino acid; d) synthesizing polypeptides encoding the CH1 variant domains of step c) to assemble a CH1 variant domain library; and e) screening the library of d) to identify CH1 domain variants with preferential CK or Cλ domain binding.

[0185] In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are wild-type. In some embodiments, one or more Cκ domains, one or more Cλ domains, and one or more CH1 domains are human (including allele functional variants). In some embodiments, the Cκ amino acid sequence in step a) is encoded by IGKC. In some embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In specific embodiments, the Cλ amino acid sequence in step a) is encoded by IGLC2. In some embodiments, the resulting CH1 domain library is designed to require interactions across the CH1-CL interface or the CH1-VH interface.

[0186] In some embodiments, the one or more CH1 amino acid residues selected for substitution (i) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cκ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of CH1:Cκ structures; (ii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures and have a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of CH1:Cλ structures; or (iii) are located at the interface with the light chain constant domain in at least 10% of a representative set of CH1:Cλ structures. κ and / or CH1:Cλ At least 10% of the representative set of structures are at the VH interface and at the CH1:C κ and / or CH1:C λ At least 90% of a representative set of structures have a fractional solvent accessible surface area greater than 10%.

[0187] The methods described herein may further include verifying that one or more substituted CH1 amino acid residues drive preferential pairing of the heavy chain relative to the λCL domain (or a light chain comprising a λCL domain) for the κCL domain (or a light chain comprising a κCL domain), and vice versa. Preferential light chain pairing can be assessed using a variety of methods, including but not limited to fluorescence activated cell sorting (FACS), LC-MS, αLISA, and SDS-PAGE. In some embodiments, the one or more CH1 domain positions selected for substitution in step c) occur at the interface of the light chain with a predetermined frequency, for example, in any given set of wild-type antibody structures, the selected CH1 domain position contacts the CL domain in at least 10% of the structures. In some embodiments, the one or more CH1 domain positions selected for substitution in step c) have a fractional solvent accessible surface area greater than about 10% in at least about 90% or more of the structures in any given set of Cκ or Cλ. In some embodiments, the one or more CH1 domain positions selected for substitution in step c) occur at the interface of a VH region with a predetermined frequency, e.g., in any given set of wild-type antibody structures, the selected CH1 domain position contacts VH in at least 10% of the structures.

[0188] By employing the methods described herein for identifying CH1 domain variants, the following CH1 domain positions were selected for substitution: 114, 116, 118, 119, 121-124, 124-143, 147-154, 160, 162-165, 167, 168, 170-172, 174, 175, 176, 178, 180, 181, 183-185, 187, 190, 191, 197, 201, 203-208, 210-214, 216, and / or 128 (according to EU numbering). Substituting any one or combination of these CH1 domain positions can result in a CH1 domain having preferential pairing for a particular CL domain. Thus, heavy chains comprising such CH1 domain variants and light chains comprising specific CL domains are more likely to form cognate pairs, i.e., there is preferential pairing between heavy chains and light chains that form cognate pairs that is driven at least in part by one or more CH1 domain substitutions.

[0189] In one embodiment, the CH1 domain variant preferentially pairs with CK, thereby driving preferential pairing of a light chain containing a CK domain and a heavy chain containing the CH1 domain variant. In another embodiment, the CH1 domain variant preferentially pairs with a Cλ domain, thereby driving preferential pairing of a light chain containing a Cλ domain and a heavy chain containing the CH1 domain variant. Certain exemplary CH1 domain substitutions are identified as promoting preferential heavy chain pairing with a κ light chain, such as 147F and / or 183R, 183K, or 183Y, while other CH1 domain substitutions are identified as promoting preferential heavy chain pairing with a λ light chain, such as 141D, 141E, 141K, 170E, 170G, 171E, 171D, 171G, 171S, 175M, 181K, 181B, 184R, 185R, 187R, 218A, or 218P. Thus, bispecific antibodies comprising such CH1 domain variants can generate improved fidelity in heavy chain-light chain pairing. In some embodiments, the bispecific antibody comprises a first heavy chain comprising CH1λ (e.g., 141D, 141E, or 141K and 170E, 170G, 171E, 171D, 171G, 171S, or 175M and / or 181K, 181B, 184R, 185R, 187R, 218A, and / or 218P) and a second heavy chain comprising CH1κ (e.g., 147F and / or 183R, 183K, or 183Y), each of which preferentially pairs with its cognate light chain. In some embodiments, the bispecific antibody contains a first heavy chain comprising CH1κ (e.g., 147F and / or 183R, 183K, or 183Y) and a second heavy chain comprising CH1λ (e.g., 141D, 141E, or 141K and 170E, 170G, 171E, 171D, 171G, 171S, or 175M and / or 181K, 181B, 184R, 185R, 187R, 218A, and / or 218P), e.g., "141D, 171E, and 185R" or "141D, 170E, and 187R," each of which is preferably paired with its cognate light chain.

[0190] Polypeptides encoding CH1 variant domains obtained using the methods described herein can be recombinantly expressed in host cells, such as eukaryotic cells. In some embodiments, the CH1 variant domains are expressed in yeast. In some embodiments, the yeast strain is Saccharomyces cerevisiae. In some embodiments, the yeast strain co-expresses one or more wild-type kappa light chains and one or more wild-type lambda light chains.

[0191] The examples provided below are intended to illustrate the present invention. These examples are not intended to constrain the present invention to any particular application or theory of operation.

[0192] Examples

[0193] Example 1: In silico selection of CH1 domain positions for diversification in a library

[0194] A set of Fab crystal structures was assembled from the Protein Data Bank (PDB) and used in a structure-guided approach to identify CH1-CL interface residues for diversification.

[0195] An initial set of 2,367 Fab crystal structures was narrowed down by selecting structures with a high percentage identity to the reference (wild-type) CH1, CK, and Cλ sequences (shown below). The reference sequence for CH1 alignment spans the appropriate CH1 (EU residues 118-215) plus a portion of the IgG1 hinge (EU residues 216-229). The CK and Cλ reference sequences span EU residue numbers 108-214 and 107A-215, respectively.

[0196] CH1 (plus top to middle hinge) reference:

[0197] ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (SEQ ID NO: 1).

[0198] Cκ reference:

[0199] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0200] Cλ reference:

[0201] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3).

[0202] If a pair of side chain atoms Residues are defined as being in “contact” if they are within a cutoff distance of . CH1 interface residues are defined as those residues that make contact with one or more Cκ or Cλ residues in a single structure.

[0203] The solvent accessible surface area (SASA) of individual heavy and light chain residues was calculated in the "free state," i.e., without pairing with a light or heavy chain, respectively. Fractional SASA was defined as the ratio of the residue SASA to the Gly-X-Gly tripeptide isolated from the model incorporating the same amino acid as the residue (i.e., X). Solvent-exposed residues were defined as those with a fractional SASA greater than 10%.

[0204] Narrowing down the initial set of crystal structures by high percentage identity resulted in the identification of a set of 183 CH1: CK structures (the "CK set") and 43 CH1: CK structures (the "Cλ set"). After accounting for gaps in the alignment due to missing amino acids in the structures, all entries in the CK set had 100% identity with the reference CH1 and CK sequences, while entries in the Cλ set had >99% identity with the reference sequences.

[0205] A structure-based sequence alignment between CK and Cλ is shown below. CH1 forms a stable interface with both CK and Cλ, despite low sequence identity between the latter domains. Conservative and semiconservative substitutions are depicted using "|" and ":", respectively, according to BLOSUM62 scoring. The sequence identity between the domains is 38.3% (41 identical residues out of 107 CK residues).

[0206] Cκ:-RTVAAP S V F I FP P SDEQ LK S G T A S V V C L L NN FYPREAKVQWKVDNALQSGNS Q E S V T

[0207] AAPSV|FPPS:E|L:::A::VCL|::FYP:V WK:D:::::|::

[0208] Cλ:GQPKAAPSV T L FP P SSEE LQ A N K A T L V C L I SD FYPGAVTVAWKADSSPVKAGV E T TT P

[0209] Cκ:EQ D SKDSTY S L S S T L T LSKADYEKHKVYACEVTHQGLSSPVT K SFNRG E C-(SEQ ID NO:2)

[0210] :::S::Y:SS L:L::|::H|Y:C|VTH|GS|VK:EC

[0211] Cλ: S K QS N-NKY A A S S Y L S LTPEQWKSHRSYSCQVTHEG—-STVE K TVAPT E CS (SEQ ID NO: 3)

[0212] The underlined amino acids represent Cκ and Cλ residues that contact the CH1 domain. This determination is based on the consensus on Fab structures focused on Cκ and Cλ. There are 25 Cκ interface residues and 26 Cλ interface residues. A 2×2 matrix was constructed that focused on positions at the interface of either Cκ or Cλ (N=28) and depended on (1) whether the residue at a given position contacts CH1 and (2) whether the amino acid at that position is identical between Cκ and Cλ (see Table 1).

[0213] Table 1. Cκ and Cλ amino acid positions at the CH1:CL interface

[0214]

[0215] Table 1 highlights a set of 14 structurally conserved CK and Cλ positions, i.e., the same EU residue numbering but different amino acid identities, that contact the CH1 domain. Table 2 lists the 14 amino acid positions (EU numbering) and indicates the amino acids present in the κ and λ light chains. Such differences in the identity of the CK and Cλ interface residues can be exploited to generate mutant CH1 domains that specifically bind only to CK or Cλ, but not both.

[0216] Table 2. Structurally conserved CH1 contact positions with non-identical amino acid residues for CK and Cλ

[0217]

[0218]

[0219] As an initial threshold for selecting library variants, a single CH1 domain position was required to meet the following criteria: 1) the position is located at the interface with the light chain constant domain in at least 10% of the structures of the Cκ set, and the residue at the position has a fractional SASA greater than 10% in at least 90% of the structures of the Cκ set; or 2) the position is located at the interface with the light chain constant domain in at least 10% of the structures of the Cλ set, and the residue at the position has a fractional SASA greater than 10% in at least 90% of the structures of the Cλ set; or 3) the position is located at the interface with the VH region in at least 10% of the structures of the CH1: Cκ set (Cκ set) or the CH1: Cλ set (Cλ set), and the residue at the position has a fractional SASA greater than 10% in at least 90% of the structures of the Cκ and / or Cλ set. The interface definition takes into account contacts between CH1 residues and any CL domain residues, including but not limited to the set of fourteen CL domain residues listed in Table 2, or contacts between CH1 residues and any VH residues.

[0220] Based on this threshold criterion, a group of 30 CH1 amino acid positions were identified for potential inclusion (after ignoring Cys220). From this larger group, a group of 25 CH1 positions were selected to vary in the library. The amino acid diversity at each position was generated by representing the degenerate NNK codons of all 20 natural amino acids (Stemmer et al., Proceedings of the National Academy of Sciences of the United States of America on October 25, 1994; 91 (22): 10747-51). Amino acid substitutions were performed separately at each of the 25 CH1 positions, and the subsets of the single substitutions were selectively combined, for example, to generate double mutants and triple mutants. The final library design consists of 89 CH1 oligonucleotides representing 25 single peaks (NNK codon diversification at a single CH1 position), 48 doublet mutants (NNK codon diversification at two CH1 positions), and 16 triplet mutants (NNK codon diversification at three CH1 positions).

[0221] Example 2: CH1 domain variant library in yeast co-expressing CK and Cλ light chains

[0222] A library of human CH1 domain variants was constructed and expressed in an engineered yeast strain co-expressing wild-type human IgG CK and CL light chains (at varying expression levels to allow subsequent selection for CL-favoring and CK-favoring CH1 substitutions).

[0223] Bidirectional expression plasmids (pAD7064 and pAD4800) were constructed, each containing the Saccharomyces cerevisiae Gal1 / Gal10 promoter region flanked by wild-type human IgG light chain kappa and lambda constant domains, and the Saccharomyces cerevisiae URA3 gene (selectable marker). Plasmids pAD7064 and pAD4800 differ in the orientation of the kappa and lambda constant domains relative to the Gal1 / 10 promoter region. Unique restriction enzyme sites (PME-I and SFI-I) were placed upstream of the kappa and lambda constant domains in each plasmid. pAD7064 and pAD4800 were digested with PME-I and SFI-I, respectively, and then transformed into engineered yeast strains along with PCR-amplified DNA inserts (ADI-26140 light chain region; Gal1 / 10 promoter region; and differentially encoded ("degenerate") ADI-26140 light chain variable region (IDT gblock) assembled into the plasmid by homologous recombination with 5' and 3' ends. The transformed yeast were plated on solid agar plates lacking URA3+ and grown at 30°C for 48 hours, after which colonies were picked and the DNA extracted and purified. After sequencing, two dual light chain DNA constructs were identified: (1) Gal10::ADI-26140-VL-CκGal1::ADI-26140-VL-Cλ (human Cλ under the control of a dominant promoter, allowing subsequent selection of Cκ-favoring CH1 substitutions); and (2) Gal10::ADI-26140-VL-CλGal1::ADI-26140-VL-Cκ (human Cκ under the control of a dominant promoter, allowing subsequent selection of Cλ-favoring CH1 substitutions). ADI-26140 is an anti-hen egg lysozyme (HEL) IgG.

[0224] For heavy chain expression, a DNA vector (pAD4466) was constructed containing the Gal1 promoter, an SFI-I ​​restriction site, the CH2-CH3 domain of human IgG heavy chain (IgG1(N297A)), and TRP1 (selectable marker).

[0225] In parallel, two independent pools of CH1 domain variant DNA fragments were generated for insertion into pAD4466. The first pool was generated using the computer design method described in Example 1. The second pool was generated by error-prone PCR (ePCR). Briefly, the mutagenic nucleotide analogs dPTP (0.01 mM) and 8-oxo-DGTP (0.01 mM) were included in the PCR reaction at dilutions of (a) 1:100 and 1:100 or (b) 1:100 and 1:10, respectively.

[0226] pAD4466 was digested with SFI-I ​​and introduced into yeast strains expressing CK and Cλ, along with PCR-amplified DNA encoding the ADI-26140 HC variable region and CH1 domain variant DNA from rational design work or ePCR. Each DNA fragment had appropriate DNA sequences at the 5' and 3' ends to guide assembly (by homologous recombination) with the digested plasmid or PCR fragment (ADI-26140 heavy chain variable region or CH1 protein domain).

[0227] Individual libraries were assembled by native Saccharomyces cerevisiae homologous recombination. Dilutions of transformed cells from each library were plated on medium lacking uracil and tryptophan to quantify the number of members in each library. The number of members in each library was greater than 10. 7 The remainder of the transformed cells were cultured in liquid medium lacking uracil and tryptophan to select for the presence of each (HC and double LC) plasmid.

[0228] Example 3: Identification of CH1 domain positions that affect light chain binding

[0229] Libraries were generated as previously described (see, e.g., WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70). Briefly, after induction and presentation of IgG, yeast cells (approximately 10^7-10^8) were stained with 1:100 dilution of goat anti-human F(ab')2κ-FITC (Southern Biotech, Birmingham, Alabama, catalog number 2062-02) and 1:100 dilution of goat anti-human F(ab')2λ-PE (Southern Biotech, Birmingham, Alabama, catalog number 2072-09) in PBSF at 4°C for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL PBSF and transferred to a filter-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and the sorting gate was determined to (1) increase λ light chains with a corresponding loss of κ light chains ( Figure 2A ) or (2) an increase in kappa light chains with a corresponding loss of lambda light chains ( Figure 2B After three rounds of selection, yeast were plated on medium lacking uracil and tryptophan to generate single isolates for sequence identification.

[0230] Individual clones expressing unique sequences were cultured in 96-well plates. After induction and presentation of IgG, approximately 2 × 10 6 Yeast cells were stained with goat anti-human F(ab')2κ-FITC (Southern Biotechnology, Birmingham, AL, catalog number 2062-02) and goat anti-human F(ab')2λ-PE (Southern Biotechnology, Birmingham, AL, catalog number 2072-09) diluted 1:100 in PBSF for 15 minutes at 4°C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.1 mL of wash buffer and evaluated on a BD FACS Canto instrument equipped with a 96-well plate processor. The ratio of the anti-κ median fluorescence intensity (MFI) to the anti-λ MFI (κ:λ ratio) of individual unique clones was calculated. Figure 3 ) were scored and then compared with a matched strain with a wild-type CH1 sequence (“parent”) to calculate FOP.

[0231] The following CH1 domain positions (EU numbering) were identified as influencing light chain binding preference, i.e., preferential binding to a kappa CL domain (or a light chain containing a kappa CL domain) or a lambda CL domain (or a light chain containing a lambda CL domain): 118, 119, 124, 126-134, 136, 139-141, 143, 145, 147-154, 163, 168, 170-172, 175-176, 181, 183, 185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218. Table 3 provides a list of CH1 sequences identified from a selection of preferred kappa light chains. The bold amino acid residues in the sequence column indicate the substitution position, i.e., the amino acid substitution that differs from the parent (SEQ ID NO: 1). A list of CH1 sequences identified from a selection of prioritized lambda light chains is provided in Table 4. Bold amino acid residues in the sequence columns indicate the positions of substitutions.

[0232] Table 3. CH1 domain sequences that preferentially bind to Cκ

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] Table 4. CH1 domain sequences that preferentially bind to Cλ

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Δ indicates amino acid deletion.

[0248] Surprisingly, it was found that some CH1 amino acid substitutions located at the VH:CH1 interface rather than at the CH1:light chain interface produced a kappa binding preference in the three-chain system. Specifically, the mutation groups K147V+P151A and P151L+N201S (SEQ ID NOs: 36 and 70, Table 3) returned kappa FOP values ​​of 18.1 and 10.4, respectively. Although position CH1:147 is located at the CH1:LC interface, CH1:201 is not (it is fully solvent exposed and is not part of any interdomain interface); therefore, the appearance of P151 substitutions in these high FOP clones suggests a potential role for this position in determining the kappa to lambda preference. Without wishing to be bound by theory, for reasons discussed below, such distal mutations are believed to affect HC:LC pairing, and therefore mutations at the VH:CH1 interface may be utilized for preferred kappa to lambda pairing.

[0249] First, P151 is part of the so-called "ball-and-socket joint" between the VH and CH1 domains (Lesk AM et al., Nature 1988 Sep 8; 335(6186): 188-90; Landolfi NF et al., J Immunol 2001 Feb 1; 166(3): 1748-54). This joint has been hypothesized to regulate intradomain flexibility through its influence on the "elbow angle" between the variable and constant domains of antibodies (Stanfield RL et al., J Mol Biol. 2006 Apr 14; 357(5): 1566-74). Substitutions in the ball-and-socket joint can have functional consequences, as in the case of anti-IFN-γ monoclonal antibodies with reduced neutralizing activity due to single amino acid substitutions in this region (Landolfi NF et al., J Immunol 2001 Feb 1; 166(3): 1748-54). This effect is attributed to the flexibility and allosteric mechanism of the change, rather than the direct change at the antigen binding interface. Secondly, it is also known that Fab with λ constant domain has a wider range of elbow angles relative to Fab with κ domain (Stanfield RL et al., Journal of Molecular Biology April 14, 2006; 357 (5): 1566-74.doi:10.1016 / J.jmb.2006.01.023.2006 January 25 electronic publication). This super flexibility is attributed to the insertion of a single residue in the so-called conversion zone between the VL and CL domains. Third, further analysis of the Fab crystal structure (Adimab unpublished data) reveals the difference between κ and λ Fab of the atomic packing in the ball joint region. Therefore, the regulation of Fab flexibility by the ball joint, together with the inherent difference between Fab with κ and λ light chains, shows a new mechanism for deriving difference κ and λ preferences by mutations at the VH:CH1 interface.

[0250] Example 4: Identification and Characterization of CH1 Domain Variants with Kappa-Preferred or Lambda-Preferred Light Chain Pairing

[0251] Based on the MFI ratio between κ and λ, clones resulting from selections with increasing CK and Cλ preferences were selected for further characterization (see Figure 4). Separate and amplify the DNA pool that mutates to each of the 20 amino acids (NNK) at each position of interest (141, 147 or 183). Use appropriate light chain alkaline strains to construct these single position targeted libraries in a manner as previously described. Construct four libraries in the case where there is a variation at position 141, 147, 183 or 147+183 of the CH1 domain. Carry out the selection of κ or λ preference as described above. As previously described, the output is sequenced, and the κ or λ preference based on FACS is quantified relative to the appropriate parent to determine the amino acid substitution that provides light chain κ or λ preferential pairing.

[0252] Several CH1 domain variants having amino acid residue substitutions at each of positions 141, 147, and 183 were identified as having pairing preferences for either a κCL domain (or a light chain containing a κCL domain) or a λCL domain (or a light chain containing a λCL domain). Substitution of D, R, or Q (compared to wild-type A) at CH1 domain position 141 increased the preferential pairing (i.e., decreased κ:λ MFI ratio) with a λCL domain (or a light chain containing a λCL domain) (see Figure 5 At CH1 domain position 147, substitution with F, I, T, Y, L, R, N, E, H, M, or Q (compared to wild-type K) increases preferential pairing with a κ CL domain (or a light chain comprising a κ CL domain) (i.e., increased κ:λ MFI ratio) (see Figure 5 Substitutions at CH1 domain position 183 to R, K, Y, W, E, F, or Q (compared to wild-type S) increase preferential pairing with a κCL domain (or a light chain containing a κCL domain) (i.e., increased κ:λ MFI ratio) (see Figure 5 ). Table 5 shows the number of observed CH1 domain variants with specific amino acid substitutions driving pairing preferences.

[0253] Table 5. Amino acid substitutions observed in CH1 domain variants with light chain preference

[0254] Amino acid substitution Observation count Light chain preference A141D 35 λ A141R 7 λ A141Q 5 λ K147F 24 κ K147I 5 κ K147T 3 κ K147Y 3 κ K147L 2 κ K147R 2 κ K147N 2 κ K147E 1 κ K147H 1 κ K147M 1 κ K147Q 1 κ S183R 19 κ S183K 11 κ S183Y 5 κ S183W 3 κ S183E 2 κ S183F 1 κ S183Q 1 κ

[0255] Next, the effects of the identified CH1 domain variants were evaluated on a control standard bispecific antibody (2 heavy chains × 2 light chains) in an IgG-like format (2 Fab regions connected to a dimeric Fc molecule at the N-terminus). VH-CH1 sequences derived from two approved clinical therapeutic antibodies: ustekinumab and panitumumab were used. 'Knob' (S354C; T366W) and 'hole' (Y349C; T366S; L368A; Y407V) mutations were introduced to promote the desired heterodimer pairing of the heavy chains. DNA plasmids were confirmed by Sanger sequencing before transfection into HEK293 cells using standard protocols.

[0256] Transfected HEK cells were cultured in CD optiCHO medium (Invitrogen) and on day 6 post-transfection, supernatants were collected and subjected to protein A-based affinity purification. Purified IgG was treated with ELISA (Genevis AB) to enzymatically cleave the Fab region from the Fc portion.

[0257] The purified Fab was subjected to LCMS to confirm the sequence of each IgG component (2 heavy chains x 2 light chains) and to determine the relative percentage of each component (see Figure 7 Briefly, purified IgG was digested with GingisKHAN to enzymatically cleave the Fab region from the Fc portion. The Fab sample was injected into a polyclonal antibody (Applied Biosystems) maintained at 65°C. The HPLC was performed on an Agilent 1100 series HPLC with an R2 10 μm column (2.1×30 mm, 0.1 mL). After injection, the sample was eluted from the column using a 0.21 minute gradient of 2-95% acetonitrile at a flow rate of 2 mL / min (mobile phase A: H2O containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid). A diverter valve was used to load a total flow of 150 μL / min into a Bruker maXis 4G mass spectrometer. The mass spectrometer was operated in positive ion mode with an m / z range of 700 to 2500. The remaining source parameters were set as follows: the capillary was set to 5500 V, the nebulizer was set to 4.0 bar, the drying gas was set to 4.0 L / min, and the drying temperature was set to 200°C. The acquired MS spectra were analyzed using Bruker Compass data analysis version 4.1. The detection of intact Fab species was confirmed based on mass measurements compared to the theoretical sequence. The relative quantification of each species was calculated based on the intensity of each species' peak compared to the sum of all peak intensities.

[0258] When both heavy chains are wild type, mispairing occurs approximately 30% of the time; however, when the heavy chain includes a CH1 variant domain as described herein, there is a significant improvement in correct pairing of the heavy and light chains (see Figure 7 and Table 6). The Pani light chain is wild type. The Uste light chain is a lambda fusion. HC1 is pani; LC1 is paniκ; HC2 is uste; LC2 is usteλ. For example, when the first heavy chain (HC1) contains K147F and S183R / K / Y and the second heavy chain contains A141D (BsAbs 10, 12, and 14, respectively), mispairing is at least reduced by half, occurring only 6.8, 10.5, or 11% of the time. In fact, a single substitution at position 141 (141D) results in a 50% reduction in mispairing, i.e., 6.1% versus 3.1% HC1-LC2 and 22.8% versus 9.9% HC2-LC1 (BsAb2). Based on this, Applicants provide exemplary CH1 domain sequences with a kappa or lambda light chain / CL domain preference in Table 7.

[0259] Table 6. Percentage of heavy chain-light chain product formation

[0260]

[0261]

[0262] Table 7. CH1 domains with kappa or lambda chain preference

[0263]

[0264] The expression and quality of the purified antibodies were assessed by size exclusion chromatography (SEC). In brief, Agilent 1100HPLC was used to monitor column chromatography (TSKgel Super SW3000 column). The column was pre-treated with highly glycosylated and aggregated IgG to minimize the possibility of antibody-column interaction and balanced with wash buffer (200mM sodium phosphate, 250mM sodium chloride pH 6.8) before use. Approximately 2-5 μg protein samples were injected onto the column, and flow rate was adjusted to 0.400 ml / min. Protein migration was monitored at a wavelength of 280nm. The total assay time was approximately 11 minutes. ChemStation software was used for data analysis. The SEC pattern confirmed that the CH1 domain replacement had no effect on the variant pattern compared to the wild type (data not shown).

[0265] The binding affinity and kinetics of the purified bispecific antibodies binding to human IL-12B (Uste) and human EGFR (Pani) were measured to confirm that the CH1 variant domain did not affect target binding (see Figures 6A-6E ). Using a 100 nM antigen The bispecific IgG samples were captured on a QKe instrument (ForteBio) on an anti-hIgG Fc sensor tip and the binding kinetics to IL12B or EGFR were measured (association rate: 180 seconds and off-rate: 180 seconds). BLI analysis was performed at 29°C using 1× kinetic buffer (ForteBio) as the assay buffer. The anti-human IgG Fc capture (AHC) biosensor (ForteBio) was first pre-soaked in assay buffer for more than five minutes. The bispecific IgG sample (5 μg / mL) was captured on the sensor for 300 seconds. The sensor was then soaked in assay buffer for 120 seconds to establish a baseline before measuring binding to IL12B or EGFR protein (100 nM concentration). The dissociation of IL12B or EGFR was measured by moving the sensor into the assay buffer for 180 seconds. The agitation for all steps was 1000 rpm. Reference subtraction, dissociation-based inter-step correction, a 1 to 1 binding model and a global fit (Rmax not connected by the sensor) were used. Kinetic parameters were generated using the Data Analysis Software version 8.2.0.7. Association rate constants (ka), dissociation rate constants (kd), and equilibrium constants (Kd) were assigned individually for each measurement. D )value.

[0266] Example 5: 141×181×218 library construction and selection

[0267] Additional CH1 amino acid substitutions that provide preferential pairing with the λCL domain were also identified. Based on previous selection data and structural analysis, a set of three CH1 positions (141, 181, and 218) were selected for additional variation. The amino acid diversity at position 141 was generated by the degenerate codon RMW representing six naturally occurring amino acids (D, T, A, E, K, and N). The amino acid diversity at positions 181 and 218 was generated by the degenerate codon NNK representing all 20 naturally occurring amino acids. The library design contains all possible combinations of amino acids at these three positions with a diversity of 2,400. Using a light chain strain with a λ light chain under the GAL10 promoter (GAL1::ADI-26140VL-Ck x GAL10::ADI-26140VL–Cl), this library was constructed as previously described. λ preference selection was performed by staining with anti-human κ-FITC and anti-human λ-PE antibodies, followed by multiple rounds of cell sorting, as previously described. The output (96 clones) was sequenced as previously described, and the FACS-based lambda preference was quantified relative to the parental strain. Wild type ("WT") and previously identified lead clone A141D were included in the analysis. Based on these data, the amino acid combination and A141D that provided the greatest improvement in light chain lambda preference relative to the parent were identified.

[0268] Figure 8 It shows that most of the output clones have a higher preference in pairing with the lambda chain as determined by the FOP value. Table 8 provides the CH1 domain substitutions and Figure 8 FOP values ​​of the λ:κ MFI ratios for the top 13 clones marked in .

[0269] Table 8. Top 13 FOP values ​​from output clones

[0270] Amino acid residues at positions 141, 181, and 218 FOP EIL 7.34 KKE 6.84 EKP 6.44 KLD 5.76 KKP 5.54 KKA 5.49 KKE 5.25 KKP 5.03 KKH 4.99 EKD 4.98 KKP 4.96

[0271] Analysis showed that substitutions at position 141 to D, K, or E, and at position 218 to L, E, D, P, A, H, S, Q, N, T, I, M, G, C, or W, paired with a substitution at position 181 to K, were frequent in the output clones and increased lambda light chain preference (increased lambda:κ MFI ratio) relative to A141D. Figure 9 Shown are individual and average FOP values ​​measured in clones with D at position 141, K at position 181, and various amino acids at position 218 of CH1. The leader CH1 sequence was cloned back into the LC stain (this process was subsequently employed in all assays) and the clones and lambda preferences were confirmed by calculating the FOP values ​​in triplicate ( Figure 10 ).

[0272] Additional analysis generated nine unique candidate CH1 sequences for mammalian IgG production (see Table 9).

[0273] Table 9. CH1 domains with kappa or lambda chain preference

[0274]

[0275]

[0276] 9 candidate CH1 sequences were cloned into mammalian expression vectors along with WT (ie, "ASK") and A141D (ie, "DSK") by standard methods. To determine the λ preference, plasmids expressing the desired heavy chain, λ light chain, and κ light chain were transfected into HEK293 cells at a 2:1:1 plasmid ratio. The transfected HEK cells were cultured and IgG was purified using a previously described protocol. Without wishing to be bound by theory, expressing approximately equal amounts of total heavy and light chain polypeptides (HC:κLC:λLC="2:1:1" here resulting in total HC:total LC=1:1) (ie, no excess HC and no excess LC) appears to have allowed the inventors to avoid various biases, resulting in visualization of the true κ or λ preference of the CH1 domain variants.

[0277] FACS-based quantification of lambda preference of mammalian-produced IgG. Figure 11 FACS plots are provided and Figure 12 Table 10 provides the FOP values ​​(λ:κ MFI) for the nine CH1 variants as well as WT and A141D (ie, "DSK"). Figure 13 It is shown that when CH1 has a D at position 141, additional substitutions at position 181 or positions 181 and 218 further improve the lambda preference (based on the lambda:kappa MFI ratio).

[0278] Table 10. FOP values ​​of 9 CH1 variants

[0279] CH1 substitutions (at 141, 181, and 218) FOP D_K_P 4.33 D_K_A 3.83 D_K_WT 3.57 K_WT_WT 2.24 E_WT_WT 2.04 K_K_WT 1.82 E_K_WT 1.70 D_WT_WT 1.61 K_K_P 1.24 K_K_E 1.18 WT_WT_WT 1.00

[0280] Additionally, LCMS data of reduced full-length IgG were used to determine the relative amounts of lambda and kappa light chains in the purified IgG samples. Figure 14 The % of species that paired with kappa light chains (LC) and the % of species that paired with lambda light chains were compared.

[0281] Analysis of these data yielded three CH1 sequences (SEQ ID NOs: 143, 142, and 141, with DKP, DKA, and DKK substitutions, respectively) with improved lambda preference relative to the parental and previously identified leader sequence A141D.

[0282] To determine whether these CH1 sequences pair with κ light chains, candidate CH1 heavy chain plasmids were transfected into HE293 cells with 1.) κ light chain or 2.) λ light chain. K147F S183R, WT, A141D, which are CH1 with κ preference, were also included as controls. The transfected HEK cells were cultured and purified by standard methods. Linked heavy chain Fab and light chain Fab were generated from purified IgG using the method described previously. The process yield was determined using standard methods and normalized to the WT process yield to calculate the "FOP" process yield. Based on the process yield FOP, when only κLC (but not λLC) was present, A141D, A141D S181K, A141D S181K K218A, and A141D S181K K218P all still bound to κLC, but more binding occurred in λLC compared to κLC ( Figure 15 The Fab Tm of κ- and λ-Fab was measured by differential scanning fluorimetry using a BioRad CFX96 RT PCR. Figure 16 For each CH1 variant, the relative gain in Tm of the λ-paired Fab ("relative λTm gain" or "net λTm gain") was calculated as defined by: [change in Tm of the λ-paired variant Fab relative to the λ-paired WT Fab ("ΔλTm")] - [change in Tm of the κ-paired variant Fab relative to the κ-paired WT Fab ("ΔκTm")] ( Figure 17 ).like Figure 17 As shown, at S181 or S181 and K218, the relative λTm gain increases with additional substitutions. Without wishing to be bound by theory, based on Figure 16 and 17 , destabilization of κLC pairing appears to contribute to the relative λTm gain and increase in pairing with λCL.

[0283] Example 6: 141×ALL library construction and selection

[0284] When paired with the substitution at position 141, build other libraries to sample the other residues in CH1 to drive λ to preferentially combine. Six new libraries (LAD11522-LAD11527) are designed to have up to three replacements (table 11) in three districts (DOR1, DOR2 and DOR3) across CH1. Six libraries together represent each possible replacement set, and the set comprises two replacements in three paid attention to domains paired with position 141. In all libraries, the amino acid diversity at position 141 is generated by degenerate codon RMW, and the amino acid diversity at other two variant positions is generated by degenerate codon NNK. Use previously described method to build libraries. As previously described, carry out the selection of λ preference.

[0285] Table 11. Library design and construction

[0286]

[0287] Starting after the second round of FACS selection, the selection output CH1 diversity was isolated and recloned into an appropriate double-stranded light chain strain to restore the reduced kappa light chain expression in the library. CH1 diversity was isolated using PCR amplification with appropriate primers and standard DNA purification. This DNA fragment pool was then electroporated with the ADI-26140 heavy chain variable region and the plasmid digested into an appropriate double-stranded light chain strain.

[0288] As previously described ( Figure 18 ) The output was sequenced and the FACS-based lambda preference was quantified relative to the parental strain. The previously identified lead clone A141D S181K K218P was included in the analysis. Based on these data, the amino acid combination with the greatest improvement in light chain lambda preference relative to the parental strain was determined.

[0289] The first 46 clones containing 28 unique CH1 sequences (Table 12) were expressed as IgG in yeast. The new CH1 sequences were compared with some of the leader sequences from the 141×181×218 series (DKP, DKA, KKE, KKP, and EKK) from Example 5 and the FOP values ​​determined by flow cytometry (λMFI:κMFI) ( Figure 19 ). corresponds to Figure 19 Of the data points marked with arrows, at least seven having CH1 sequences SEQ ID NO: 155, 157, 159, 162, 163, 164, or 165 showed FOP values ​​equal to or higher than the value of the tested 141×181×218 leader sequence.

[0290] Table 12. 28 unique CH1 sequences with lambda preference from 141×ALL sequences

[0291]

[0292]

[0293]

[0294] Example 7: 141×(170 / 171)×(185 / 187) series of constructs and screening

[0295] Analysis of the results in Example 6 yielded four new positions / residues of interest, including F170, P171, V185, and T187. Based on the frequently observed amino acids at positions 170, 171, 185, and 187, as well as 141 that produced high FOP values ​​in previous studies (e.g., frequent E and D at position 141; frequent E at position 170 or 171 in the 141×ALL output; and frequent R at positions 185 and / or 187 when position 141 was substituted and independently substituted by position 171), 14 unique CH1 domain variants (Table 13) with a maximum of three amino acid substitutions per CH1 domain were rationally designed as candidates for the leading lambda-preferred substitution group. The 14 leader sequences in Table 13 include "A141E"; V185R; T187R" (SEQ ID NO: 163) and "A141E; P171E; V185R (SEQ ID NO: 159)", which were tested in Example 6.

[0296] Table 13. New CH1 sequences from the 141×(170 / 171)×(185×187) series

[0297]

[0298]

[0299] As described above, a heavy chain containing one of the 14 CH1 domain variant sequences was cloned into mammalian (HEK) cells that co-express kappa and lambda light chains (heavy chain (HC): lambda light chain (LC): kappa LC ratio = 2:1:1, i.e., the HC:LC ratio was always 1:1). Wild-type (ADI-26140 heavy chain), "A141D" variant, and "A141D_S181K_K218P" variant were also included as controls. The same assay as described above was used to determine lambda preference.

[0300] The λMFI to κMFI ratio was assessed by flow cytometry. Figure 20-22 The FOP values ​​and individual FACS plots for the 14 leader sequences are provided in Table 14 (the numbers in each figure are the ranking # shown in Table 14). Among the 14 leader sequences, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed even higher FOP values ​​than the leader sequence "A141D_S181K_K218P" identified in Example 5. Many other variants of the 14 leader sequences also showed higher FOP values ​​compared to "A141D", and all 14 leader sequences showed higher FOP values ​​compared to the wild type.

[0301] Table 14. FOP values ​​of 14 CH1 variant leader sequences and controls (ranking based on FOP value)

[0302]

[0303]

[0304] The amount of kappa and lambda LC was quantified for each sample using LCMS (Table 15 and Figure 23 ). Similar to the results of the FACS-based λ preference assessment, "A141D_P171E_V185R" and "A141D_F170E_T187R" showed even higher λ chain % and even lower κ chain % compared to the leader sequence "A141D_S181K_K218P" identified in Example 6. Many other variants among the 14 leader sequences also showed higher λ% and lower κ% compared to "A141D", and all 14 leader sequences showed higher λ% and lower κ% compared to the wild type.

[0305] Table 15. λ LC% and κ LC% measured by LCMS (with FOP values ​​in Table 14)

[0306] Substitution in CH1 %κLC %λLC score A141D_P171E_V185R 4％ 96％ 4.71 A141D_F170E_T187R 6％ 94％ 3.29 A141D_S181K_K218P 9％ 91％ 2.90 A141E_V185R_T187R 10％ 90％ 2.30 A141E_P171E_V185R 10％ 90％ 2.29 A141D_F170E_V185R 15％ 85％ 2.18 A141D_V185R_T187R 15％ 85％ 2.11 A141E_F170E_T187R 12％ 88％ 2.00 A141D_V185R 18％ 82％ 1.76 A141E_V185R 20％ 80％ 1.70 A141D_P171E_T187R 19％ 81％ 1.68 A141E_P171E_T187R 20％ 80％ 1.60 A141D 28％ 72％ 1.47 A141D_T187R 31％ 69％ 1.37 A141E_F170E_V185R 24％ 76％ 1.31 A141E_T187R 27％ 73％ 1.18 WT 40％ 60％ 1.00

[0307] To determine whether the first two λ-biased CH1 variants ("A141D_P171E_V185R" and "A141D_F170E_T187R") pair with κ light chains, CH1 variant heavy chain plasmids were transfected into HEK293 cells with either 1.) a κ light chain or 2.) a λ light chain (heavy chain:light chain ratio = 1:1). K147F S183R and WT, which are CH1 variants with a κ bias, were also included as controls. The transfected HEK cells were cultured, and IgG was purified using a protein A column using standard methods. The process yield (mg / L) was determined using standard methods and normalized to the WT process yield. Based on the normalized process yields, when only κLC (but not λLC) was present, “A141D_P171E_V185R” and “A141D_F170E_T187R” still bound to κLC, but more binding occurred in λLC compared to κLC ( Figure 30 ).

[0308] The process yield of Fab format was also evaluated. IgG with CH1 variant heavy chains was produced and purified using the same method. K147F S183R, WT, A141D and A141D S181K K218P as CH1 with κ preference were also included as controls. Using standard methods, linked heavy chain Fab and light chain Fab were generated from purified IgG via papain enzymatic digestion and CH1 column purification. Normalized Fab digests were calculated as the % recovery of Fab recovered from IgG digests normalized to the parent recovery of each light chain (amount of recovered Fab / amount of IgG in the digest). Process yield was determined using standard methods and normalized to WT process yield. Figure 15 Consistent with the data, the process yields of λLC for “A141D” and “A141D S181K K218P” were higher than those for κLC, and “K147F S183R” showed an extremely high κ preference ( Figure 31 When only κCH1 (but not λCH1) was present, “A141D_P171E_V185R” and “A141D_F170E_T187R” still bound to κCH1, but the yields obtained with λLC were significantly higher than those obtained with κLC ( Figure 31 ). Adding “P171E_V185R” or “F170E_T187R” to the “A141D” mutation further enhanced the λ preference of “A141D”.

[0309] Example 8: Structural analysis of the “A141D” and “K147F S183R” variants

[0310] method

[0311] Crystallization and structure determination of wild-type CH1-Cλ of panirumab

[0312] 6.5 mg / ml of panitumumab wild-type CH1-constant lambda (Cλ) Fab protein was centrifuged at 14,000×g for 5 minutes at 4° C. 305 nL of protein was mixed with 150 nL of reservoir droplet and 50 nL of seed solution and equilibrated with 40 ul of reservoir solution in an MRC 3-well plate at 20° C. Seeds identified from the BCS screen (molecular size) were used for microseed matrix screening (MMS) (D'Arcy, A., Villard, F., and Marsh, M. (2007) "An automated microseed matrix-screening method for protein crystallization," Acta Crystallogr D Biol Crystallogr 63, 550-554.) crystallization experiments to obtain crystals grown in 0.1 M phosphate / citrate pH 5.5 and 36% (v / v) PEG Smear Low and transferred to 0.1 M phosphate / citrate pH 5.5, 38% PEG Smear Low and 4% glycerol, and then flash frozen in liquid nitrogen. Crystals were collected at 100 K at Diamond Light Source I03, Didcot, England, equipped with an Eiger2 XE16M detector (DECTRIS). Diffraction data. The data sets were integrated in autoPROC (Vonrhein, C. et al. (2011) “Data processing and analysis with the autoPROC toolbox” Acta Crystallographica D 67, 293-302.) using XDS (Kabsch W. (2010) “XDS” Acta Crystallographica Section D—Biological Crystallography 66, 125-132.) and scaled using Aimless of the CCP4 software package (Evans PR and Murshudov, GN (2013) “How good are my data and what is the resolution” Acta Crystallographica Section D—Biological Crystallography 69, 1204-1214.) (Winn MD et al. (2011) “Overview of the CCP4suite and current developments” developments)” Acta Crystallographica Section D - Biological Crystallography 67, 235-242.235-242.). The crystals are composed of two molecules per asymmetric unit (ASU) in the P1211 space group.The structure was solved using the automated molecular replacement system MoRDA (Vagin A. and Lebedev A. (2015) “MoRDa, an automatic molecular replacement pipeline” Acta Crystallographica A.A71, s19.) (incorporated into MOLREP (Vagin A., Teplyakov A. (1997) “MOLREP: an automated program for molecular replacement”). The system was used to refine the crystal structure of a macromolecular crystal using Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for refinement of macromolecular crystal structures, Acta Crystallographica D - Biological Crystallography, 67, 355-367). The system selected entries 5N7W and 5SX4 of the Protein Data Bank (Berman HM et al. (2000) "The Protein Data Bank", Nucleic Acids Research, 28) as initial search models. Automated model building was performed using BUCCANEER software (Cowtan K. (2006) “The Buccaneer software for automated model building. 1. Tracing protein chains” Acta Crystallographica D62, 1002-1011).The model was refined by manual refinement in Coot (Emsley P., Lohkamp, ​​B., Scott, WG, and Cowtan K. (2010) “Features and development of Coot.” Acta Crystallographica D, 66, 486-501.) and Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F., and Vagin, AA (2011) REFMAC5 for therefinement of macromolecular crystal structures. Acta Crystallographica D, 67, 355-367.) and Buster (Bricogne G, Blanc E, Brandl M, Flensburg C, Keller P, Paciorek W, Roversi P, Sharff A, Smart O, Vonrhein C, Womack T. (2011). Refinements to final R and R in BUSTER version 2.11.7. Global Phasing Ltd, Cambridge, United Kingdom. 游离 14.5% and 16.9% respectively ( Figure 32 ).

[0313] Crystallization and structure determination of panitumumab A141D CH1-Cλ, wild-type CH1-Cκ, and K147F-S183R CH1-Cκ

[0314] Panitumumab A141D CH1-Cλ, panitumumab wild-type CH1-constant kappa (Cκ), and panitumumab K147F-S183R CH1-Cκ Fab were centrifuged at 14,000 × g for 5 minutes at 4°C. For panitumumab A141D CH1-Cλ and K147F-S183R CH1-Cκ, 200 nL of 10.0 mg / ml Fab was mixed with 150 nL of reservoir droplet, and 50 nL of seed solution was equilibrated with 40 ul of reservoir solution. Seed crystals identified from BCS screening were used in MMS experiments to find the optimal crystallization conditions. 0.1M phosphate / citrate buffer pH 5.5 and 36% (v / v) PEG Smear Low were used for panitumumab A141D CH1-Cλ and 0.1M sodium acetate pH 4.5, wherein 30% v / v PEG Smear Low was used for panitumumab K147F-S183R CH1-Cκ. 150nL of 19.2mg / ml wild-type CH1-Cκ was mixed with 150nL reservoir droplets and added to 40ul reservoir solution and screened using a PACT kit (molecular size). Final crystallization conditions consisted of 0.1MMES pH 6.0 and 0.2M calcium chloride dihydrate with 20% w / v PEG 6000. The crystals were transferred to cryogenic solutions consisting of: 0.1 M phosphate / citrate buffer pH 5.5, 38% PEG Smear Low, 4% glycerol; 0.07 M MES, pH 6.0, 21% PEG6000, 0.2 M CaCl2, 23.5% glycerol; and 0.1 M NaAc pH 4.5, 32.5% PEG Smear Low, 25% glycerol for panitumumab A141D CH1-Cλ, wild-type CH1-Cκ, and K147F-S183R CH1-Cκ, respectively. All crystals were flash frozen in liquid nitrogen and collected at 100 K at Diamond Light Source Station I03, Didcot, England, equipped with an Eiger2 XE 16M detector (Decotes). High-resolution crystallographic data. The data were indexed and integrated in iMOSFLM (Battye, TGG, Kontogiannis, L., Johnson, O., Powell, HR and Leslie, AG (2011). iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallographica Section D: Biological Crystallography, 67(4), 271-281.) and scaled and merged by using AIMLESS (Evans PR and Murshudov, GN (2013) “How good are my data and what is the resolution?” Acta Crystallographica Section D—Biological Crystallography 69, 1204-1214.) of the CCP4 suite (Winn MD et al. (2011) “Overview and current developments of the CCP4 suite.” Acta Crystallographica Section D—Biological Crystallography 67, 235-242.).

[0315] The panitumumab A141D-CH1-Cλ structure was solved by molecular replacement using the wild-type CH1-Cλ crystal structure as a search model. Several rounds of anisotropic B-factor and simple restraint refinement were performed in Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F., and Vagin, AA (2011) REFMAC5 for Refining Macromolecular Crystal Structures. Acta Crystallographica D - Biological Crystallography 67, 355-367). Fuzziness factors were applied in the final rounds of refinement. The positional occupancies of A141D CH1-Cλ were assigned based on the occupancies of wild-type CH1-Cλ and manually adjusted in Coot during iterative refinement (Emsley P., Lohkamp, ​​B., Scott, W.G. And, and Cowtan K. (2010) “Features and Development of Coot.” Acta Crystallographica D—Biological Crystallography 66, 486–501.). R and R of the final structure solved in P1211 with 2 molecules per ASU 游离 The values ​​were 15.2% and 17.0% ( Figure 33 ).

[0316] The panitumumab wild-type CH1-Cκ and K147F-S183R-CH1-Cκ structures were solved by molecular replacement with Phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC, and Read, RJ (2007). Phaser crystallographic software. Journal of Applied Crystallography, 40(4), 658-674.) using coordinates of the panitumumab Fab fragment in complex with EGFR (PDB code 5SX4) and the solved wild-type CH1-Cκ structure, respectively, followed by iterative manual model building using Coot (Emsley, P., Lohkamp, ​​B., Scott, W.G., and Cowtan et al.). K. (2010) "Characterization and Development of Coot" Acta Crystallographica D - Biological Crystallography 66, 486-501.) and automatic refinement in Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for Refining Macromolecular Crystal Structures, Acta Crystallographica D - Biological Crystallography 67, 355-367.). Translational non-crystallographic symmetry was observed for the wild-type CH1-Cκ structure, so the structure was solved in ASU in a lower space group (P1211) with 6 Fab molecules. The structure was refined to a final R and R 游离 The values ​​were 19.8% and 23.2% ( Figure 34 The K147F-S183R CH1-Cκ structure was solved in the P31 space group with one molecule per ASU to the final R and R 游离 The values ​​were 19.8% and 23.3% ( Figure 35 ).

[0317] Structural analysis and interpretation

[0318] λLC preference mediated by HC-A141D

[0319] Without wishing to be bound by theory, the enhanced lambda preference of panitumumab A141D CH1-Cλ may be mediated by the interchain hydrogen bond formed between the side chain carboxyl group of HC-Asp141 and the side chain hydroxyl group of λLC-Thr116 ( Figure 36 C), which cannot form a cleavage site with HC-Ala141 in panitumumab wild-type CH1-Cλ ( Figure 36A). The κLC region surrounding HC-Ala141 consists of hydrophobic residues Phe116, Phe118, and Leu135, while κLC-Phe116 is replaced by the polar residue Thr116 in λLC ( Figure 36 B). Therefore, the introduction of charge by the A141D mutation can reduce κ preference by disrupting the hydrophobicity of the CH1-κLC interface, while stabilizing the CH1-λLC pairing through hydrogen bonding with λLC-Thr116. Additionally, without wishing to be bound by theory, κ preference may be further reduced by steric hindrance between HC-Asp141 and κLC-Phe116, as shown by an alignment of panitumumab A141D CH1-Cλ and wild-type CH1-κLC ( Figure 36 D).

[0320] In the hydrogen bond between HC-Asp141 and λLC-Thr116, the bond is formed between the hydrogen acceptor atom (O) in the side chain of Asp141 and the hydrogen donor atom (H) in the side chain of Thr116. Therefore, another amino acid having a hydrogen acceptor atom in its side chain can also form a hydrogen bond with Thr116 of λLC, providing a λ preference. Based on the fact that the side chain of glutamic acid also has a hydrogen acceptor atom (O) and glutamic acid is similar to aspartic acid in size and shape, glutamic acid is likely to form a hydrogen bond with Thr116 of λLC, while causing the following: Figure 36 The steric hindrance with κLC shown in D provides an overall λ preference. In fact, the A141E substitution provides a strong λ preference as demonstrated in the above examples, confirming the applicant's structural analysis.

[0321] κLC preference mediated by HC-K147F-S183R

[0322] The observed κ preference of panitumumab K147F-S183R CH1-Cκ can be mediated by two new hydrogen bonds at the CH1 and Cκ interface. In the panitumumab wild-type CH1-Cκ structure, a hydrogen bond network coordinated by HC-Lys147 and HC-Asp148 isolates HC-Gln175, contributing to the baseline κ pairing preference ( Figure 37 A). One explanation is that substitution of CH1HC-Lys147 with phenylalanine at this position disrupts this network and frees the HC-Gln175 side chain, which interacts with κLC via hydrogen bonding to the carboxamide oxygen of κLC-Gln160, thus increasing κ preference ( Figure 37 B). Additionally, without wishing to be bound by theory, the HC-S183R substitution results in an additional hydrogen bond between the guanidinium group of the HC-Arg183 side chain and the hydroxyl group of κLC-Thr178 ( Figure 37 B. Figure 38C). In contrast, without wishing to be bound by theory, the hydrogen bonding observed at position HC 183 between HC-Ser183 and λLC-Tyr178 of panitumumab wild-type CH1-Cλ is eliminated in the simulated pairing of K147F-S183R CH1 and λLC by the severe steric hindrance of HC-Arg183 and λLC-Tyr178 side chains, destabilizing the λ pairing in favor of κLC ( Figure 38 B and 38D).

[0323] In the hydrogen bond between HC-Arg183 and κLC-Thr178, the bond is formed between the hydrogen donor atom (H) in the side chain of Arg183 and the hydrogen acceptor atom (O) in the side chain of Thr178. Therefore, another amino acid having a hydrogen donor atom in the side chain can also form a hydrogen bond with Thr178 of κLC, providing a κ preference. Larger side chains such as the side chain of Arg can help generate steric hindrance with Tyr178 of λLC, thereby providing additional κ preference. For example, the side chains of both lysine and tryptophan have large side chains containing a hydrogen donor atom (H). Therefore, lysine and tryptophan may form a hydrogen bond with Thr178 of κLC and may undergo a reaction such as Figure 38 The steric hindrance with λLC shown in D provides a κ preference overall. The side chain of threonine can also act as a hydrogen donor through the H atom of -OH. Therefore, the applicant further envisions that amino acids with relatively large side chains that can act as hydrogen acceptors can also form hydrogen bonds with Thr178 of κLC to provide a κ preference. For example, when placed at residue 183 of HC, glutamic acid, glutamine, histidine or tyrosine with relatively large side chains (with hydrogen acceptor atoms) can also provide a κ preference. In fact, most of these newly proposed amino acid substitutions at residue 183 were actually identified as κ preference in Example 3 (see Table 3).

[0324] As described above, replacing Lys147 with Phe disrupts the hydrogen bond between Lys147 and Gln175, thereby freeing Gln175 to form a hydrogen bond with Gln160 of κ LC and thus favoring κ preference. Therefore, replacing Lys147 with another amino acid, such as alanine, glycine, isoleucine, leucine, or valine, whose side chain does not contain a hydrogen donor or acceptor atom, may also favor κ preference. In fact, most of these newly proposed amino acid substitutions at residue 147 were actually identified as κ-biased in Example 3 (see Table 3). Sequence Listing <110> Adimab Limited Liability Company <120> CH1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising the same <130> 1160430.002413 <150> 62 / 908,367 <151> 2019-09-30 <160> 189 <170> PatentIn 3.5 Edition <210> 1 <211> 112 <212> PRT <213> Homo sapiens <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 <210> 2 <211> 107 <212> PRT <213> Homo sapiens <400> 2 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu<000​​​​​​​​​​​​​​​​​​​​​​​​Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 [[ID=z0]]Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 4 <211> 101 <212> PRT[[ID=2y]] <213> Artificial Sequence <220> <223> SAD9611_P01_B08 <400> 4 Gly Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Phe Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser It should be noted that there seems to be an error in your original text where "Glu Lys Thr Val Ala Pro Thr Glu Cys Ser" has an incorrect line numbering in the original (should be ID=20 instead of ID=z0). I've translated it as is but this might need to be corrected in the source for a more accurate representation. Also, the "y" in "ID=2y" is likely a mistake. 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 5 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_D09 <400> 5 Ala Ser Thr Lys Gly Pro His Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Ile Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 6 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_H09 <400> 6 Ala Ser Thr Lys Gly Pro Arg Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 7 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_B09 <400> 7 Ala Ser Thr Lys Gly Pro Ser Val Ala Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> SAD9611_P01_E08 <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Val Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu 85 90 95 Lys Val Glu Pro Lys 100 <210> 9 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_H07 <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro His Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 050 55 60 Leu Trp Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 10 [[ID=?6]]<211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_F08 <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Phe Val Ile Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 It should be noted that there may be an error in the original text where "1050" in line is likely a typo. I translated it as "050" for consistency with the format. If this is incorrect, please provide the correct text for a more accurate translation. Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 11 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_D07 <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Ile Val Thr Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Gln 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 12 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_C07 <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 13 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_E09 <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Ser Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 14 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_F09 <400> 14 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Tyr 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr �5 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 15 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_A07<00​​​​​​​​​ Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Gln 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 16 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_C09 <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Val Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gly Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 17 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_B07 <400> 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Ile Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gly Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 18 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_G09 <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Gly Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Phe Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 19 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_C08 <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Glu Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 20 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_G08 <400> 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Tyr Ser Val Val Thr Val Pro Ser Ile Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 21 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P01_G07 <400> 21 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Trp Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 22 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_D10 [[ID=2...]]<400> 22 Ala Ser Thr Lys Gly Pro Glu Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Val Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 [[ID=4...]]Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 23 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_D12 <400> 23 Ala Ser Thr Lys Gly Pro Leu Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Phe Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 24 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_B10 <400> 24 Ala Ser Thr Lys Gly Pro Val Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Tyr Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 ④5 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 25[[ID=​​​​​​​​​​​Ala Ser Thr Lys Gly Pro Ser Val Thr Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 26 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_E12 <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Leu Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Glu Gly Gly Thr Ala Ala Leu Ser Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 27 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_F10 <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Pro Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Glu Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 28 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_A10 <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ala Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Leu Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 29 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_C12 <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Ile Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Tyr Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 30 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_H10 <400> 30 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Asn Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Leu Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 31 <211> 101 <21​​​​​​​​​​​​​Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Thr Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 050 55 60 Leu Phe Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 32 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_D11 <400> 32 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Val Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys [[ID=!1]]85 90 95 Lys Val Glu Pro Lys 100 <210> 33 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_E10 <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 [[ID=3!]]Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr It seems there might be a small error in the text you provided. The "85 90 95" in ID=11 was likely misformatted as "!1" in the original text. I've corrected it in the translation for clarity. If this is not what you intended, please let me know.65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 34 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_B12 <400> 34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gly Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 35 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_F11 <400> 35 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Gln Asp Tyr 20 2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Val Asp Tyr 20 25 30 Phe Ala Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 37 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_A12 <400> 37 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Tyr Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Gly 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 38 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_F12 <400> 38 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Met Gln Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gly Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 39 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_G12 <400> 39 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Tyr Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Ala Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Tyr Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 40 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_B11 <400> 40 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Tyr Tyr Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Ala Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 41 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_H11 <400> 41 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Lys Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 42 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_C10 <400> 42 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Gln Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 43 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P02_E11 <400> 43 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Gln Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Arg Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 44 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_C03 <400> 44 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Ile Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 45 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_G03 <400> 45 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Val Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45<00​​​​​​​​​​​​​​​​​ <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_F03 <400> 46 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 47 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_H01 <400> 47 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Ala 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 48 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_C01 <400> 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Ser Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 49 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_A01 <400> 49 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Gln Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 50 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_D02 <400> 50 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Cys 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 51 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_H03 <400> 51 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Ser 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser [[ID=3​​​​​​​​​​​​​​​​​​​​ <220> <223> SAD9610_P01_B03 <400> 52 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Cys 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Gln 100 <210> 53 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_H02 <400> 53 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp His 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys 100 <210> 54 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_E02 <400> 54 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp His 20 25 30 Leu Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 55 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_B02[[ID=...]] <400> 55 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Leu Pro Glu Pro Met Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 56 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_A03 <400> 56 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Pro Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 57 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_E03 <400> 57 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Ser Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60[[ID=..]] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Gly Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 58 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_D01 <400> 58<00..307>Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Gly Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 59 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_C02 <400> 59 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Asn Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys<00023३9>85 90 95 Lys Val Glu Pro Lys 100 <210> 60 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_B01 <400> 60 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr<00023५२>20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Arg Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 61 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P01_G01 <400> 61 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Ser Tyr Lys Pro Ser Asn Thr Arg Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 62 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_E05 <400> 62 Ala Ser Thr Lys Gly Pro Ser Val Leu Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 63 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_C04 <400> 63 Ala Ser Thr Lys Gly Pro Ser Val Leu Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Gly Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 二十 二十五 三十 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 三十五 四十 四十五 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 五十 五十五 六十 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 六十五 七十 七十五 八十 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu<000​​​​​​​​​​​​​​​​​​​​​​​​Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Cys 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 65 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_F05 <400> 65 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Pro Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Ser Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 66 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_D05 <400> 66 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Val Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 67 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_C06 <400> 67 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 68 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_C05 <400> 68 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 69 [[ID=4३]]<211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_H06 <400> 69 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Arg 85 90 95 Lys Val Glu Pro Lys 100 <210> 70 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_A06 <400> 70 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Leu Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Ser Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 71 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_F06 <400> 71 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Ser Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 72 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_E04 <400> 72 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Leu Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 73 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P02_B05 <400> 73 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 74 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_F12 <400> 74 Ala Ser Thr Lys Gly Pro Val Val Val Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Ser 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Ile Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 75 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_D12 <400> 75 Ala Ser Thr Lys Gly Pro Ser Val Phe Gly Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 77 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_C12 <400> 77 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Gln Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 78 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_H12 <400> 78 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Thr Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 79 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_C10 <400> 79 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Asn Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val Phe Thr Asn Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60[[ID=2,8]] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 80 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_A11 <400> 80 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Asp 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 81 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_A10 <400> 81 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Thr 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Pro 100 <210> 82 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_B11 <400> 82 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ala Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 83 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_G12 <400> 83 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 His Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 84 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_F11 <400> 84 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ile Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 85 <211> 101 <212> PRT <213> Artificial Sequence It should be noted that there is a possible error in the original text where "PRT" in line 11 and 53 seems to be an incorrect tag. Usually, in patent sequences, more standard amino acid sequence notations are used. Also, in the translation, the number "36" in the line with ID 36 might be a formatting or input error in the original. If this is a real number in the sequence, it should be translated as "36" like other numbers in the same context. <220> <223> SAD9611_P02_E10 <400> 85 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Pro Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 86 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_A12 <400> 86 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Val Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 87 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_B10 <400> 87 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Val Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 甘氨酸 缬氨酸 组氨酸 苏氨酸 苯丙氨酸 脯氨酸 丙氨酸 缬氨酸 亮氨酸 谷氨酰胺 丝氨酸 丝氨酸 甘氨酸 亮氨酸 酪氨酸 丝氨酸 50 55 60 亮氨酸 丝氨酸 丝氨酸 缬氨酸 缬氨酸 苏氨酸 缬氨酸 脯氨酸 丝氨酸 丝氨酸 丝氨酸 亮氨酸 甘氨酸 苏氨酸 谷氨酰胺 苏氨酸 65 70 75 80 酪氨酸 异亮氨酸 半胱氨酸 天冬酰胺 缬氨酸 天冬酰胺 组氨酸 赖氨酸 脯氨酸 丝氨酸 天冬酰胺 苏氨酸 赖氨酸 缬氨酸 天冬氨酸 赖氨酸 85 90 95 赖氨酸 缬氨酸 谷氨酸 脯氨酸 赖氨酸 `100` <210> 88 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_C11 <400> 88 丙氨酸 丝氨酸 苏氨酸 赖氨酸 甘氨酸 脯氨酸 丝氨酸 缬氨酸 苯丙氨酸 脯氨酸 亮氨酸 丙氨酸 脯氨酸 丝氨酸 丝氨酸 赖氨酸 1 5 10 15 丝氨酸 苏氨酸 丝氨酸 甘氨酸 甘氨酸 苏氨酸 丙氨酸 谷氨酸 亮氨酸 甘氨酸 半胱氨酸 亮氨酸 缬氨酸 赖氨酸 天冬氨酸 酪氨酸 20 25 3'0 苯丙氨酸 脯氨酸 谷氨酸 脯氨酸 缬氨酸 苏氨酸 缬氨酸 丝氨酸 色氨酸 天冬酰胺 丝氨酸 甘氨酸 丙氨酸 亮氨酸 苏氨酸 丝氨酸 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 89 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_H10 <400> 89 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 90 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_B12 <400> 90 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Arg Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 91[[ID=4​​​​​​​​​​​​Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Leu Val Asp Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 92 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_E11 <400> 92 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Ser Val Glu Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 93 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9611_P02_D10 <400> 93 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Val Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Ser Val Tyr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 94 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_F07 <400> 94 Ala Arg Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 95 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_B07 <400> 95 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Ser Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30[[ID=2,8]] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Thr Val Ser Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 96 <211> 101 <212> PRT[[ID=,54]] <213> Artificial Sequence <220> <223> SAD9613_P01_A09 <400> 96 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Asn Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ser Thr Ser Gly Gly Thr Ala Met Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 98 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_G07 <400> 98 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Thr Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 99 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_B09 <400> 99 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Asp Ser Leu 1 5 10 15 Asn Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 100 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_E07 <400> 100 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Asp Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Asp Ser Ser Gly Leu Tyr Val 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 101 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_A08 <400> 101 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Glu 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Arg Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 102 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_H09 <400> 102 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Pro 1 5 10 15 Ser Thr Ser Gly Gly Ala Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 103 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_D07 <400> 103 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Leu Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 104 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_A07 <400> 104 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Asn Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 105 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_H08 <400> 105 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Pro Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 106 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_F08 <400> 106 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Val Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 107 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_D08 <400> 107 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser<00�3343>50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 108 <211> 101 <2I2> PRT<00Q3353><213> Artificial Sequence <220> <223> SAD9613_P01_E09 <400> 108 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Thr Val Glu Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 109 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_C09 <400> 109 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val Val Thr Thr Asn Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 110 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9613_P01_H07 <400> 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Leu Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 111 <211> 101 <212> PRT <213> Artificial Sequence <220>​​​​​​​​​​​​​​​​​​​​​​​​​Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 112 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_B04 <400> 112 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 113 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_G06 <400> 113 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 114 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_G04 <400> 114 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Ala Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 115 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD96A0_P02_C06 <400> 115 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Gly Pro Lys 100 <210> 116 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_G05 <400> 116 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Met Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 117 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_C04 <400> 117 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Val Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asn Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 118 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9610_P02_E05 <400> 118 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Thr 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210>​ <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_A01 <400> 119 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Pro Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 120 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_G01 <400> 120 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Val Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 9​​​​​​​​​​​​​​​​​​​​​​​​​​ Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu 85 90 95 Lys Val Glu Pro Lys 100 <210> 122 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_D03 <400> 122 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser<050 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys 100 <210> 123 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_E01 <400> 123 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Gly Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 124 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_H03 <400> 124 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Val Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Gly Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 125 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD9612_P01_F02 <400> 125 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Gly Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser [[ID=2,5]][

[50] ] [

[55] ] [

[60] ] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 126 <211> 101 <212> PRT `<213> Artificial Sequence <y220> <223> S 4 AD9612_P01_A02 <400> 126 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Val Ser Thr Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Thr Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 [[ID=2*]] <210> 128 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P01_F07 <400> 128 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys [[ID=**]]1 5 10 15 Ser Thr Ser Gly Gly Thr Tyr Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser It seems there are some incorrect line numbers in the provided translation request. I've tried my best to translate accurately based on the given text. If you have any further clarifications or corrections, please let me know.50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 129 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P01_C07 <400> 129 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Lys Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Asp Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 130 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P01_A07 <400> 130 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ser Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Ser Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 131 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P02_G08 <400> 131 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Trp Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 132 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P02_D08 <400> 132 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Thr Ser Gly Gly Thr Arg Asp Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 65, 70, 75, 80 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 85 90 95 Val Glu Pro Asn 100 <210> 133 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> SAD10791_P02_F07 <400> 133 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Leu Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 [[ID=1十七]]Lys Val Glu Pro Lys 100 <210> 134 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A140I; A141D <400> 134 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ile Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser It should be noted that there seems to be a small error in your original text where "Lys Val Glu Pro Lys" was misspelled as "Lys Val Glu Pro Lys" in the translation. It should be "Lys Val Glu Pro Lys" without the extra "十七" in the translation. I've corrected it in the translation above.50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 135 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A140V; A141D <400> 135 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Val Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 136 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> S183Y <400> 136 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 [[ID=३३]]Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Tyr Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 137 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> K147F; S183R <400> 137 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Phe Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Arg Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 138 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> K147F; S183K <400> 138 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Phe Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Lys Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <Synthetic 1> 139 <Synthetic 2> 101 <Synthetic 3> PRT <Synthetic 4> Artificial Sequence <Synthetic 5> <Synthetic 6> K147F S183Y <Synthetic 7> 139 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Phe Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Tyr Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 140 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141D <400> 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 141 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141D; S181K <400> 141 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 142 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141D; S181K; K218A <400> 142 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Ala 100 <210> 143 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141D; S181K; K218P <400> 143 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Asp Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Pro 100 <210> 144 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141E <400> 144 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 145 <211> 10l <212> PRT <213> Artificial Sequence <220> <223> A141E; S181K <400> 145 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Glu Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 146 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141K <400> 146 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 147 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141K; S181K <400> 147 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Light Val Glu Pro Light 100 <210> 148 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141K; S181K; K218E <400> 148 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys<* 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95[[ID=*28]] Lys Val Glu Pro Glu 100 <210> 149 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> A141K; S181K; K218P <400> 149[[ID=*46]] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 It seems there are some tags that might be misaligned in your original text. I've translated it as accurately as possible while keeping those tags intact. If you have any further clarification or correction regarding the text, please let me know.Ser Thr Ser Gly Gly Thr Ala Lys Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Lys 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Pro 100 <210> 150 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> P127G; G138R; A141T; F170G; S176R; S181L <400> 150 Ala Ser Thr Lys Gly Pro Ser Val Phe Gly Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Arg Thr Ala Thr Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Gly Pro Ala Val Leu Gln Arg Ser Gly Leu Tyr Leu 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys 100 <210> 151 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> S131R; A141E...

Claims

1. A heavy chain constant region 1 (CH1) domain variant polypeptide, wherein the CH1 domain variant polypeptide is paired with a polypeptide comprising a kappa light chain constant region (CL) domain, wherein the amino acid sequence of the CH1 domain variant polypeptide is SEQ ID NO:

137. 2 . The CH1 domain variant polypeptide of claim 1 , wherein the κCL domain comprises a T at position 178 according to EU numbering. 3 . The CH1 domain variant polypeptide of claim 1 , wherein the κCL domain comprises a Q at position 160 and a T at position 178 according to EU numbering.

4. The CH1 domain variant polypeptide of claim 1, wherein the κCL domain comprises the amino acid residues at the following positions according to EU numbering: F at position 116, D at position 122, Q at position 124, S at position 127, T at position 129, S at position 131, N at position 137, N at position 138, Q at position 160, S at position 162, D at position 167, S at position 174, T at position 178, and T at position 180.

5. The CH1 domain variant polypeptide according to claim 1, wherein: (i) the κCL domain is a wild-type κCL domain; and / or (ii) the amino acid sequence of the κCL domain is SEQ ID NO:

2. The CH1 domain variant polypeptide according to claim 1 , further comprising a CH2 domain and a CH3 domain.

7. The CH1 domain variant polypeptide according to claim 6, wherein: (i) the CH2 domain is a wild-type CH2 domain; and / or (ii) the CH3 domain is a wild-type CH3 domain.

8. The CH1 domain variant polypeptide according to claim 6, wherein: (i) the CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; and / or (ii) the CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain. 9 . The CH1 domain variant polypeptide according to claim 6 , further comprising a variable region.

10. An antibody or antibody fragment comprising a first heavy chain polypeptide and a first light chain polypeptide, wherein: (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises a variable region and a first heavy chain constant region, wherein the first heavy chain constant region comprises a first CH1 domain, and wherein the amino acid sequence of the first CH1 domain is SEQ ID NO: 137; and (c) the first light chain polypeptide comprises a variable region and a first light chain constant region, wherein the first light chain constant region comprises a first CL domain, and the first CL domain is a kappa CL domain paired with the first CH1 domain.

11. The antibody or antibody fragment according to claim 10, wherein: (i) the first CL domain is a wild-type kappa CL domain; and / or (ii) The amino acid sequence of the first CL domain is SEQ ID NO:

2.

12. The antibody or antibody fragment of claim 10, wherein the first heavy chain constant region further comprises a first CH2 domain and / or a first CH3 domain.

13. The antibody or antibody fragment according to claim 12, wherein: (i) the first CH2 domain is a wild-type CH2 domain; and / or (ii) the first CH3 domain is a wild-type CH3 domain.

14. The antibody or antibody fragment according to claim 12, wherein: (i) the first CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; and / or (ii) the first CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain.

15. The antibody or antibody fragment of claim 10, further comprising a second heavy chain polypeptide and a second light chain polypeptide, wherein: (a) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; (b) the second heavy chain polypeptide comprises a variable region and a second heavy chain constant region, wherein the second heavy chain constant region comprises a second CH1 domain, wherein the amino acid sequence of the second CH1 domain is different from the amino acid sequence of the first CH1 domain; and (c) the second light chain polypeptide comprises a variable region and a second light chain constant region, wherein the second light chain constant region comprises a second CL domain, and the second CL domain is a lambda CL domain paired with the second CH1 domain.

16. The antibody or antibody fragment according to claim 15, wherein the amino acid sequence of the second CH1 domain is any one of SEQ ID NOs: 140-189 and 74-135. 17 . The antibody or antibody fragment of claim 15 , wherein the amino acid sequence of the second CH1 domain is SEQ ID NO: 188, 186 or 143.

18. The antibody or antibody fragment according to claim 15, comprising one or more of features (i)-(vi): (i) the first CL domain is a wild-type κ CL domain; (ii) the second CL domain is a wild-type λ CL domain; (iii) the first CL domain is a human κ CL domain; (iv) the second CL domain is a human lambda CL domain; (v) the amino acid sequence of the first CL domain is SEQ ID NO: 2; and / or (vi) The amino acid sequence of the second CL domain is SEQ ID NO:

3.

19. The antibody or antibody fragment of claim 15, wherein the second heavy chain constant region further comprises a second CH2 domain and / or a second CH3 domain.

20. The antibody or antibody fragment according to claim 19, comprising one or more of features (i)-(iv): (i) the first CH2 domain is a wild-type CH2 domain; (ii) the first CH3 domain is a wild-type CH3 domain; (iii) the second CH2 domain is a wild-type CH2 domain; and / or (iv) the second CH3 domain is a wild-type CH3 domain.

21. The antibody or antibody fragment of claim 19, wherein: (i) the first CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; (ii) the first CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain; (iii) the second CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; and / or (iv) the second CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain.

22. An antibody or antibody fragment comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide and a second light chain polypeptide, wherein: (a) the first heavy chain polypeptide and the first light chain polypeptide form a first cognate pair; (b) the first heavy chain polypeptide comprises, in the direction from N-terminus to C-terminus, a variable region and a first heavy chain constant region, wherein the first heavy chain constant region comprises, in the direction from N-terminus to C-terminus, a first CH1 domain, a first CH2 domain and a first CH3 domain, and wherein the amino acid sequence of the first CH1 domain is SEQ ID NO: 137; (c) the first light chain polypeptide comprises a variable region and a first light chain constant region in the direction from N-terminus to C-terminus, wherein the first light chain constant region comprises a first CL domain, and the first CL domain is a κCL domain paired with the first CH1 domain; (d) the second heavy chain polypeptide and the second light chain polypeptide form a second cognate pair; (e) the second heavy chain polypeptide comprises a variable region and a second heavy chain constant region in the direction from the N-terminus to the C-terminus, wherein the second heavy chain constant region comprises a second CH1 domain, a second CH2 domain, and a second CH3 domain in the direction from the N-terminus to the C-terminus, and wherein the amino acid sequence of the second CH1 domain is any one of SEQ ID NOs: 140-189 and 74-135; and (f) the second light chain polypeptide comprises a variable region and a second light chain constant region in the direction from N-terminus to C-terminus, wherein the second light chain constant region comprises a second CL domain, and the second CL domain is a lambda CL domain paired with the second CH1 domain.

23. The antibody or antibody fragment of claim 22, wherein the amino acid sequence of the second CH1 domain is SEQ ID NO: 188, 186 or 143.

24. The antibody or antibody fragment of claim 22, wherein: (i) the κCL domain is a wild-type κCL domain; (ii) the λCL domain is a wild-type λCL domain; (iii) the amino acid sequence of the κCL domain is SEQ ID NO: 2; and / or (iv) The amino acid sequence of the λCL domain is SEQ ID NO:

3.

25. The antibody or antibody fragment of claim 22, comprising one or more of features (i)-(iv): (i) the first CH2 domain is a wild-type CH2 domain; (ii) the first CH3 domain is a wild-type CH3 domain; (iii) the second CH2 domain is a wild-type CH2 domain; and / or (iv) the second CH3 domain is a wild-type CH3 domain.

26. The antibody or antibody fragment of claim 22, wherein: (i) the first CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; (ii) the first CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain; (iii) the second CH2 domain comprises one or more amino acid substitutions relative to a wild-type CH2 domain; and / or (iv) the second CH3 domain comprises one or more amino acid substitutions relative to a wild-type CH3 domain.

27. The antibody or antibody fragment of any one of claims 15 to 26 which is multispecific.

28. The antibody or antibody fragment of any one of claims 15 to 26 which is bispecific.

29. The antibody or antibody fragment according to claim 27, wherein the structure of the antibody or antibody fragment is according to any one of the following: (I) A structure comprising: (I-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a first heavy chain variable region (VH), a first CH1 domain, a first CH2 domain and a first CH3 domain; (I-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a first light chain variable region (VL) and a first CL domain; (I-iii) a second heavy chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (I-iv) a second light chain polypeptide, which comprises a second VL domain and a second CL domain in the direction from the N-terminus to the C-terminus; (II) A structure comprising: (II-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (II-ii) a first light chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a third VL, a first VL and a first CL domain; (II-iii) a second heavy chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a fourth VH, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (II-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VL, a second VL, and a second CL domain; (III) A structure comprising: (III-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (III-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VL and a first CL domain; (III-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VL, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (III-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VH, a second VL and a second CL domain; (IV) A structure comprising: (IV-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (IV-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VL and a first CL domain; (IV-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VH, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (IV-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VL, a second VL, and a second CL domain; (V) A structure comprising: (V-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (V-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VL and a first CL domain; (V-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VL, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (V-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VH, a second VL, and a second CL domain; (VI) The structure according to (I), wherein: (VI-i) the first heavy chain polypeptide further comprises a single chain variable fragment (scFv) at the C-terminus of the first CH3 domain, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second heavy chain polypeptide further comprises a scFv at the C-terminus of the second CH3 domain, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus; (VI-ii) the first light chain polypeptide further comprises a scFv at the C-terminus of the first CL domain, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second light chain polypeptide further comprises a scFv at the C-terminus of the second CL domain, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus; or (VI-iii) the first heavy chain polypeptide further comprises a scFv at the N-terminus, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second heavy chain polypeptide further comprises a scFv at the N-terminus, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus.

30. The antibody or antibody fragment of claim 28, wherein the structure of the antibody or antibody fragment is according to any one of the following: (I) A structure comprising: (I-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a first heavy chain variable region (VH), a first CH1 domain, a first CH2 domain and a first CH3 domain; (I-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a first light chain variable region (VL) and a first CL domain; (I-iii) a second heavy chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (I-iv) a second light chain polypeptide, which comprises a second VL domain and a second CL domain in the direction from the N-terminus to the C-terminus; (II) A structure comprising: (II-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (II-ii) a first light chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a third VL, a first VL and a first CL domain; (II-iii) a second heavy chain polypeptide, which comprises, in the direction from the N-terminus to the C-terminus, a fourth VH, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (II-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VL, a second VL, and a second CL domain; (III) A structure comprising: (III-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (III-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VL and a first CL domain; (III-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VL, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (III-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VH, a second VL and a second CL domain; (IV) A structure comprising: (IV-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (IV-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VL and a first CL domain; (IV-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VH, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (IV-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VL, a second VL, and a second CL domain; (V) A structure comprising: (V-i) a first heavy chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VH, a first VH, a first CH1 domain, a first CH2 domain and a first CH3 domain; (V-ii) a first light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a third VL, a first VL and a first CL domain; (V-iii) a second heavy chain polypeptide comprising, in the direction from the N-terminus to the C-terminus, a fourth VL, a second VH, a second CH1 domain, a second CH2 domain, and a second CH3 domain; and (V-iv) a second light chain polypeptide, which comprises, in the direction from N-terminus to C-terminus, a fourth VH, a second VL, and a second CL domain; (VI) The structure according to (I), wherein: (VI-i) the first heavy chain polypeptide further comprises a single chain variable fragment (scFv) at the C-terminus of the first CH3 domain, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second heavy chain polypeptide further comprises a scFv at the C-terminus of the second CH3 domain, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus; (VI-ii) the first light chain polypeptide further comprises a scFv at the C-terminus of the first CL domain, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second light chain polypeptide further comprises a scFv at the C-terminus of the second CL domain, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus; or (VI-iii) the first heavy chain polypeptide further comprises a scFv at the N-terminus, wherein the scFv comprises a third VH and a third VL in the direction from the N-terminus to the C-terminus or comprises a third VL and a third VH in the direction from the N-terminus to the C-terminus, and the second heavy chain polypeptide further comprises a scFv at the N-terminus, wherein the scFv comprises a fourth VH and a fourth VL in the direction from the N-terminus to the C-terminus or comprises a fourth VL and a fourth VH in the direction from the N-terminus to the C-terminus.

31. A composition comprising the antibody or antibody fragment according to any one of claims 10 to 26.

32. A composition comprising the antibody or antibody fragment according to any one of claims 27 or 28.

33. A composition comprising the antibody or antibody fragment according to any one of claims 29 or 30.

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