Stabilized single immunoglobulin variable domains

Abstract

The present disclosure relates to single immunoglobulin variable domains having amino acid substitutions that result in improved thermal stability, cellular expression and other biophysical properties. The present disclosure relates to single immunoglobulin variable domains having the amino acid sequence of a human heavy chain V gene portion (IGHV) of an antibody, wherein the IGHV amino acid sequence includes one or more amino acid substitutions that result in one or more of increased cellular expression, increased thermal stability, decreased dimerization, and decreased light chain pairing, compared to a wild-type IGHV sequence lacking the one or more amino acid substitutions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 298,051, filed January 10, 2022, which is incorporated by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (21-1467-WO_ST26_Sequence_Listing.xml; size: 960,098 bytes; created on January 9, 2023) are incorporated herein by reference in their entirety.

[0003] Field of the Disclosure The present disclosure relates generally to single immunoglobulin variable domains with amino acid substitutions that result in improved biophysical properties. [Background technology]

[0004] background Immunoglobulin therapeutics have become a large and growing segment of the pharmaceutical sector. Given their high specificity for a single target, minimal off-target cross-reactivity and generally good biophysical behavior, immunoglobulin G (IgG) antibodies in particular are powerful tools to intervene in a highly specific manner in various disease processes. IgG typically consists of the amino acid sequences of two heavy chains (HC) and two light chains (LC) of either kappa or lambda isotypes that assemble into a heterotetramer. Once assembled, IgG consists of two main subunits that perform different functions: the crystallizable fragment (Fc) and the antigen-binding fragment (Fab).

[0005] The Fab region of natural IgG is highly diverse, encompassing two variable domains, variable heavy (VH) and variable light (VL), from the HC and LC, which are further diversified by recombinant VDJ (VH) or VJ (VL) linkages, as well as hypersome mutations to achieve an almost unlimited diversity that is utilized to optimize interactions with target antigens. Fab also contains CH1 / CL domains from the HC and LC, respectively, which are disulfide-bonded and are present to stabilize the VH / VL pairing. The VH domain, especially the HC complementarity determining region (HCDR) 3, is the most diverse region of the antibody based on the complexity of the VDJ bond, and thus typically drives the specificity of the antibody / antigen interaction.

[0006] IgG thermodynamics is relatively complex. The Fab and Fc subunits are thermodynamically distinct from each other. Demarest SJ & Glaser SM, Curr Opin Drug Discov (2007) 11:675-87. Typically, IgG-Fc reaches a thermal unfolding midpoint (T) at approximately 70°C. m ) and the CH3 domain unfolds at 70–85°C depending on the IgG subclass. Demarest SJ, et al., J Biol Chem (2006) 281:30755-67; ​​Garber E & Demarest SJ, Biochem Biophys Res Commun (2007) 355:751-7.). The domains in an IgG Fab that encompass a kappa LC (VH, Vkappa, CH1, Ckappa) are thermodynamically coupled and unfold cooperatively (Garber & Demarest 2007 (supra); Toughiri R, et al., MAbs (2016) 8:1276-85), whereas a Fab with a lambda LC typically unfolds using two independent transitions, VH / Vlambda and CH1 / Clambda, with each subunit highly stabilized by heterodimeric interactions of the partner domains.

[0007] The ability to isolate VH domains for use as therapeutics offers both advantages and disadvantages over traditional IgG antibody therapeutics. Given the relatively small size of VH domains (approximately 14 kDa) compared to full-length antibodies (approximately 150 kDa) and the fact that the VH domain drives both antigen specificity and the majority of antibody binding strength, VH domains have the theoretical utility of being used as single domain binders for a variety of antigens (Holt LJ, Herring C, et al., Trends Biotechnol (2003) 21:484-90). This allows for the use of small modular binding units that do not require multi-chain heterodimerization to achieve binding events. On the other hand, especially for kappa-containing Fabs, removal of the VH domain from their Fab subunits reduces the Tm by approximately 20-25°C, which can result in significant challenges related to their thermal stability and folding (Michaelson JS, et al., MAbs (2009) 1:128-41; Demarest & Garber 2007 (supra); Kim et al., Biochem Biophys Acta (2014) 1844:1983-2001. 2014), making VH domains difficult to use as therapeutics due to their lower expression and reduced pharmacokinetic profile compared to full Fabs or antibodies. Therefore, optimization is typically required for VH domains to be used as therapeutic moieties independent of full IgG.

[0008] Thus, there remains a need in the art to find substitutions for the VH germline family, VH1, VH2, VH3, VH4, VH5, VH6 and VH7, that can be used to improve their biophysical properties, including thermostability and / or expression. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Demarest SJ, et al., J Biol Chem(2006)281:30755-67 [Non-Patent Document 2] Garber E&Demarest SJ,Biochem Biophys Res Commun(2007)355:751-7 [Non-Patent Document 3] Garber & Demarest 2007 (above); Toughiri R, et al., MAbs (2016) 8:1276-85) [Non-Patent Document 4] Holt LJ, Herring C, et al., Trends Biotechnol (2003) 21:484-90 [Non-Patent Document 5] Kim et al.,Biochem Biophys Acta(2014)1844:1983-2001.2014 Summary of the Invention

[0010] Abstract In various embodiments, the disclosure relates to a single immunoglobulin variable domain having an amino acid sequence of a human heavy chain V gene portion (IGHV) of an antibody, wherein the IGHV amino acid sequence comprises one or more amino acid substitutions that result in one or more of increased cellular expression, increased thermostability, decreased dimerization, and decreased light chain pairing compared to a wild-type IGHV sequence lacking the one or more amino acid substitutions. The single-chain immunoglobulin variable domain may also comprise a D gene sequence and / or a J gene sequence.

[0011] In another aspect, the disclosure relates to a single immunoglobulin variable domain comprising an amino acid sequence of a framework region of a human heavy chain V gene portion (IGHV) of an antibody, wherein the IGHV amino acid sequence includes one or more amino acid substitutions described herein or a combination thereof. The framework sequence may comprise a J gene sequence.

[0012] In another aspect, the present disclosure relates to at least one framework sequence selected from FR1, FR2, FR3, and FR4 of a single immunoglobulin heavy chain variable domain, wherein the framework sequence comprises at least one of the substitutions or combinations thereof described herein.

[0013] In various aspects of the disclosure, the one or more substitutions are at the following amino acids according to the Kabat numbering system: 1E, 2A, 5Q, 10Q, 10T, 14E, 15G, 16D, 16Q, 19I, 23K, 23Q, 23Y, 25F, 25Y, 28D, 28E, 28K, 28N, 28R, 30K, 30S, 31K, 33P, 35A, 35G, 35S, 37F, 37Y, 37H, 39R, 40P , 44D, 45E, 48I, 49A, 52E, 52D, 55E, 56E, 60A, 60D, 65D, 68E, 73D, 73P, 74E, 76K, 76N, 77Q, 82bD, 82bN, 83D, 83K, 83L, 83Q, 83T, 84E, 84P, 84Y, 85K, 85R, 85S, 85T, 89I, 105D, 107I, 107Y, 110I, and 110V. Substitutions may also include non-naturally occurring disulfide bonds involving at least one cysteine ​​residue at a non-naturally occurring amino acid position, for example, a non-naturally occurring disulfide bond may be present between two cysteine ​​residues at positions 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50 according to the Kabat numbering system.

[0014] Also, in various aspects of the disclosure, substitutions may include one of the following amino acid combinations according to the Kabat numbering system: 5Q / 23Q 10Q / 48I / 84E 10T / 82bD 10T / 82bD 10T / 82bN 10T / 84P 15G / 37Y 15G / 44D 15G / 85S <h2 style=";text-align:left;direction:ltr">15G / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16D / 37F<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16D / 37Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16D / 39R / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16D / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16D / 110I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 23Q / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 37Y / 48I / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 37Y / 48I / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 37Y / 76N / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 37Y / 76N / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 39R / 45E / 76N / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 39R / 48I / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 39R / 48I / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 39R / 76N / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 39R / 76N / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 48I / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 48I / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 49A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 49A / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 55E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 55E / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 76N / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28D / 76N / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 49A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 49A / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 49A / 55E / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 49A / 55E / 84E / 10T / 82bN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 55E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 55E / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37F / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 10T / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 10T / 82bD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 39R / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 39R / 45E / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 44D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 49A / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 85S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 83T 39R / 28D 39R / 45E 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 39R / 83T 39R / 45E / 48I 39R / 45E / 49A / 74E 39R / 45E / 82bD / 84P 44D / 85S 44D / 83T 45E / 82bD / 84P 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 45E / 82bD / 84P 49A / 84E 82bD / 84P 82bN / 84P 83T / 44D In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0015] In another embodiment of the disclosure, the single immunoglobulin variable domain (or its framework region(s)) may have the origin of a human germline gene selected from germline family 1, germline family 2, germline family 3, germline family 4, germline family 5 or germline family 7.

[0016] As an example of a human germline sequence, germline gene family 1 may include germline gene family members 1-2 (SEQ ID NO:1), 1-3 (SEQ ID NO:2), 1-8 (SEQ ID NO:3), 1-18 (SEQ ID NO:4), 1-24 (SEQ ID NO:5), 1-45 (SEQ ID NO:6), 1-46 (SEQ ID NO:7), 1-58 (SEQ ID NO:8), 1-69 (SEQ ID NO:9), and 1-69.2 (SEQ ID NO:10), and alleles thereof, and the single immunoglobulin variable domain (or framework region(s) thereof) may include one or more of the following substitutions: 10Q, 16D, 16Q, 25Y, 25F, 37F, 37Y, 39R, 45E, 48I, 84E, 84P, 110V, and 110I. Additionally, the single immunoglobulin variable domain (or framework region(s) thereof) may include one of the following combinations of substitutions: 10Q / 48I / 84E 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 37F / 48I 37Y / 48I 39R / 45E / 48I 39R / 48I

[0017] In additional embodiments of the disclosure having a human germline gene family 1 origin, the single immunoglobulin variable domain (or its framework region(s)) may comprise one of the following substitution combinations: 17C / 82aC / 10Q / 48I / 84E 17C / 82aC / 16D 17C / 82aC / 16D / 37F 17C / 82aC / 16D / 37Y 17C / 82aC / 16D / 37Y / 39R 17C / 82aC / 16D / 39R 17C / 82aC / 16D / 39R / 48I 17C / 82aC / 16D / 48I 17C / 82aC / 37F 17C / 82aC / 37Y 17C / 82aC / 37Y / 48I 17C / 82aC / 39R 17C / 82aC / 39R / 45E / 48I 17C / 82aC / 39R / 48I 17C / 82aC / 84E 34C / 78C / 16D 34C / 78C / 37F 34C / 78C / 84E 34C / 78C / 16D / 37F 34C / 78C / 16D / 48I 34C / 78C / 10Q / 48I / 84E

[0018] In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0019] As another example of a human germline sequence, germline gene family 2 can include germline gene family members 2-5 (SEQ ID NO:11), 2-26 (SEQ ID NO:12), and 2-70 (SEQ ID NO:13), and alleles thereof, and a single immunoglobulin variable domain (or framework region(s) thereof) can include one or more of the following substitutions: 15G, 16D, 37Y, 37H, 39R, 44D, 45E, 65D, 73D, 73P, 83L, 83Q, 83K, 83T, 84Y, 85R, 85S, 85K, 85T, 89I, 105D, and 107I.

[0020] In additional embodiments of the disclosure having a human germline gene family 2 origin, the single immunoglobulin variable domain (or its framework region(s)) may comprise one of the following substitution combinations: 1.15G / 37Y 15G / 44D 15G / 85S 15G / 83T 37Y / 39R / 45E / 83T 37Y / 39R / 83T 37Y / 44D 37Y / 85S 37Y / 83T 39R / 83T 44D / 85S 44D / 83

[0021] Moreover, in additional embodiments of the disclosure having a human germline gene family 2 origin, the single immunoglobulin variable domain (and its framework regions) may comprise one of the following combinations of substitutions: 19C / 81C / 15G 19C / 81C / 15G / 37Y 19C / 81C / 15G / 44D 19C / 81C / 15G / 85S 19C / 81C / 15G / 83T 19C / 81C / 37Y 19C / 81C / 37Y / 39R / 83T 19C / 81C / 37Y / 39R / 45E / 83T 19C / 81C / 37Y / 44D 19C / 81C / 37Y / 83T 19C / 81C / 37Y / 85S 19C / 81C / 39R / 83T 19C / 81C / 44D 19C / 81C / 44D / 85S 19C / 81C / 85S 19C / 81C / 83T 19C / 81C / 83T / 44D In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0022] As another example of a human germline sequence, germline gene family 3 includes germline gene family members 3-7 (SEQ ID NO:14), 3-9 (SEQ ID NO:15), 3-11 (SEQ ID NO:16), 3-13 (SEQ ID NO:17), 3-15 (SEQ ID NO:18), 3-20 (SEQ ID NO:19), 3-21 (SEQ ID NO:20), 3-23 (SEQ ID NO:21), 3-30 (SEQ ID NO:22), 3-33 (SEQ ID NO:23), 3-43 (SEQ ID NO:24), 3-48 (SEQ ID NO:25), 3-49 (SEQ ID NO:26), 3-53 (SEQ ID NO:27), 3-64 (SEQ ID NO:28), 3-66 (SEQ ID NO:29), 3-72 (SEQ ID NO:30), 3-73 (SEQ ID NO:31), 3-74 (SEQ ID NO:32), 3-d (SEQ ID NO:33), and 3-NL1 (SEQ ID NO:34), and alleles thereof, wherein the single immunoglobulin variable domain (or framework region(s) thereof) may comprise one or more of the following substitutions: 2A, 5Q, 14E, 23K, 23Q, 23Y, 28D, 28E, 28N, 28K, 28R, 30K, 30S, 31K, 33P, 35G, 35A, 35S, 37Y, 39R, 40P, 45E, 49A, 52E, 52D, 55E, 56E, 74E, 76K, 77Q, 82bD, 84E, 84P, 110V, and 110I.

[0023] In additional embodiments of the disclosure having a human germline gene family 3 origin, the single immunoglobulin variable domain (and its framework region(s)) may comprise one of the following substitution combinations: 5Q / 23Q 23Q / 77Q 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28K / 49A 28K / 49A / 55E / 84E 28K / 49A / 77Q 28K / 55E 28K / 55E / 74E 37Y / 49A / 74E 39R / 45E / 49A / 74E 39R / 49A / 84E 39R / 84E 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 49A / 84E

[0024] Moreover, in additional embodiments of the disclosure having a human germline gene family 3 origin, the single immunoglobulin variable domain (and its framework regions) may comprise one of the following combinations of substitutions: 23C / 77C / 28K / 49A 23C / 77C / 28D / 49A 23C / 77C / 28K / 55E 23C / 77C / 28K / 55E / 74E 23C / 77C / 28K / 49A / 55E / 84E 23C / 77C / 37Y / 49A / 74E 23C / 77C / 39R / 45E / 49A / 74E 23C / 77C / 39R / 49A / 74E 23C / 77C / 39R / 49A / 84E 23C / 77C / 39R / 49A / 84E 23C / 77C / 49A / 55E / 84E 34C / 78C / 28D 34C / 78C / 28K 34C / 78C / 49A 34C / 78C / 55E 34C / 78C / 74E 34C / 78C / 77Q 34C / 78C / 84E In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0025] As another example of a human germline sequence, germline gene family 4 can include germline gene family members 4-4 (SEQ ID NO:35), 4-28 (SEQ ID NO:36), 4-30-1 (SEQ ID NO:37), 4-30-2 (SEQ ID NO:38), 4-30-4 (SEQ ID NO:39), 4-31 (SEQ ID NO:40), 4-34 (SEQ ID NO:41), 4-38-2 (SEQ ID NO:42), 4-39 (SEQ ID NO:43), 4-59 (SEQ ID NO:44) and 4-61 (SEQ ID NO:45), 4-b (SEQ ID NO:46), and alleles thereof, and a single immunoglobulin variable domain (or framework region(s) thereof) can include one or more of the following substitutions: 1E, 10Q, 10T, 15G, 19I, 37Y, 39R, 45E, 82bD, 82bN, 84P, 107I, and 107Y.

[0026] In additional embodiments of the disclosure having a human germline gene family 4 origin, the single immunoglobulin variable domain (and its framework region(s)) may comprise one of the following substitution combinations: 10T / 82bN 10T / 84P 10T / 82bD 37Y (and / or 39R) / 82bN / 84P 37Y (and / or 39R) / 10T / 84P 37Y (and / or 39R) / 10T / 82bD 37Y (and / or 39R) / 10T / 82bN 39R / 45E / 82bD / 84P 45E / 82bD / 84P

[0027] Moreover, in additional embodiments of the disclosure having a human germline gene family 4 origin, the single immunoglobulin variable domain (and its framework regions) may comprise one of the following combinations of substitutions: 17C / 82aC / 10T 17C / 82aC / 10T / 82bN 17C / 82aC / 10T / 82bD 17C / 82aC / 82bN / 84P 17C / 82aC / 37Y(and / or 39R) / 10T / 82bD 17C / 82aC / 37Y(and / or 39R) / 10T / 84P 17C / 82aC / 37Y(and / or 39R) / 82bD / 84P 23C / 77C / 10T / 84P 23C / 77C / 39R / 45E / 82bD / 84P 23C / 77C / 45E / 82bD / 84P 23C / 77C / 82bD / 84P 23C / 77C / 82bN / 84P 23C / 77C / 37Y(and / or 39R) / 10T / 82bD 23C / 77C / 37Y(and / or 39R) / 10T / 82bN 23C / 77C / 37Y(and / or 39R) / 10T / 84P 23C / 77C / 37Y(and / or 39R) / 82bD / 84P 23C / 77C / 37Y(and / or 39R) / 82bD / 84P In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0028] As another example of a human germline sequence, germline gene family 5 can include germline gene family members 5-51 (SEQ ID NO:47) and 5-a (SEQ ID NO:48), and alleles thereof; a single immunoglobulin variable domain (or its framework region(s)) can include one or more of the following substitutions: 28D, 37Y, 39R, 45E, 48I, 60D, 60A, 68E, 76N, 83D, and 84E.

[0029] In additional embodiments of the disclosure having a human germline gene family 5 origin, the single immunoglobulin variable domain (and its framework region(s)) may comprise one of the following substitution combinations: 1.39R / 28D 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 28D / 48I / 84E 28D / 76N / 83D 28D / 76N / 84E 28D / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 39R / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 37Y / 48I / 83D 28D / 39R / 45E / 76N / 84E In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0030] As another example of a human germline sequence, germline gene family 6 can include germline gene family member 6-1 (SEQ ID NO:49) and its alleles.

[0031] As another example of a human germline sequence, germline gene family 7 can include germline gene family member 7-4-1 (SEQ ID NO:50) and its alleles.

[0032] In embodiments of the disclosure having a human germline gene family 7 origin, the single immunoglobulin variable domain (and its framework region(s)) may comprise one of the following substitution combinations: 17C / 82aC / 39R 17C / 82aC / 39R / 45E 17C / 82aC / 37Y 35C / 50C / 39R 35C / 50C / 39R / 45E 35C / 50C / 37Y In each of the above combinations, the combination may include, if not already present, at least one of 39R, 45E, and 37Y.

[0033] In another aspect, the present disclosure relates to polynucleotides encoding a single immunoglobulin variable domain of the present disclosure, any framework region(s) thereof.

[0034] In another aspect, the disclosure relates to a pharma- ceutically acceptable composition comprising a single immunoglobulin variable domain, any framework region(s) thereof.

[0035] In another aspect, the present disclosure relates to a VH domain library comprising a plurality of the single immunoglobulin variable domains disclosed herein.

[0036] In another aspect, the present disclosure relates to a polynucleotide library comprising a plurality of polynucleotides encoding a plurality of the single immunoglobulin variable domains disclosed herein.

[0037] In another aspect, the present disclosure relates to a method for identifying an antigen-binding molecule, the method comprising contacting a single immunoglobulin variable domain library of the present disclosure with a target, and (ii) identifying a single immunoglobulin variable domain of the library that binds to the target.

[0038] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which: [Brief description of the drawings]

[0039] [Figure 1A]FIG. 1A-H show V gene amino acid sequences for the most commonly observed alleles of functional human IGHV genes from several human antibody germlines. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 1H] Same as above.

[0040] [Figure 2A] Figures 2A-2O show the amino acid sequences of disulfide-stabilized full-length VH domains according to the present disclosure for germline gene family members VH1-8, VH1-18, VH1-69.2, VH3-9, VH3-11, 3-15, VH3-20, VH3-21, VH-30, VH3-53, VH4-34, VH4-39, and VH7-4-1. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above. [Figure 2M] Same as above. [Figure 2N] Same as above. [Figure 2O] Same as above.

[0041] [Figure 3A]3A-3N show the amino acid sequences of several modified full-length VH domains according to the present disclosure for germline gene family members VH3-20, VH3-21, VH3-15, VH1-69.2 and VH4-39. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above. [Figure 3L] Same as above. [Figure 3M] Same as above. [Figure 3N] Same as above.

[0042] [Figure 4A] Figures 4A-4F show the amino acid sequences of modified full-length VH domains of the present disclosure having selected combinations of amino acid substitutions in the full-length VH from members of germline families 1, 3 and 4 shown in Figures 2A-2M and 3A-3E, designated as Opt1 and Opt2 designs according to the present disclosure. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above.

[0043] [Figure 5A] 5A-5I show the V gene amino acid sequences of modified VH domains according to the present disclosure. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above.

[0044] [Figure 6] FIG. 6 shows a summary of amino acid substitutions and fold improvement in expression potency for selected sequences in germline families VH family 1 and VH family 3 according to the present disclosure.

[0045] [Figure 7A] 7A-7L show the amino acid sequences of each of the wild-type and modified amino acid sequences of the germline family in FIG. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 7I] Same as above. [Figure 7J] Same as above. [Figure 7K] Same as above. [Figure 7L] Same as above.

[0046] [Figure 8] 1 shows a summary of amino acid substitutions, expression potencies and thermal melting temperatures (Tm) for selected sequences in germline family VH4 members 4-34 and 4-39 according to the present disclosure.

[0047] [Figure 9]1 shows a summary of amino acid substitutions for selected wild-type and modified sequences in germline VH4 members 4-4, 4-28, 4-30-1, 4-30-2, 4-30-4, 4-31, 4-34, 4-38, 4-59 and 4-61 according to the present disclosure.

[0048] [Figure 10A] 10A-10N show the amino acid sequences for the wild-type and modified amino acid sequences for the germline VH4 sequences of FIG. 8 and FIG. 9, respectively. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 10G] Same as above. [Figure 10H] Same as above. [Figure 10I] Same as above. [Figure 10J] Same as above. [Figure 10K] Same as above. [Figure 10L] Same as above. [Figure 10M] Same as above. [Figure 10N] Same as above.

[0049] [Figure 11] 1 shows a summary of selected wild-type and modified amino acid substitutions (some including I37Y) in germline VH2 members 2-5 and 2-26 according to the present disclosure. [Figure 12] 1 shows a summary of selected wild-type and modified amino acid substitutions (some including I37Y) in germline VH2 members 2-5 and 2-26 according to the present disclosure.

[0050] [Figure 13A] 13A-13H show the amino acid sequences of the wild-type and modified amino acid sequences of the germline VH2 member 2-5 and 2-26 sequences of FIG. 11 and FIG. 12, respectively. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above.

[0051] [Figure 14] 1 shows a summary of amino acid substitutions of selected wild-type and modified sequences in germline family VH5 member 5-51. [Figure 15] 1 shows a summary of amino acid substitutions of selected wild-type and modified sequences in germline family VH5 member 5-51.

[0052] [Figure 16A] 16A-16F show the amino acid sequences for the wild-type and modified amino acid sequences for the VH5-51 sequences of FIG. 14 and FIG. 15, respectively. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 16F] Same as above.

[0053] [Figure 17] FIG. 17 shows an overview of substitutions in germline family members 1-8, 3-30, and 4-34, reflecting the effect of the 37Y variant according to the present disclosure.

[0054] [Figure 18A] 18A to 18C show the amino acid sequences of each of the amino acid sequences in FIG. [Figure 18B] Same as above. [Figure 18C] Same as above.

[0055] [Figure 19] Panels A and B show size exclusion chromatography (SEC) of the Gr6 human VH domain with and without various substitutions designed to reduce dimerization.

[0056] [Figure 20A] 20A-20U show the amino acid sequences of several germline family members that have been modified with substitutions according to the present disclosure. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 20E] Same as above. [Figure 20F] Same as above. [Figure 20G] Same as above. [Figure 20H] Same as above. [Figure 20I] Same as above. [Figure 20J] Same as above. [Figure 20K] Same as above. [Figure 20L] Same as above. [Figure 20M] Same as above. [Figure 20N] Same as above. [Figure 20O] Same as above. [Figure 20P] Same as above. [Figure 20Q] Same as above. [Figure 20R] Same as above. [Figure 20S] Same as above. [Figure 20T] Same as above. [Figure 20U] Same as above.

[0057] [Figure 21A] Figures 21A-H show an overview of amino acid substitutions and combinations thereof in VH families 1-69.2, 3-15, 3-21, 4-39, and 3-20, along with expression and Tm data. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 21E] Same as above. [Figure 21F] Same as above. [Figure 21G] Same as above. [Fig. 21H] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] explanation The present disclosure relates to the design and characterization of single immunoglobulin variable domains having substitutions within the variable regions that result in one or more of improved thermal stability, improved cellular expression, reduced dimerization, and reduced light chain pairing.

[0059] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0060] Prior to describing the various aspects of the present disclosure, certain terms are defined. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0061] As utilized in accordance with the present disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0062] The term "amino acid" or "residue" as used in this application refers to the group of naturally occurring carboxy α-amino acids including alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cyan, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0063] The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody as described herein.

[0064] "Antibody" refers to a glycoprotein that contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds and has a structure substantially similar to that of a native antibody structure. For example, a native IgG class antibody is a heterotetrameric glycoprotein of about 150 kilodaltons (kD) composed of two light chains and two heavy chains disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) followed by a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0065] "Germline" as used herein refers to DNA-encoded amino acid sequences that are transmitted between generations. Human antibody germline genes and polypeptide sequences, including wild-type functional VDJ gene segments, can be found on the ImMunnoGeneTics (IMGT®) website (http: / / www.imgt.org / ). IMGT® is a global reference in immunogenetics and immunoinformatics, an integrated knowledge resource, specifically dedicated to immunoglobulins (IG) or antibodies. IMGT® provides common access to sequence, genomic and structural immunogenetic data. IMGT® works closely with EBI (Europe), DDBJ (Japan) and NCBI (USA). See also Barker, et al., The IPD-IMGT / HLA database, Nucleic Acids Research, gkac1011, November 2022, https: / / doi.org / 10.1093 / nar / gkac1011.

[0066] Many gene family members have one or several known polymorphisms (referred to by IMGT® as -*01, -*02, etc., e.g., "3-64-*01"). Unless otherwise indicated, for each of the V gene sequences identified in this disclosure, the *01 allele is shown as representative for the family members.

[0067] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have a similar structure, with each full-length domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). Although a single full-length VH or VL domain may be sufficient to confer antigen-binding specificity, the disclosure herein focuses on the VH domain and, in some embodiments, its V gene portion.

[0068] The term "complementarity determining region(s)" or "CDR(s)" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six CDRs: three full-length VH (HCDR1, HCDR2, HCDR3) and three full-length VL (LCDR1, LCDR2, LCDR3).

[0069] "Framework" or "FR" refers to variable domain residues other than the CDR residues. The FR of a full-length variable domain generally consists of four FR regions: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the following sequence in either VH or VL: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For simplicity in the context of the VH domain described herein, references to FR1, FR2, FR3 and FR4 are intended to refer to the FR regions of the VH domain (with the understanding that the VL domain also has FRs).

[0070] As used herein, "IGHV" refers to the amino acid sequence of the V gene portion of a full-length VH, including FR1, CDR1, FR2, CDR2, and FR3. In some examples, the V gene encodes some amino acids of CDR3. The V gene portion is recombinantly fused to one of approximately 23 functional D chains and one of six J chains to form a mature full-length VH domain. The HCDR3 region is the most diverse region of the full-length VH domain, consisting of sequences from the V gene, D chain, and J chain, and contains significant diversity generated by insertions, deletions, and mutations that occur at the junction during recombination. The J chain encompasses the latter portion of HCDR3 and the entire FR4. The FR4 regions of the six J chains are fairly well conserved (i.e., there is little diversity), and are shown here with the amino acids of FR4 underlined. JH1 AEYFQHWGQGTLVTVSS (SEQ ID NO:51) JH2 YWYFDLWGRGTLVTVSS (SEQ ID NO:52) JH3 DAFDVWGQGTMVTVSS (SEQ ID NO:53) JH4 YFDYWGQGTLVTVSS (SEQ ID NO:54) JH5 NWFDSWGQGTLVTVSS (SEQ ID NO:55) JH6 YYYYYGMDVWGQGTTVTVSS (SEQ ID NO:56)

[0071] As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in variable domains are numbered herein with the "Kabat numbering system" to assign positions for any variable region sequence, without reliance on experimental data beyond the sequence itself. According to the Kabat numbering system, CDR1 includes amino acids 23-35 (including amino acids 31a and 31b, if present), CDR2 includes amino acids 50-58 (including amino acids 52a, 52b and 52c, if present), and CDR3 includes amino acids 93-102 (including amino acids 100a, 100b, 100c, 100d, 100e, 100f, 100g, 100h, 100i, 100j, 100k and 100l, if present) (see, e.g., North et al 2013, J Mol Biol. 2011 406(2):228-256). Many of the VH sequences of the present disclosure encompass different lengths as a result of variability in the length of the CDRs, and therefore positions with lowercase letters (a, b, c, etc.) are used according to the Kabat numbering system. For example, many of the sequences of this disclosure do not have an amino acid at one or more of positions 31a, 31b, 52a, 52b, and 52c, 100a, 100b, 100c, 100d, 100e, 100f, 100g, 100h, 100i, 100j, 100k, and 100l. Accordingly, some of the tables and figures disclosed herein reflect positions within the Kabat numbering system that do not have an amino acid at that position (shown herein as a "·" or blank at that position).

[0072] The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system, however, it is well within the skill of one in the art to convert the numbering of the sequences in the sequence listing to the Kabat numbering system, and vice versa.

[0073] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms.

[0074] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides.

[0075] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0076] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a pharma- ceutical acceptable carrier and a therapeutically effective amount of an immunotoxin fusion protein of the present disclosure.

[0077] As used herein, the term "pharmaceutical acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of one or more heavy chain variable domains of the present disclosure.

[0078] Turning now to various aspects of the present disclosure, the inventors have identified approaches to modify the biophysical properties of single chain VH domains from several human immunoglobulin germline sequences. Substitutions of the VH domains can result in improved biophysical properties and, alone or in combination, enhance the therapeutic utility of the VH domains for human and non-human medicine.

[0079] 1A-1H show functional germline wild type IGHV amino acid sequences from several VH genes. These WT IGHV sequences have been modified to provide exemplary modified IGHV sequences according to the present disclosure. The variable domain germline sequences shown in FIG. 1A-1H include FR1, CDR1, FR2, CDR2 and FR3, regions from amino acids 1-94 or 95 (according to the Kabat numbering system), encoding regions that encode optimized sequence variants. The sequences do not include CDR3 or FR4, because these segments are derived from the D and J chains as a result of homologous recombination that generates diversity, and are therefore highly variable across antibodies. Thus, although CDR3 and FR4 may be present in the full-length VH embodiments of the present disclosure and may themselves act on the stability of the full-length VH domain, some embodied improvements of the present disclosure are independent of CDR3 and FR4, except for those variants having substitutions at amino acids 102, 105, 107 or 110, for example. Examples of these full-length sequences are shown, for example, in Figures 2A-2O and 3A-3N. Where FR4 amino acids are shown, they are intended to be representative of the six J segments available in the human genome. However, some embodiments of the present disclosure include only the V gene portion of the full-length VH, even though D and J chain sequences are shown as part of the sequences disclosed herein.

[0080] In a first approach to modifying IGHV domains according to the present disclosure, several human germline-derived IGHV sequences were modified to introduce cysteine ​​residues and create novel cysteine ​​bonds between residues. In a second approach, IGHV sequences were modified to substitute amino acids at various positions. In a third approach, a combination of both novel cysteine ​​bonds and other modified amino acids was introduced. Each approach can be used on IGHV sequences and full-length VH domains across one or more germline families to modify at least one of the following properties of the domain: thermostability, cellular expression, VH dimerization, and light chain pairing.

[0081] According to one or more of the approaches identified herein, one or more substitutions introduce cysteine ​​residues that generate one or more new disulfide bonds in the IGHV sequence or full-length VH. In certain embodiments, the IGHV sequence or full-length VH of the present disclosure contains cysteine ​​residues at the following positions in combination (according to the Kabat numbering system): positions 2 and 102; positions 17 and 82a; positions 19 and 81; positions 23 and 77; positions 34 and 78; positions 35 and 50, which result in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C. The cysteine ​​bonds between these positions can conformationally lock down and stabilize the modified VH domain. For example, Figures 2A-2O show several VH domains of the disclosure that have novel disulfide bond-forming cysteine ​​residues, which may be referred to herein as "cys clamp(s)." Additionally, several other sets of figures herein include one of these sets of cysteine ​​substitutions as further described herein.

[0082] In another approach to modifying and / or improving the biophysical properties of the VH domains of the present disclosure, VH domains from several human germline families were modified to provide the following amino acids (according to the Kabat numbering system): 1E, 2A, 5Q, 10Q, 10T, 14E, 15G, 16D, 16Q, 19I, 23K, 23Q, 23Y, 25F, 25Y, 28D, 28E, 28K, 28N, 28R, 30K, 30S, 31K. , 33P, 35A, 35G, 35S, 37F, 37Y, 37H, 39R, 40P, 44D, 45E, 48I, 49A, 52E, 52D, 55E, 56E, 60A, 60D, 65D, 68E, 73D, 73P, 74E, 76 K, 76N, 77Q, 82bD, 82bN, 83D, 83K, 83L, 83Q, 83T, 84E, 84P, 84Y, 85K, 85R, 85S, 85T, 89I, 105D, 107I, 107Y, 110I, and 110V

[0083] Moreover, combinations of two or more of these (or other) amino acids can be used to alter and / or improve the biophysical properties of the VH domain. In various embodiments of the disclosure, combinations can include, for example: 5Q / 23Q 10Q / 48I / 84E 10T / 82bD 10T / 82bD 10T / 82bN 10T / 84P 15G / 37Y 15G / 44D 15G / 85S 15G / 83T 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 23Q / 77Q 28D / 37Y / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 39R / 45E / 76N / 84E 28D / 39R / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 48I / 83D 28D / 48I / 84E 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28D / 76N / 83D 28D / 76N / 84E 28K / 49A 28K / 49A / 77Q 28K / 49A / 55E / 84E <h2 style=";text-align:left;direction:ltr">28K / 49A / 55E / 84E / 10T / 82bN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 55E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 28K / 55E / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37F / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 10T / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 10T / 82bD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y(または39R) / 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 39R / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 39R / 45E / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 44D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 49A / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 85S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 37Y / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 28D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 45E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 60A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 60D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 68E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 76N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 83D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 45E / 48I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 45E / 49A / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39R / 45E / 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 44D / 85S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 44D / 83T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 45E / 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 55E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 55E / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 55E / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 74E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 74E / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 77Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 77Q / 55E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 77Q / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 45E / 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 49A / 84E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 82bD / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 82bN / 84P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 83T / 44D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] In some embodiments of the modified IGHV sequences and VH domains of the present disclosure, position 39 is modified to arginine (39R), which may result in increased solubility and decreased tendency to pair with VL domains. Figures 3A-3N show some VH domains of the present disclosure with selected amino acid substitutions according to the present disclosure. In some aspects, the modified IGHV sequences and VH domains of the present disclosure include a substitution of position 37 to tyrosine (37Y), which reduces light chain pairing and dimerization with other VH domains. In some aspects of the present disclosure, the modified IGHV sequences and VH domains include one or both of 39R and 37Y. Furthermore, adding 37Y to a VH domain may have a neutral or positive effect on expression and stability across VH domains from multiple VH families. Thus, each of the IGHV sequences and VH domains according to the present disclosure may include 37Y and / or 39R, if not already present.

[0085] In some embodiments of the germline sequences described herein, an amino acid that may be modified in one IGHV sequence or VH domain is natural in another IGHV sequence or VH domain. For example, amino acid 49 in the germline VH IGHV3-7 sequence in Figure 1C is an alanine, while the germline amino acid sequence at position 49 in IGHV 3-9 is a serine, which is modified to an alanine in the stabilizing variant as shown in Figure 5D. Additionally, the sequences shown in all figures use the -*01 allele as a representative of all family polymorphisms readily available from the IMGT® database.

[0086] Combining the above approaches may result in further improvement of the properties of the VH domain.Thus, any one or more of the above non-cysteine ​​substitutions or combinations thereof can be combined with any one of the cysteine ​​combinations (cys clamp).In a particular example, any one of the above combinations of cysteine ​​residues can be further combined with any one or more of the amino acid substitutions and combinations thereof of the present disclosure, which may include any of the above combinations.

[0087] Also included in the combination may be 39R and 37Y, if not already included. The combination results in an IGHV sequence or VH domain having one of the following cys clamps: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C, in combination with one or more single amino acid substitutions or combinations thereof, as disclosed herein.

[0088] IGHV sequences and VH domains from several human antibody germlines are suitable for substitution to provide improved properties according to various embodiments of the disclosure, including, for example, VH Family 1, VH Family 2, VH Family 3, VH Family 4, VH Family 5, and VH Family 7. Further examples of substitutions, particularly human antibody germlines, are provided below.

[0089] Examples of substitutions for germline family 1 Examples of IGHV sequences include members of germline V gene family 1, such as germline family gene members 1-2 (SEQ ID NO:1), 1-3 (SEQ ID NO:2), 1-8 (SEQ ID NO:3), 1-18 (SEQ ID NO:4), 1-24 (SEQ ID NO:5), 1-45 (SEQ ID NO:6), 1-46 (SEQ ID NO:7), 1-58 (SEQ ID NO:8), 1-69 (SEQ ID NO:9), and 1-69.2 (also known as 1-f) (SEQ ID NO:10), and alleles thereof.

[0090] In various embodiments of the present disclosure, members of germline family 1 can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, resulting in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C.

[0091] Additionally, exemplary Family 1 substitutions may include one or more of the following: 10Q, 16D, 16Q, 25Y, 25F, 37F, 37Y, 39R, 45E, 48I, 84E, 84P, 110V, and 110I.

[0092] Exemplary Family 1 substitution combinations include, but are not limited to, the following: 10Q / 48I / 84E 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 37F / 48I 37Y / 48I 39R / 45E / 48I 39R / 48I

[0093] Additionally, Family 1 substitutions include either 17C / 82aC or 34C / 78C along with other single or multiple substitutions, providing the following exemplary substitution combinations: 17C / 82aC / 10Q / 48I / 84E 17C / 82aC / 16D 17C / 82aC / 16D / 37F 17C / 82aC / 16D / 37Y 17C / 82aC / 16D / 37Y / 39R 17C / 82aC / 16D / 39R 17C / 82aC / 16D / 39R / 48I 17C / 82aC / 16D / 48I 17C / 82aC / 37F 17C / 82aC / 37Y 17C / 82aC / 37Y / 48I 17C / 82aC / 39R 17C / 82aC / 39R / 45E / 48I 17C / 82aC / 39R / 48I 17C / 82aC / 84E 34C / 78C / 16D 34C / 78C / 37F 34C / 78C / 84E 34C / 78C / 16D / 37F 34C / 78C / 16D / 48I 34C / 78C / 10Q / 48I / 84E

[0094] As described herein, each of the combinations may include, or if not already included, one or more of 37Y, 39R, and 45E.

[0095] Examples of substitutions for germline family 2 Examples of IGHV sequences include members of germline V gene family 2, such as germline family gene members 2-5 (SEQ ID NO:11), 2-26 (SEQ ID NO:12), and 2-70 (SEQ ID NO:13), and alleles thereof.

[0096] In various embodiments of the present disclosure, members of germline family 2 can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, resulting in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C.

[0097] Additionally, exemplary Family 2 substitutions can include one or more of the following: 15G, 16D, 37Y, 37H, 39R, 44D, 45E, 65D, 73D, 73P, 83L, 83Q, 83K, 83T, 84Y, 85R, 85S, 85K, 85T, 89I, 105D, 107I.

[0098] Exemplary Family 2 substitution combinations include, but are not limited to, the following: 15G / 37Y 15G / 44D 15G / 85S 15G / 83T 37Y / 39R / 45E / 83T 37Y / 39R / 83T 37Y / 44D 37Y / 85S 37Y / 83T 39R / 83T 44D / 85S 44D / 83T

[0099] Additionally, Family 2 substitutions include 19C / 82C along with other single or multiple substitutions, providing the following exemplary substitution combinations: 19C / 81C / 15G 19C / 81C / 15G / 37Y 19C / 81C / 15G / 44D 19C / 81C / 15G / 85S 19C / 81C / 15G / 83T 19C / 81C / 37Y 19C / 81C / 37Y / 39R / 83T 19C / 81C / 37Y / 39R / 45E / 83T 19C / 81C / 37Y / 44D 19C / 81C / 37Y / 83T 19C / 81C / 37Y / 85S 19C / 81C / 39R / 83T 19C / 81C / 44D 19C / 81C / 44D / 85S 19C / 81C / 85S 19C / 81C / 83T 19C / 81C / 83T / 44D

[0100] As described herein, each of the combinations may include, if not already included, one or more of 37Y, 39R, and 45E.

[0101] Examples of substitutions for germline family 3 Exemplary VH domains of the disclosure include members of germline V gene family 3, such as germline family gene members 3-7 (SEQ ID NO:14), 3-9 (SEQ ID NO:15), 3-11 (SEQ ID NO:16), 3-13 (SEQ ID NO:17), 3-15 (SEQ ID NO:18), 3-20 (SEQ ID NO:19), 3-21 (SEQ ID NO:20), 3-23 (SEQ ID NO:21), 3-30 (SEQ ID NO:22), 3-33 (SEQ ID NO:23), 3-43 (SEQ ID NO:24), 3-48 (SEQ ID NO:25), 3-49 (SEQ ID NO:26), 3-53 (SEQ ID NO:27), 3-64 (SEQ ID NO:28), 3-66 (SEQ ID NO:29), 3-72 (SEQ ID NO:30), 3-73 (SEQ ID NO:31), 3-74 (SEQ ID NO:32), 3-d (SEQ ID NO:33), and 3-NL1 (SEQ ID NO:34), and alleles thereof.

[0102] In various embodiments of the present disclosure, germline family 3 members can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, resulting in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C.

[0103] Additionally, exemplary Family 3 substitutions may include one or more of the following: 2A, 5Q, 14E, 23K, 23Q, 23Y, 28D, 28E, 28N, 28K, 28R, 30K, 30S, 31K, 33P, 35G, 35A, 35S, 37Y, 39R, 40P, 45E, 49A, 52E, 52D, 55E, 56E, 74E, 76K, 77Q, 82bD, 84E, 84P, 110V, 110I.

[0104] Exemplary Family 3 substitution combinations include, but are not limited to, the following: 5Q / 23Q 23Q / 77Q 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28K / 49A 28K / 49A / 55E / 84E 28K / 49A / 77Q 28K / 55E 28K / 55E / 74E 37Y / 49A / 74E 39R / 45E / 49A / 74E 39R / 49A / 84E 39R / 84E 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 49A / 84E

[0105] Additionally, Family 3 substitutions include either 23C / 77C with other single or multiple substitutions, providing the following exemplary substitution combinations: 23C / 77C / 28K / 49A 23C / 77C / 28D / 49A 23C / 77C / 28K / 55E 23C / 77C / 28K / 55E / 74E 23C / 77C / 28K / 49A / 55E / 84E 23C / 77C / 37Y / 49A / 74E 23C / 77C / 39R / 45E / 49A / 74E 23C / 77C / 39R / 49A / 74E 23C / 77C / 39R / 49A / 84E 23C / 77C / 39R / 49A / 84E 23C / 77C / 49A / 55E / 84E 34C / 78C / 28D 34C / 78C / 28K 34C / 78C / 49A 34C / 78C / 55E 34C / 78C / 74E 34C / 78C / 77Q 34C / 78C / 84E

[0106] As described herein, each of the combinations may include, if not already included, one or more of 37Y, 39R or 45E.

[0107] Examples of substitutions for germline family 4 Exemplary VH domains of the present disclosure include members of germline V gene family 4, e.g., germline family gene members include 4-4 (SEQ ID NO:35), 4-28 (SEQ ID NO:36), 4-30-1 (SEQ ID NO:37), 4-30-2 (SEQ ID NO:38), 4-30-4 (SEQ ID NO:39), 4-31 (SEQ ID NO:40), 4-34 (SEQ ID NO:41), 4-38-2 (SEQ ID NO:42), 4-39 (SEQ ID NO:43), 4-59 (SEQ ID NO:44) and 4-61 (SEQ ID NO:45), 4-b (SEQ ID NO:46), and alleles thereof.

[0108] In various embodiments of the present disclosure, germline family 4 members can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, resulting in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C.

[0109] Additionally, exemplary Family 4 substitutions can include one or more of the following: 1E, 10Q, 10T, 15G, 19I, 82bD, 82bN, 84P, 107I, 107Y, and combinations thereof.

[0110] Exemplary Family 4 substitution combinations include: 10T / 82bN 10T / 84P 10T / 82bD 37Y (and / or 39R) / 82bN / 84P 37Y (and / or 39R) / 10T / 84P 37Y (and / or 39R) / 10T / 82bD 37Y (and / or 39R) / 10T / 82bN 39R / 45E / 82bD / 84P 45E / 82bD / 84P 82bN / 84P 82bD / 84P

[0111] Additionally, Family 4 substitutions include either 17C / 82aC or 23C / 77C along with other single or multiple substitutions, providing the following exemplary substitution combinations: 17C / 82aC / 10T 17C / 82aC / 10T / 82bN 17C / 82aC / 10T / 82bD 17C / 82aC / 82bN / 84P 17C / 82aC / 37Y(and / or 39R) / 10T / 82bD 17C / 82aC / 37Y(and / or 39R) / 10T / 84P 17C / 82aC / 37Y(and / or 39R) / 82bD / 84P 23C / 77C / 10T / 84P 23C / 77C / 39R / 45E / 82bD / 84P 23C / 77C / 45E / 82bD / 84P 23C / 77C / 82bD / 84P 23C / 77C / 82bN / 84P 23C / 77C / 37Y(and / or 39R) / 10T / 82bD 23C / 77C / 37Y(and / or 39R) / 10T / 82bN 23C / 77C / 37Y(and / or 39R) / 10T / 84P 23C / 77C / 37Y(and / or 39R) / 82bD / 84P 23C / 77C / 37Y(and / or 39R) / 82bD / 84P In each of the exemplary Family 4 combinations, the combination may also include, if not already present, one or more of 37Y, 39R and 45E.

[0112] Examples of substitutions for germline family 5 Exemplary VH domains of the present disclosure include members of germline V gene family 5, such as germline family gene members 5-51 (SEQ ID NO:47) and 5-a (also known as 5-10) (SEQ ID NO:48), and alleles thereof.

[0113] In various embodiments of the present disclosure, germline family 5 members can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, resulting in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C.

[0114] Additionally, exemplary Family 5 substitutions can include one or more of the following: 28D, 37Y, 39R, 45E, 48I, 60A, 60D, 68E, 76N, 83D, and 84E, alone, in combination, or in combination with one of the cys clamps described herein.

[0115] Exemplary Family 5 substitution combinations include, but are not limited to, the following: 39R / 28D 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 28D / 48I / 84E 28D / 76N / 83D 28D / 76N / 84E 28D / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 39R / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 37Y / 48I / 83D 28D / 39R / 45E / 76N / 84E

[0116] In each of the exemplary Family 5 combinations, the combination may also include, if not already present, one or more of 37Y, 39R and 45E, and one of the cys clamps described herein.

[0117] Examples of substitutions for germline family 7 Exemplary VH domains of the disclosure include members of germline V gene family 7, such as germline family gene member 7-4-1 (SEQ ID NO:50).

[0118] In various embodiments of the disclosure, members of germline family 7 can be modified to contain combinations of cysteine ​​residues at the following positions (according to the Kabat numbering system): 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50, which result in the following amino acid combinations: 2C / 102C; 17C / 82aC; 19C / 81C; 23C / 77C; 34C / 78C; and 35C / 50C, which can be combined with one or more of 37Y, 39R, and 45E.

[0119] Exemplary Family 7 substitution combinations include, but are not limited to, the following: 17C / 82aC / 39R 17C / 82aC / 39R / 45E 17C / 82aC / 37Y 35C / 50C / 39R 35C / 50C / 39R / 45E 35C / 50C / 37Y

[0120] In other embodiments of the present disclosure, the human antibody germline family 6 member may be modified with any of the aforementioned amino acid substitutions or substitutions thereof.

[0121] Table 1 provides an overview of single amino acid substitutions in specific gene families that provided improved expression and / or stability for several VH domains of the present disclosure. [Table 1]

[0122] Table 2 provides an overview of exemplary combinations of amino acids, particularly from the germline family, that provide improved stability and / or expression of several VH domains of the present disclosure. [Table 2-1] [Table 2-2] [Table 2-3]

[0123] Further embodiments of the present disclosure include only framework sections (FR1, FR2.FR3 or FR4) or sections of IGHV sequence or VH domain. For example, one or more framework sections are of germline family members modified according to the present disclosure. Furthermore, the present disclosure includes IGHV or full-length VH, such that the CDRs may be the same or different from the CDRs of the IGHV sequence or VH domain identified herein. Thus, aspects of the present disclosure relate to a polypeptide that includes one framework region or two, three or four framework regions of a human heavy chain V gene portion (IGHV) or full-length VH of an antibody, where the IGHV amino acid sequence or full-length VH includes one or more amino acid substitutions that result in improved biophysical properties, such as increased thermal stability, increased cellular expression, and reduced VH dimerization and light chain pairing, compared to a wild-type IGHV sequence lacking one or more amino acid substitutions. The IGHV sequence may also include a framework portion of the J chain. The polypeptide may include any one of the amino acid substitutions described above or a combination thereof. As long as one of the modified amino acids falls within one of the CDRs of the IGHV or VH domain, the remainder of the CDR may be the same or different from those identified in the sequences disclosed herein.

[0124] In some of the figures, the CDR3 of some amino acid sequences (amino acid positions 93-102, including amino acids 100a, 100b, 100c, 100d, 100e, 100f, 100g, 100h, 100i, 100j, 100k, and 100l according to the Kabat numbering system) are identified with an "X" amino acid. Consideration of CDR3 across several germlines reflects that the CDR3 sequences have only a limited amount of homology. As an example, for the VH domains in Figures 2A-2O: (a) a minimum of 6% and a maximum of 50% identity between the HCDR3s with an average identity of around 25% across all sequences; (b) The minimum length was 12 residues, the maximum length was 21 residues, and the average length was 14.6 residues.

[0125] Similarly, with respect to the VH domains of Figures 3A-3N: (a) a minimum of 6% and a maximum of 50% identity between the HCDR3s with an average identity of around 25% across all sequences; (b) The minimum length is 12 residues, the maximum length is 21 residues, and the average length is 14.6 residues.

[0126] These data indicate that the stabilizing effects observed as a result of the various VH domain substitutions tested (see, e.g., Example 1) were not HCDR3 dependent. Instead, the data indicate that the amino acid substitutions disclosed herein were surprisingly and unexpectedly stabilizing for each of their germline families, regardless of CDR3. Some of the variable domain portions of the germline origin modified VH domains of the present disclosure shown in Figures 1A-1H and 5A-5I include amino acids 1-94 or 95 (according to the Kabat numbering system) and represent V genes.

[0127] The VH substitutions of the present disclosure have been shown to improve at least the stability and / or expression of VH domains originating from multiple germline origins.Therefore, such substitutions are not limited to a specific VH domain amino acid sequence, but can instead be useful across a wide range of germline and sequence.Furthermore, the VH substitutions described herein can result in increased stability and / or expression regardless of CDR and their corresponding antigen or epitope.Therefore, the VH substitutions described herein are suitable for use with any VH domain, regardless of germline and regardless of CDR.

[0128] In other aspects of the disclosure, substitutions may be used in sequences that are similar but not identical to the IGHV sequences or full-length VH domains described herein. For example, the substitutions described herein may be used in sequences that are at least 50%, 60, 70%, 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95% or 99% identical to the IGV sequences or full-length VH domains described herein, with the CDRs excluded from determining percent identity. For example, the substitutions of the present disclosure may be used in IGVH sequences or VH domains having at least 50%, 60, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95% or 99% identity to any one of the framework portions of SEQ ID NOs: 1-50 and 76-627 and their alleles, along with other IGHV and VH domains of human antibody germline sequences.

[0129] The IGVH sequences and VH domains of the present disclosure can be synthesized or expressed by methods known in the art. For example, the IGVH sequences and VH domains of the present disclosure can be synthesized or expressed in genetically engineered animals, such as mice, rats, rabbits, and cows, where the VH locus or separate transgenes are replaced, and the endogenous heavy and light chain, lambda and kappa loci are inactivated or cannot express endogenous heavy and light chain genes (Bruggeman et al., Human Antibody Production in Transgenic Animals Arch. Immunol. Ther. Exp. 63, 101-108 (2015). https: / / doi.org / 10.1007 / s00005-014-0322-x). Furthermore, the VH domains of the present disclosure can be incorporated into a polypeptide library display system to allow for the selection and engineering of sequences with the biophysical properties and therapeutic relevance described herein. Display systems include, for example, phage display, the HuARG™ mammalian display system (Kielczewska, A. et al. Development of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence-based affinity maturation. J Biol Chem 298, 101533, doi:10.1016 / j.jbc.2021.101533(2022)); ribozyme display, yeast surface display, bacterial display, and mammalian display.

[0130] In some embodiments, the IGVH sequences and VH domains disclosed herein may be combined with other VH domains in sequences (5'-3' or 3'-5') to provide stabilized molecules that bind to one or more molecular targets that may be associated with the control or regulation of biological processes, such as processes associated with the treatment of human and non-human diseases. Thus, the IGVH sequences and VH domains disclosed herein may be formulated with pharma- ceutically acceptable carriers, excipients or stabilizers as pharmaceutical compositions. In certain embodiments, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration, using methods known in the art. The term "pharma-ceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and other therapeutic agents as appropriate. Such pharma-ceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium, and the like. These and additional known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005), and "Physician's Desk Reference", 60th ed., Medical Economics, Montvale, NJ (2005). A pharma-ceutically acceptable carrier suitable for the desired or required mode of administration, solubility and / or stability can be selected. EXAMPLES

[0131] The following examples illustrate certain embodiments of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0132] Example 1 - Disulfide Stabilization The first approach is to identify potential novel disulfides that can be used to stabilize VH domains of different germline families. Homology models were generated for eight diverse VH sequences representing VH families 1-5 by identifying the most appropriate crystal structure (considering resolution and sequence similarity) and modifying any non-germline residues to germline using RosettaScript. All VH coordinates were originally complexed within the multi-domain context of an antibody Fab.

[0133] The starting VH structure was diversified by building two homology models from either a single structure or two separate structures for in silico mutagenesis. Computational prediction of possible stabilizing disulfide bonds was performed in silico by modifying two residues at a time to cys and evaluating all combinations in the structure based on geometric constraints and then evaluating them based on energy functions (Gaurav et al., Nature 538:7625(2016):329-335). The results were classified based on the disulfide score (dslf_fa13), and models showing a score less than -0.3 were considered for experimental testing. Table 3 shows the starting structures of the eight frameworks built based on crystal structures deposited in the Protein Data Bank (PDB). [Table 3-1] [Table 3-2]

[0134] A large number of disulfide pairs were experimentally evaluated. All VH domains tested had unique HCDR3 and bound to different antigens. The purpose of using VH domains with diverse HCDR3 sets was to test the generalizability of the results obtained for each new disulfide, regardless of HCDR3 sequence.

[0135] For testing, the nucleotide sequence encoding the VH domain sequence was first cloned into a mammalian expression plasmid. The plasmid contained a CMV promoter-driven open reading frame and a BGH polyA tail. A mouse IgG signal peptide was used to drive secretion. The VH domain was recombinantly fused to human IgG1-Fc at the hinge region.

[0136] Cloning and plasmid production were performed using standard molecular biology methods. Secreted proteins were produced by transfecting the plasmids into HEK293 cells for transient expression using the Thermofisher Expi293 system. Supernatants for protein characterization were collected by centrifugation and then filtered. Determination of VH-Fc protein titers was performed on a GatorBio biointerferometry instrument using a Protein A chip supplied by the manufacturer and purified VH-Fc as a standard. Alternatively, for VH-His tagged proteins, titers were performed (1) in a similar manner on a GatorBio instrument using an anti-His tag chip and purified human PD1-His tagged protein as a standard, or (2) by performing SDS-PAGE analysis on HEK293 supernatants and quantifying protein levels using densitometry and using purified VH-His tagged protein as a standard. For stability measurements, mammalian supernatants were analyzed using differential scanning fluorimetry (DSF) using a QuantStudio3 according to the manufacturer's protocol (Applied Biosystems), and fluorescence versus temperature curves were analyzed using Applied Biosystem's Protein Thermal Shift™ software version 1.4.

[0137] Five novel disulfides were tested (Figures 2A-O) and consistently demonstrated the ability to improve the expression yield of poorly expressed VH domains and / or improve the stability of the VH domain, with some selectivity for individual disulfide pairs for each of the germline families (Table 4).

[0138] In the germlines of the VH1 family, several disulfide pairs improve expression and stability. One particular disulfide, 17C-82aC, appears to be superior in improving both expression and stability of three different VH1 family member germlines (Table 4). When the VH1-69.2 germline was tested for two VH domains that bind different antigens and contain significantly different HCDR3 residues, the 17C-82aC disulfide was superior in both VHs. The 35C-50C disulfide also increased the stability of all of the VH1 germlines tested. The 23C-77C and 19C-81C disulfides improved the stability of the majority of the VH1 germlines (Table 4).

[0139] In the germline of the VH3 family, some disulfides improve expression and stability. One particular disulfide, 23C-77C, is superior in improving both expression and stability of all seven VH3 family member germlines tested (Table 4). The VH3-20 germline was tested for two VH domains that bind different antigens and contain significantly different HCDR3 residues, and the 23C-77C disulfide improves the expression and stability of both VHs. The 17C-82aC, 19C-81C and 35C-50C disulfides improved the stability of most of the VH3 germlines (Table 4).

[0140] For the disulfide engineered set of VH domains tested: (1) There is a minimum of 0% and a maximum of 50% identity between HCDR3s, with one outlier pair having 81% identity (for this outlier pair, one is VH1-8 and the other is VH3-20). There is an average of nearly 25% identity across all HCDR3 sequences, which sets the sequences very far apart from each other. (2) The minimum length was 6 residues, the maximum length was 17 residues, and the average length was 12.2 residues.

[0141] The data obtained on the CDR3 composition of the various VH domains tested indicates that the designed substitutions (see, e.g., Examples 1 and 2, also described above) were stabilizing for each of those germline families, and that the construct design and observed stabilizing effect were not HCDR3-dependent.

[0142] Two VH4 family member germlines were tested, and the results were different for each VH4 member: the 23C-77C, 35C-50C, and 2C-102C disulfides significantly improve expression of the VH4-34 germline, while the 17C-82aC and 19C-81C disulfides significantly improve expression of the VH4-39 germline (Table 4).

[0143] Finally, the VH7 family consists of one germline member, VH7-4-1. Both the 17C-82aC and 35C-50C disulfides are involved in expression and T values ​​above 10°C. m This resulted in a substantial increase in

[0144] Table 4 shows the expression titers, change in expression titers relative to wild type, and results of stability experiments for some of the disulfide-stabilized VH domains of the present disclosure. The amino acid sequences of the VH domains summarized in Table 4 are provided in Figures 2A-2O. [Table 4-1] [Table 4-2] [Table 4-3] The CH2 domain of the Fc unfolds at a Tm of approximately 71° C., thus preventing the ability to quantify VH Tms with improved stability above 71° C. Disulfides likely improve stability, but in unmodified molecules with Tms above 71° C., the effect of disulfides has been difficult to characterize.

[0145] Example 2 - Discovery of stabilizing variants Computational design was utilized to identify additional residues where amino acid substitutions could result in increased stability. The same homology model used in Example 1 was utilized to generate libraries of primarily single amino acid variants and a small number of combinatorial variants. The energy of these homology models was then minimized in Rosetta software using existing protocols in RosettaScripts (Froning, K., et al. Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics. Nat Commun 11, 2330 (2020).). In silico site saturation mutagenesis was performed in which each position in the protein was replaced with all possible amino acids (except cys). Each point mutation was compared to the score of the WT sequence to calculate the energy difference (ΔE). The average scores of the target sequences were then sorted by value to rank the mutations for experimental testing.

[0146] VH domain-IgG1Fc variants were generated using the same methodology as described in Example 1. Approximately 200 variants were generated and screened across three VH families containing five different germlines (VH3-15, VH3-20, VH3-21, VH1-69.2, and VH4-39) (Figures 3A-N). A subset of these variants were found to improve expression of each domain and were consistent with the thermostability data (DSF T) of some molecules. m ) are shown in Table 5. [Table 5-1] [Table 5-2]

[0147] Example 3 - Combinatorial design specific to each germline VH gene family to generically stabilize VH domains Based on the data from Examples 1 and 2, two sets of combinatorial designs were generated for germline gene families 1, 3 and 4. The specific combinatorial designs are shown in Table 6. [Table 6]

[0148] Nine distinct VH domains with unique HCDR3s were tested with two design combinations specific for each germline. The nine individual germlines included three VH1 families (one VH1-8 and two VH1-69.2 with different HCDR3s), five VH3 families (one VH3-11, one VH3-15, two VH3-20 with different HCDR3s, and one VH3-48) and one VH4 family (VH4-39) germlines. Molecules were synthesized as gblocks by IDT and cloned into an expression vector with a C-terminal 8x histidine tag. Constructs were His-tagged at the C-terminus for purification.

[0149] Expression plasmids were transfected in duplicate into HEK293 cells and supernatants were collected as described above. After dilution 1:20 in PBS buffer, the supernatants were titrated using a GatorBio biointerferometer. A purified his-tagged 15 kDa V class Ig-fold protein was used to generate a standard curve. For DSF experiments, proteins were affinity purified by incubation with His60 Nickel resin (Takara), washing with neutral pH buffer containing 10–30 mM imidazole buffer, and elution using 200–400 mM imidazole. The eluted proteins were used directly for DSF measurements as described above.

[0150] Both the VH1 Opt1 and VH1 Opt2 designs significantly improved both the expression and thermal stability of the VH domains tested (Figures 4A-F).

[0151] One of the wild type VH1-69.2 VH domains was very poorly expressed and undetectable in the expression supernatant (lower limit of quantification approximately 1 μg / mL), whereas both the VH1 Opt1 and VH1 Opt2 variants showed significantly improved expression at approximately 100 μg / mL. The other VH1 domains also showed significant increases in both expression and thermostability (Table 7).

[0152] Both the VH3 Opt1 and VH3 Opt2 designs resulted in a significant increase in the thermal stability of all VH3 domains and improved expression of all but one VH3 domain (Table 7).

[0153] One VH4 germline molecule evaluated did not express as a WT molecule, but expressed well with the optimized VH4_Opt1 designed mutations, including 17C-82aC (Table 7).

[0154] Table 7 shows the expression titers, the change in expression titers relative to wild type, and the results of stability experiments for several disulfide-stabilized VH domains containing additional substitutions according to the present disclosure. [Table 7]

[0155] Similar methods were used to identify additional stabilizing sequences for VH Family 1 and VH Family 3, as shown in Figure 6 and Figure 7A-L. Family 1 members were modified according to option 2 in Table 6. Family 3 members were modified according to option 1 in Table 6. Figure 6 provides stability and expression data for each variant, along with a summary of each sequence substitution. Figure 7A-L provide sequence information for each variant.

[0156] The increased expression and thermal stability of each domain was primarily that observed for standard antibodies. For example, the measurable Tm values ​​of the optimized VH domains ranged from 73 to 89 °C, which places them in the same or higher thermal stability range as native antibody Fab domains. Overall, these enhanced designs represent a general stability / expression solution for VH domains that can be used within scaffolds for recombinantly derived libraries used in phage, yeast or mammalian display as well as therapeutic antibody-like modalities.

[0157] Example 4 - VH germline family 4 variants VH4 family members 4-34 and 4-39 were modified with one of the disulfide pairs 17C / 82aC or 23C / 77C and one or more of the following amino acid substitutions 10T, 23Q, 49A, 82bN, 82bD and 84P. A summary of the substitutions together with their effect on VH stability and expression (determined according to Example 1) is shown in Figure 8.

[0158] Figure 9 shows a summary of the substitutions and their effects on molecular stability and expression (determined according to Example 1) for germline family 4 VH family members 4-4, 4-28, 4-30-1, 4-30-2, 4-30-4, 4-31, 4-34, 4-38, 4-59 and 4-61. Each variant contains 23C / 77C along with 82aD and 84P.

[0159] The amino acid sequences of the variants summarized in Figures 8 and 9 are shown in Figures 10A-N.

[0160] Example 5 - VH Germline Family 2 Germline Variants The VH family 2 member VH2-5 (parent) with the existing 39R substitution was further modified with one of the following substitutions: 15G, 16D, 17D, 25D, 37Y, 44D, 44G, 44P, 65D, 71M, 73D, 73P, 83L, 83Q, 83T, 84Y, 85R, 85S, 85K, V85T, 89I, 105D, 107I, 107Y, or a combination of substitutions 17C / 82aC, 19C / 81C, and 23C / 77C. A summary of the substitutions along with expression data is shown in Figure 11.

[0161] Figure 12 shows VH family 2 member V2-5 with the existing 39R and 19C / 81C substitutions alone or in combination with several other substitutions including 15G, 37Y, 44D, 83T, 85S, 15G / 37Y, 15G / 44D, 15G / 83T, 15G / 85S, 37Y / 44D, 37Y / 83T, 37Y / 85S, 44D / 83T, and 44D / 85S. Expression data is shown for each variant.

[0162] The examples that include the 37Y substitution reflect that, when present, 37Y reduces the dimerization of the VH domain. In particular, variants with 19C / 81C and one of the following combinations avoid dimerization: 15G / 37Y and 37Y / D83T. These examples show a comparison of the 37Y / 83T variant with WT 39Q and substitution 39R. Both sequences avoid dimerization. These data show that, when 37Y is present, 39R is not necessary to eliminate homodimerization of the VH domain. The variant 19C / 81C / 37Y / 83T appears to have the most significant improvement in expression over the WT sequence.

[0163] FIG. 12 also shows data demonstrating improved expression of VH family 2-26 variant 19C / 81C / 37Y / 83T relative to the germline sequence.

[0164] The amino acid sequences of the variants in Figures 11 and 12 are shown in Figures 13A to 13H.

[0165] Example 6 - VH germline family 5 variants VH family 5 members with a pre-existing 39R substitution in VH5-51 were further modified with one of the following substitutions: 8D, 8S, 9D, 9P, 10K, 10Q, 17P, 28D, 35A, 35T, 37Y, 40P, 40Q, 47Y, 47Q, 48I, 58E, 60D, 60A, 68E, 74R, 76N, 76Q, 77V, 83D, 83T, 84E, 89V, 89I, 110I or substitution combinations 17C / 82aC, 19C / 81C, 23C / 77C or 35C / 50C. A summary of the substitutions along with expression data is shown in Figure 14.

[0166] FIG. 15 shows two sets of expression data for germline and optimized variants of VH5-51 modified at 39R and containing the following substitutions 28D / 48I / 84E, S28D / 76N / K83D, 28D / 39R / 76N / 84E and 28D / 48I / 83D.

[0167] 16A-16F provide the sequences of the VH family 5 variants of FIG.

[0168] Example 7: Reducing constitutive dimerization of VH domains

[0169] In nature, the majority of antibody VH domains, including human VH, heterodimerize with the VL domain from the antibody LC to form complete antigen-binding fragments or Fabs. However, antibody VH and VL domains are highly homologous in structure and use similar residue positions to fill residues within the VH / VL interface. Given the homology, the tendency for VH domains to homodimerize using residues at the VH / VL interface has been shown to be present in fully human VH domains derived from phage display libraries (Baral TN, Chao, SY, Li, S, et al., 2012 Crystal structure of a human single domain antibody dimer formed through VH-VH non-covalent interactions. PLoS One 7, e30149; "Gr6 homodimer"). This VH domain forms a constitutive homodimer whose structure has been elucidated. In the structure, residues that typically form interactions with antibody VL domains are buried within the VH dimerization interface and are also on the periphery of the VH dimerization interface, including positions 35, 37, 45, 49, and 91 (according to the Kabat numbering system).

[0170] The published structure of the Gr6 VH homodimer (PDB code: 3QYC) was evaluated for residue positions within the framework that are distal to the complementarity determining regions (CDRs) and involved in homodimer interactions. Two residue positions fit this description. The first was Kabat position 37, which is a valine or isoleucine in all human VH germlines. The second was Kabat position 45, which is canonically a leucine in all human VH germlines. These two residues were selected for a Rosetta software-based computational-based screen for residues that destabilize the Gr6 VH homodimer while having minimal impact on the stability of monomeric Gr6.

[0171] The Kabat residue valine 37 in the 3QYC structure (residue 39 in the Gr6 structure) was computationally mutated to all possible amino acids and the calculated stability of the mutants was compared to the wild-type protein. This calculation was performed for the VH homodimer as well as the VH monomer (Table 8). The structure of the monomer was created by removing one of the chains in the 3QYC crystal structure. During the energy calculations, residues near the site of the mutation were allowed to adopt alternative conformations to accommodate the mutation. Substitutions V37Y and V37F were interesting because they were predicted to mostly destabilize the homodimer (>10 kcal / mol) without destabilizing the monomer. Substitutions V37P and V37R were also predicted to destabilize the dimer, but also the monomer. A computational scan of all possible point mutations was also performed for Kabat residue leucine 45 (residue 47 in the Gr6 structure), which is also buried in the homodimer interface. At this position, no substitutions were predicted to significantly destabilize the homodimer without perturbing the stability of the monomer. However, substitutions to create charge-charge repulsions within the interface at position 45 were more destabilizing to the dimer compared to the monomer based on Rosetta energy calculations. Table 8 shows the effect of residue substitutions at VH Kabat positions 37 and 45 as measured using Rosetta. [Table 8]

[0172] The effect of substitutions at residues 37 and 45 on Gr6 VH homodimerization was evaluated. A mammalian expression plasmid encoding the Gr6 VH domain with a C-terminal 8x-histidine tag was generated as described elsewhere herein. Variants were generated by DNA synthesis and cloning into the mammalian expression plasmid. The plasmid was then transfected into 25 mL of Expi293 cells as described above, which were then cultured for 5 days before harvesting. Protein was purified from Expi293 supernatant using His60 Nickel resin (Takara; Cat. No. 635657) and an AKTA Pure instrument (Cytiva). After elution, protein was analyzed by HPLC (Thermo Vanquish FLEX) using a Zenix-C SEC 150 column with 3 μm particle size and 150 Å pore size resin (Sepax Technologies). Low protein molecular weight (LMW) standards (Cell Mosaic Inc.) were used in parallel. The running buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 6.8, and the flow rate was 1 mL / min at 25°C.

[0173] HPLC analysis demonstrated that the 37Y mutation significantly reduced the level of dimerization by the Gr6 protein. Based on molecular weight standards, Gr6 ran at a molecular weight slightly greater than 30 kDa, consistent with the formation of homodimers (Figure 19, panel A). Substitution of L45E did not affect VH dimerization, as the VH protein eluted from the HPLC column simultaneously with the unmodified Gr6 protein (Figure 19, panel A). Substitution of V37 with F or Y resulted in Gr6 protein eluting at a molecular weight consistent with the monomer (Figure 19, panel B). The V37Y variant eluted slightly later than the V37F protein. The V37F protein may exist in a monomer / dimer equilibrium. The V37Y protein eluted at a molecular weight that more closely matched that of the monomer (Figure 19, panel B). V37R was also evaluated. It had a negative effect on both the expression and biophysical properties of Gr6.

[0174] 37F and 37Y were consistently shown to be stabilized by Rosetta across multiple VH germline monomers. 37Y proved to be one of the most stabilizing single substitutions for both the VH1 and VH2 family germlines tested here and is an essential piece of the VH2 family combinatorial design. Notably, when assessing the monomer / dimer propensity of the TTX017-v13-VH2-5 VH protein, the molecule was essentially dimeric. Stabilizing combinatorial designs lacking the 37Y substitution maintained this dimeric state, whereas combinatorial designs containing the 37Y substitution became monomeric.

[0175] To evaluate whether the 37Y variant is suitable for addition to additional VH1, VH3 and VH4 family members, we measured the effect on the VH domains from each family. We found that for the VH1-8, VH3-20 and VH4-34 variants with existing stabilization designs, adding 37Y did not affect expression and in some cases improved expression. For VH1-8 and VH3-20, which could be assessed for their oligomeric state by size exclusion chromatography, the VHs containing 37Y behaved as monomers. Figure 17 provides an overview of the amino acid replacement strategies of the germline members tested. The complete amino acid sequences are provided in Figures 18A-C.

[0176] Example 8: Effect of CDR3 on the stability of modified VH domains

[0177] To confirm that the sequence of the CDR3 does not affect the effect of stabilizing substitutions on the VH domain as described herein, the expression and stability of a VH molecule with identical sequences except for the CDR3 was determined.

[0178] In VH family 1-69.2, VH molecules ITS050-M022 and ITS045-M070 (Figure 4B) have identical sequences except for CDR3 (not shown) and have affinities for different targets. Each molecule was tested with two different sets of substitution combinations shown in Table 9 below. [Table 9]

[0179] In the VH 3-20 family, the VH molecules ITS051-M019 and ITS045-M069 have identical V gene sequences except for CDR3 and FR4 (different J chains) and have affinities for different targets. Each molecule was tested with two different sets of substitution combinations shown in Table 10. [Table 10]

[0180] Example 9 Figures 20A-20U show some examples of modified human germline IGHV sequences with combinations of substitutions according to the present disclosure. These include: Germline Family 1 Members Modified with 17C / 82aC,39R,48I 17C / 82aC,39R,45E,48I 17C / 82aC,39Y,48I Germline family 3 members modified with 23C / 77C, 39R, 49A, 74E 23C / 77C, 39R, 45E, 49A, 74E 23C / 77C,37Y,49A,74E Germline Family 4 Members Modified with 23C / 77C,39R,82bD,84P 23C / 77C,39R,45E,82bD,84P 23C / 77C,37Y,82bD,84P Germline Family 2 Members Modified with 19C / 81C, 37Y, 39R, 83T 19C / 81C, 37Y, 39R, 45E, 83T 19C / 81C,37Y,83T Germline family 5 members modified with 28D, 39R, 76N, 84E 28D, 39R, 45E, 76N, 84E 28D, 37Y, 76N, 84E, and Germline family 7 members modified with 39R, 17C (paired with natural C at 82a) 39R, 45E, 17C (paired with natural C at 82a) 37Y, 17C (paired with natural C in 82a).

[0181] Example 10 Figures 21A-H show summaries of additional examples of modified VH domains of the present disclosure in germline family members 1-69.2, 3-15, 3-21, 4-39 and 3-20, along with expression and / or Tm data for each exemplary molecule.

[0182] Having described the invention in detail and with reference to specific embodiments thereof, it will be apparent that substitutions and modifications are possible without departing from the scope of the invention as defined in the appended claims. More specifically, although certain aspects of the invention are identified herein as being particularly advantageous, it is contemplated that the invention is not necessarily limited to these particular aspects of the invention.

Claims

1. 1. A single immunoglobulin variable domain comprising an amino acid sequence of a human heavy chain V gene portion (IGHV) of an antibody, wherein said IGHV amino acid sequence comprises one or more amino acid substitutions that result in increased thermal stability compared to a wild-type IGHV sequence lacking said one or more amino acid substitutions.

2. A single immunoglobulin variable domain as described in claim 1, which includes a non-natural disulfide bond including at least one cysteine ​​residue at an amino acid position that does not occur in nature.

3. A single immunoglobulin variable domain as described in claim 2, wherein the non-natural disulfide bond is present between two cysteine ​​residues located at positions 23 and 77; 17 and 82a; and 19 and 81 according to the Kabat numbering system.

4. 4. The single immunoglobulin variable domain of claim 3, further comprising a D gene sequence.

5. 4. The single immunoglobulin variable domain of claim 3, further comprising a J gene sequence.

6. The one or more substitutions are at any of the following amino acids according to the Kabat numbering system: 1E, 2A, 5Q, 10Q, 10T, 14E, 15G, 16D, 16Q, 19I, 23K, 23Q, 23Y, 25F, 25Y, 28D, 28E, 28K, 28N, 28R, 30K, 30S, 31K, 33P, 35A, 35G, 35S, 37F, 37Y, 37H, 39R, 40P, 44D, 45E, 48I, 49A, 2. The single immunoglobulin variable domain of claim 1, comprising at least one of: 52E, 52D, 55E, 56E, 60A, 60D, 65D, 68E, 73D, 73P, 74E, 76K, 76N, 77Q, 82bD, 82bN, 83D, 83K, 83L, 83Q, 83T, 84E, 84P, 84Y, 85K, 85R, 85S, 85T, 891, 105D, 1071, 107Y, 1101, and 110V.

7. 7. The single immunoglobulin variable domain of claim 6, comprising one of the following amino acid combinations according to the Kabat numbering system: 5Q / 23Q 10Q / 48I / 84E 10T / 82bD 10T / 82bD 10T / 82bN 10T / 84P 15G / 37Y 15G / 44D 15G / 85S 15G / 83T 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 23Q / 77Q 28D / 37Y / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 39R / 45E / 76N / 84E 28D / 39R / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 48I / 83D 28D / 48I / 84E 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28D / 76N / 83D 28D / 76N / 84E 28K / 49A 28K / 49A / 77Q 28K / 49A / 55E / 84E 28K / 49A / 55E / 84E / 10T / 82bN 28K / 55E 28K / 55E / 74E 37F / 48I 37Y (or 39R) / 10T / 84P 37Y (or 39R) / 10T / 82bD 37Y (or 39R) / 82bD / 84P 37Y / 39R / 83T 37Y / 39R / 45E / 83T 37Y / 44D 37Y / 48I 37Y / 49A / 74E 37Y / 85S 37Y / 83T 39R / 28D 39R / 45E 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 39R / 83T 39R / 45E / 48I 39R / 45E / 49A / 74E 39R / 45E / 82bD / 84P 44D / 85S 44D / 83T 45E / 82bD / 84P 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 45E / 82bD / 84P 49A / 84E 82bD / 84P 82bN / 84P 83T / 44D.

8. 8. The single immunoglobulin variable domain of claim 7, further comprising, if not already present, at least one of 39R, 45E and 37Y.

9. 2. The single immunoglobulin variable domain of claim 1, having a human germline genetic origin selected from germline family 1, germline family 2, germline family 3, germline family 4, germline family 5 or germline family 7.

10. 10. The single immunoglobulin variable domain of claim 9, wherein the germline gene family 1 comprises germline gene family members 1-2 (SEQ ID NO:1), 1-3 (SEQ ID NO:2), 1-8 (SEQ ID NO:3), 1-18 (SEQ ID NO:4), 1-24 (SEQ ID NO:5), 1-45 (SEQ ID NO:6), 1-46 (SEQ ID NO:7), 1-58 (SEQ ID NO:8), 1-69 (SEQ ID NO:9), and 1-69.2 (SEQ ID NO:10) and alleles thereof.

11. 11. The single immunoglobulin variable domain of claim 10, comprising one or more of the following substitutions: 10Q, 16D, 16Q, 25Y, 25F, 37F, 37Y, 39R, 45E, 48I, 84E, 84P, 110V, and 110I.

12. 12. The single immunoglobulin variable domain of claim 11, comprising one or more of the following substitution combinations: 10Q / 48I / 84E 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 37F / 48I 37Y / 48I 39R / 45E / 48I 39R / 48I.

13. 12. The single immunoglobulin variable domain of claim 11, comprising one of the following substitution combinations: 17C / 82aC / 10Q / 48I / 84E 17C / 82aC / 16D 17C / 82aC / 16D / 37F 17C / 82aC / 16D / 37Y 17C / 82aC / 16D / 37Y / 39R 17C / 82aC / 16D / 39R 17C / 82aC / 16D / 39R / 48I 17C / 82aC / 16D / 48I 17C / 82aC / 37F 17C / 82aC / 37Y 17C / 82aC / 37Y / 48I 17C / 82aC / 39R 17C / 82aC / 39R / 45E / 48I 17C / 82aC / 39R / 48I 17C / 82aC / 84E 34C / 78C / 16D 34C / 78C / 37F 34C / 78C / 84E 34C / 78C / 16D / 37F 34C / 78C / 16D / 48I 34C / 78C / 10Q / 48I / 84E.

14. 13. The single immunoglobulin variable domain of claim 12, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

15. 10. The single immunoglobulin variable domain of claim 9, wherein the germline gene family 2 comprises germline gene family members 2-5 (SEQ ID NO:11), 2-26 (SEQ ID NO:12), and 2-70 (SEQ ID NO:13) and alleles thereof.

16. 16. The single immunoglobulin variable domain of claim 15, comprising one or more of the following substitutions: 15G, 16D, 37Y, 37H, 39R, 44D, 45E, 65D, 73D, 73P, 83L, 83Q, 83K, 83T, 84Y, 85R, 85S, 85K, 85T, 891, 105D, and 1071.

17. 17. The single immunoglobulin variable domain of claim 16, comprising one of the following substitution combinations: 15G / 37Y 15G / 44D 15G / 85S 15G / 83T 37Y / 39R / 45E / 83T 37Y / 39R / 83T 37Y / 44D 37Y / 85S 37Y / 83T 39R / 83T 44D / 85S 44D / 83.

18. 17. The single immunoglobulin variable domain of claim 16, comprising one of the following substitution combinations: 19C / 81C / 15G 19C / 81C / 15G / 37Y 19C / 81C / 15G / 44D 19C / 81C / 15G / 85S 19C / 81C / 15G / 83T 19C / 81C / 37Y 19C / 81C / 37Y / 39R / 83T 19C / 81C / 37Y / 39R / 45E / 83T 19C / 81C / 37Y / 44D 19C / 81C / 37Y / 83T 19C / 81C / 37Y / 85S 19C / 81C / 39R / 83T 19C / 81C / 44D 19C / 81C / 44D / 85S 19C / 81C / 85S 19C / 81C / 83T 19C / 81C / 83T / 44D.

19. 18. The single immunoglobulin variable domain of claim 17, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

20. 10. The single immunoglobulin variable domain of claim 9, wherein the germline gene family 3 comprises germline gene family members 3-7 (SEQ ID NO:14), 3-9 (SEQ ID NO:15), 3-11 (SEQ ID NO:16), 3-13 (SEQ ID NO:17), 3-15 (SEQ ID NO:18), 3-20 (SEQ ID NO:19), 3-21 (SEQ ID NO:20), 3-23 (SEQ ID NO:21), 3-30 (SEQ ID NO:22), 3-33 (SEQ ID NO:23), 3-43 (SEQ ID NO:24), 3-48 (SEQ ID NO:25), 3-49 (SEQ ID NO:26), 3-53 (SEQ ID NO:27), 3-64 (SEQ ID NO:28), 3-66 (SEQ ID NO:29), 3-72 (SEQ ID NO:30), 3-73 (SEQ ID NO:31), 3-74 (SEQ ID NO:32), 3-d (SEQ ID NO:33), and 3-NL1 (SEQ ID NO:34), and alleles thereof.

21. 21. The single immunoglobulin variable domain of claim 20, comprising one or more of the following substitutions: 2A, 5Q, 14E, 23K, 23Q, 23Y, 28D, 28E, 28N, 28K, 28R, 30K, 30S, 31K, 33P, 35G, 35A, 35S, 37Y, 39R, 40P, 45E, 49A, 52E, 52D, 55E, 56E, 74E, 76K, 77Q, 82bD, 84E, 84P, 110V, and 110I.

22. 21. The single immunoglobulin variable domain of claim 20, comprising one of the following substitution combinations: 5Q / 23Q 23Q / 77Q 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28K / 49A 28K / 49A / 55E / 84E 28K / 49A / 77Q 28K / 55E 28K / 55E / 74E 37Y / 49A / 74E 39R / 45E / 49A / 74E 39R / 49A / 84E 39R / 84E 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 49A / 84E.

23. 21. The single immunoglobulin variable domain of claim 20, comprising one of the following substitution combinations: 23C / 77C / 28K / 49A 23C / 77C / 28D / 49A 23C / 77C / 28K / 55E 23C / 77C / 28K / 55E / 74E 23C / 77C / 28K / 49A / 55E / 84E 23C / 77C / 37Y / 49A / 74E 23C / 77C / 39R / 45E / 49A / 74E 23C / 77C / 39R / 49A / 74E 23C / 77C / 39R / 49A / 84E 23C / 77C / 39R / 49A / 84E 23C / 77C / 49A / 55E / 84E 34C / 78C / 28D 34C / 78C / 28K 34C / 78C / 49A 34C / 78C / 55E 34C / 78C / 74E 34C / 78C / 77Q 34C / 78C / 84E.

24. 23. The single immunoglobulin variable domain of claim 22, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45G.

25. 10. The single immunoglobulin variable domain of claim 9, wherein the germline gene family 4 comprises germline gene family members 4-4 (SEQ ID NO:35), 4-28 (SEQ ID NO:36), 4-30-1 (SEQ ID NO:37), 4-30-2 (SEQ ID NO:38), 4-30-4 (SEQ ID NO:39), 4-31 (SEQ ID NO:40), 4-34 (SEQ ID NO:41), 4-38-2 (SEQ ID NO:42), 4-39 (SEQ ID NO:43), 4-59 (SEQ ID NO:44), and 4-61 (SEQ ID NO:45), 4-b (SEQ ID NO:46), and alleles thereof.

26. 26. The single immunoglobulin variable domain of claim 25, comprising one or more of the following substitutions: 1E, 10Q, 10T, 15G, 19I, 37Y, 39R, 45E, 82bD, 82bN, 84P, 107I, and 107Y.

27. 26. The single immunoglobulin variable domain of claim 25, comprising one of the following substitution combinations: 10T / 82bN 10T / 84P 10T / 82bD 37Y (and / or 39R) / 82bN / 84P 37Y (and / or 39R) / 10T / 84P 37Y (and / or 39R) / 10T / 82bD 37Y (and / or 39R) / 10T / 82bN 39R / 45E / 82bD / 84P 45E / 82bD / 84P.

28. 26. The single immunoglobulin variable domain of claim 25, comprising one of the following substitution combinations: 17C / 82aC / 10T 17C / 82aC / 10T / 82bN 17C / 82aC / 10T / 82bD 17C / 82aC / 82bN / 84P 17C / 82aC / 37Y (and / or 39R) / 10T / 82bD 17C / 82aC / 37Y (and / or 39R) / 10T / 84P 17C / 82aC / 37Y (and / or 39R) / 82bD / 84P 23C / 77C / 10T / 84P 23C / 77C / 39R / 45E / 82bD / 84P 23C / 77C / 45E / 82bD / 84P 23C / 77C / 82bD / 84P 23C / 77C / 82bN / 84P 23C / 77C / 37Y (and / or 39R) / 10T / 82bD 23C / 77C / 37Y (and / or 39R) / 10T / 82bN 23C / 77C / 37Y (and / or 39R) / 10T / 84P 23C / 77C / 37Y (and / or 39R) / 82bD / 84P 23C / 77C / 37Y (and / or 39R) / 82bD / 84P.

29. 28. The single immunoglobulin variable domain of claim 27, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

30. 10. The single immunoglobulin variable domain of claim 9, wherein the germline gene family 5 comprises germline gene family members 5-51 (SEQ ID NO:47) and 5-a (SEQ ID NO:48), and alleles thereof.

31. 31. The single immunoglobulin variable domain of claim 30, comprising one or more of the following substitutions: 28D, 37Y, 39R, 45E, 48I, 60D, 60A, 68E, 76N, 83D, and 84E.

32. 31. The single immunoglobulin variable domain of claim 30, comprising one of the following substitution combinations: 39R / 28D 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 28D / 48I / 84E 28D / 76N / 83D 28D / 76N / 84E 28D / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 39R / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 37Y / 48I / 83D 28D / 39R / 45E / 76N / 84E.

33. 33. The single immunoglobulin variable domain of claim 32, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

34. 10. The single immunoglobulin variable domain of claim 9, wherein the germline genes include germline gene family member 6-1 (SEQ ID NO:49) and alleles thereof.

35. 10. The single immunoglobulin variable domain of claim 9, wherein the germline genes include germline gene family member 7-4-1 (SEQ ID NO: 50) and alleles thereof.

36. 36. The single immunoglobulin variable domain of claim 35, comprising one of the following substitution combinations: 17C / 82aC / 39R 17C / 82aC / 39R / 45E 17C / 82aC / 37Y 35C / 50C / 39R 35C / 50C / 39R / 45E 35C / 50C / 37Y.

37. 37. The single immunoglobulin variable domain of claim 36, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

38. A polynucleotide encoding the single immunoglobulin variable domain of claim 1.

39. A pharmaceutically acceptable composition comprising a single immunoglobulin variable domain according to claim 1.

40. A polypeptide comprising at least one framework sequence selected from FR1, FR2, FR3 and FR4 of a single immunoglobulin variable domain according to claim 4, wherein the framework sequence comprises at least one of the substitutions or combinations thereof.

41. A VH domain library comprising a plurality of single immunoglobulin variable domains described in claim 1.

42. A polynucleotide library comprising a plurality of polynucleotides encoding a plurality of single immunoglobulin variable domains described in claim 1.

43. A method for identifying an antigen-binding molecule, comprising: (i) contacting the single immunoglobulin variable domain library of claim 42 with a target; and (ii) identifying a single immunoglobulin variable domain of said library that binds to said target.

44. 1. A single immunoglobulin variable domain comprising an amino acid sequence of a framework region of a human heavy chain V gene portion (IGHV) of an antibody, wherein the IGHV amino acid sequence has one or more amino acid substitutions or combinations thereof: 1E, 2A, 5Q, 10Q, 10T, 14E, 15G, 16D, 16Q, 19I, 23K, 23Q, 23Y, 25F, 25Y, 28D, 28E, 28K, 2 8N, 28R, 30K, 30S, 31K, 33P, 35A, 35G, 35S, 37F, 37Y, 37H, 39R, 40P, 44D, 45E, 48I, 49A, 52E, 52D, 55E, 56E, 60A, 60D, 65D, 68E, 73D, 73P, 74E, 76K, 76N, 77Q, 82bD, 82bN, 83D, 8 3K, 83L, 83Q, 83T, 84E, 84P, 84Y, 85K, 85R, 85S, 85T, 89I, 105D, 107I, 107Y, 110I, 110V A single immunoglobulin variable domain, comprising:

45. 45. The single immunoglobulin variable domain of claim 44, comprising one of the following amino acid combinations according to the Kabat numbering system: 5Q / 23Q 10Q / 48I / 84E 10T / 82bD 10T / 82bD 10T / 82bN 10T / 84P 15G / 37Y 15G / 44D 15G / 85S 15G / 83T 16D / 37F 16D / 37Y 16D / 39R / 48I 16D / 48I 16D / 110I 23Q / 77Q 28D / 37Y / 48I / 83D 28D / 37Y / 48I / 84E 28D / 37Y / 76N / 83D 28D / 37Y / 76N / 84E 28D / 39R / 45E / 76N / 84E 28D / 39R / 48I / 83D 28D / 39R / 48I / 84E 28D / 39R / 76N / 83D 28D / 39R / 76N / 84E 28D / 48I / 83D 28D / 48I / 84E 28D / 49A 28D / 49A / 77Q 28D / 55E 28D / 55E / 74E 28D / 76N / 83D 28D / 76N / 84E 28K / 49A 28K / 49A / 77Q 28K / 49A / 55E / 84E 28K / 49A / 55E / 84E / 10T / 82bN 28K / 55E 28K / 55E / 74E 37F / 48I 37Y (or 39R) / 10T / 84P 37Y (or 39R) / 10T / 82bD 37Y (or 39R) / 82bD / 84P 37Y / 39R / 83T 37Y / 39R / 45E / 83T 37Y / 44D 37Y / 48I 37Y / 49A / 74E 37Y / 85S 37Y / 83T 39R / 28D 39R / 45E 39R / 48I 39R / 60A 39R / 60D 39R / 68E 39R / 76N 39R / 83D 39R / 84E 39R / 83T 39R / 45E / 48I 39R / 45E / 49A / 74E 39R / 45E / 82bD / 84P 44D / 85S 44D / 83T 45E / 82bD / 84P 49A / 55E 49A / 55E / 77Q 49A / 55E / 84E 49A / 74E 49A / 74E / 77Q 49A / 77Q 49A / 77Q / 55E 49A / 77Q / 84E 45E / 82bD / 84P 49A / 84E 82bD / 84P 82bN / 84P 83T / 44D.

46. 45. The single immunoglobulin variable domain of claim 44, further comprising a non-naturally occurring disulfide bond comprising at least one cysteine ​​residue at a non-naturally occurring amino acid position.

47. 47. The single immunoglobulin variable domain of claim 46, wherein the non-native disulfide bond is between two cysteine ​​residues at positions 2 and 102; 17 and 82a; 19 and 81; 23 and 77; 34 and 78; 35 and 50 according to the Kabat numbering system.

48. 47. The single immunoglobulin variable domain of claim 46, wherein the combination includes, if not already included, at least one of 37Y, 39R and 45E.

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