Compounds and methods for extending the half-life of biological molecules - Patents.com

Abstract

The present invention relates to variants of OB-fold proteins, particularly variants of the Sac7d family, that can be used to bind human and mouse serum albumin and increase the serum half-life of associated members.

Description

[Technical field]

[0001] The present invention is in the field of extending the plasma half-life of proteins, and in particular relates to the development of novel conjugates to albumin that can be used for such purposes. [Background technology]

[0002] Introduction To transfer laboratory experiments to in vivo environment to evaluate the safety and efficacy of molecules, additional parameters need to be taken into account.For products exposed to systemic circulation, the most important change is the effect of the residence time of the product in bloodstream.It is necessary to control the residence time in bloodstream, and this can be achieved by using albumin, the most abundant circulating protein, as a temporary carrier.This can be achieved by temporarily linking the compound of interest to serum albumin by a specific ligand that is fused or conjugated to the compound. Summary of the Invention

[0003] The present invention relates to polypeptides comprising variants of Aho7c that bind to Sac7d protein or proteins of the Sac7d family, in particular albumin, and their use, in particular to extend the half-life of molecules (including peptides or polypeptides) bound thereto in vivo.

[0004] The variant binds to both human and mouse serum albumin, which is beneficial for speeding up the clinical development of products and the transition from animals to humans. Indeed, most of the conjugates for serum albumin do not cross-react between mice and humans. Furthermore, affinity is important, thus ensuring strong binding (and low turnover) of the variant to serum albumin (hence increasing the serum half-life of the biomolecule associated with the variant). The variant also binds to an epitope on serum albumin that does not interfere with the binding of serum albumin to FcRs, thus reusing the complex serum albumin / variant associated with the biomolecule, which proves to be interesting for increasing the serum half-life of the variant associated with the biomolecule. As the variant is a small protein, it does not reduce the biological activity of the biomolecule itself to which it is associated.

[0005] In particular, in certain embodiments, the variants are based on the Sac7d protein and include SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65. In certain embodiments, the variants are based on the Ahoc protein and include SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86. In the event of any discrepancy between the specification and the sequence listing, the sequences set forth in the specification shall control.

[0006] In particular, the variant comprises SEQ ID NO:38 or amino acids 1 to 54 of SEQ ID NO:38.

[0007] In particular, variants include sequences selected from SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, and amino acids 1 to 57 of these sequences.

[0008] In particular, variants include sequences selected from SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 86, and amino acids 1 to 55 of these sequences.

[0009] The invention also relates to nucleic acids encoding the variants, polypeptides comprising the variants and active agents, methods of making the variants alone or in combination with active agents, and methods of use.

[0010] In particular, the present invention relates to: - A polypeptide as disclosed herein which is composed of a variant of a member of the Sac7d family that binds to human and mouse serum albumin. - A polypeptide as disclosed herein, in which a variant of a member of the Sac7d family that binds human and mouse serum albumin is conjugated to an organic molecule. - A polypeptide as disclosed herein, in which a variant of a member of the Sac7d family that binds human and mouse serum albumin is conjugated to another polypeptide. - A polypeptide as disclosed herein, wherein the other polypeptide is another variant of a Sac7d family protein. - A nucleic acid molecule encoding a polypeptide disclosed herein. - An expression vector comprising a nucleic acid molecule disclosed herein. - A host cell comprising a nucleic acid molecule disclosed herein, or an expression vector disclosed herein. - A pharmaceutical composition comprising a polypeptide as disclosed herein, a nucleic acid as disclosed herein, an expression vector as disclosed herein, or a host cell as disclosed herein, and a pharma- ceutically acceptable carrier. - a method for producing a polypeptide as disclosed herein, comprising the steps of: (a) culturing a cell culture in which the cells are transformed with an expression vector disclosed herein; and (b) Producing the polypeptide A method comprising: - A polypeptide as disclosed herein as a medicament. - A polypeptide as disclosed herein for use in the treatment or prevention of a disease, wherein the variant is associated with an active substance for the treatment or prevention of the disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention It should be noted that the above sequences may contain a methionine at their N-terminus, but it is also possible to carry out the invention without the N-terminus of each sequence containing such a methionine as described. Similarly, amino acids located at the termini of the protein may be omitted. In particular, the amino acids located after residue L58 ​​of Sac7d (residues located after L60 of SEQ ID NO: 16) may be omitted. As a result, variants may contain amino acids 1-57, 1-58, 1-59, 1-60, 1-61, 1-62 or 1-63 of any of SEQ ID NO: 45 to SEQ ID NO: 65.

[0012] When based on the Aho7c scaffold, the variant may comprise amino acids 1-55, 1-56, 1-57, or 1-58 of any of SEQ ID NOs:66-86.

[0013] Such variants can be fused to biomolecules and / or conjugated to small chemical entities and can extend the plasma half-life of the molecule, thus increasing efficacy or bioavailability, or allowing the use of smaller doses. Exemplary molecules requiring such half-life extension include small chemical molecules (e.g. cytotoxic agents for cancer such as auristatins or doxorubicin), peptides (insulin analogues such as GLP-1), and Sac7d from Sulfolobus acidocaldarius. (and analogues such as Aho7c or Sso7d) derivatives, antibody mimetics such as affibodies, affilins, affimers, alphabodies, anticalins, avimers, DARPins, finomers, Kunits domain peptides, monobodies, Z domain of protein A, gamma B crystals, ubiquitin, cystatins, lipocalins, A domains of membrane proteins, ankyrin repeat motives, SH3 domain of Fyn, Kunitz domains of protease inhibitors, 10th type III domain of fibronectin, 3- or 4-helix bundle proteins, armadillo repeat domains, leucine rich repeat domains, PDZ domains, SUMO or SUMO-like domains, immunoglobulin-like domains, phosphotyrosine binding domains, pleckstrin homology domains, src homology 2 domains or synthetic peptide ligands, antibody fragments (including Fab, VHH, ScFv), enzymes, therapeutic nucleic acids such as antisense oligonucleotides, siRNA, shRNA.

[0014] This is particularly interesting in the case of biomolecules aimed at neutralizing circulating molecules such as cytokines (e.g. IL-17, TNF-α...), growth factors, hormones, antimicrobial peptides, complement factors, coagulation factors, etc.; as well as interacting with circulating cells such as immune cells (T-lymphocytes, macrophages, antigen presenting cells...) towards neutralization, recruitment or activation; as well as in the case of biomolecules that require diffusion to tissues and organs and therefore benefit from a prolonged exposure in the vasculature to enhance extravasation and / or tissue uptake, e.g. in the case of brain uptake, liver, lung or bone targeting; as well as a long residence time in the circulation favors a higher diffusion to solid tumors, leading to enhanced antitumor activity (e.g. when neutralizing PD-L1 or EGF-R); as well as in the case of molecules with antibacterial or antiviral activity to treat sepsis due to more common infections.

[0015] Depending on the biomolecule to which the variant is fused, the fusion product can be used to treat cancer, metabolic diseases (such as diabetes), neurological diseases, inflammatory and autoimmune diseases (such as rheumatoid arthritis, psoriasis, lupus or multiple sclerosis), or enzyme deficiencies, particularly in the brain.

[0016] A biomolecule (polypeptide or organic molecule) is preferably biologically active. This indicates that the biomolecule interacts with a molecule (receptor, ligand, antigen, etc.) present in the subject's body and advantageously provides a beneficial effect to the subject (cure, relief, reduction of symptoms, reduction of markers related to the disease to be treated (e.g. reduction of viral load in the case of a viral infection). In fact, an active substance is a substance or mixture of substances intended for use in the manufacture of a medicinal (or diagnostic product) that, when used in its manufacture, is the active ingredient of a product intended to exert a pharmacological, immunological or metabolic effect aimed at restoring, correcting or modifying a physiological function or to perform a medical diagnosis.

[0017] It should be noted that although this specification discloses variants of Sac7d, the teachings are applicable to other proteins of the Sac7d family. The teachings are also applicable to other OB-fold domains, as disclosed in WO2007139397. The present invention is also applicable to the SH3 domain, a small protein domain of about 60 amino acid residues, originally described as a conserved sequence in the viral adaptor protein v-Crk and described under PF00018 in the PFAM database. The SH3 domain has a characteristic β-barrel fold consisting of five or six β-strands arranged as two closely packed antiparallel β-sheets. The linker region may contain a short helix. It should be noted that the OB-fold domain and the SH3 domain share homology, and considering the knowledge of the sequences and structures of these domains, it is possible to determine which amino acids in either the OB-fold domain or the SH3 domain correspond to the amino acids disclosed below for Sac7d.

[0018] WO2012069654 describes the generation of albumin-binding DARPins (containing ancrin repeats) to improve the pharmacokinetics or pharmacodynamics of a molecule of interest.

[0019] EP1930342 describes the isolation of OB-fold protein variants.

[0020] WO2012150314 describes the possibility of transferring the binding ability of one Sac7d family protein to a target of interest from another Sac7d family protein.

[0021] EP3483180 describes Sac7d variant-antibody conjugates.

[0022] Robinson et al. (Nature, 1998 vol. 392, no. 6672, 202-205) describe Sac7d and its binding to DNA.

[0023] Description of variants based on the Sac7d family consensus sequence (SEQ ID NO: 16) Variants for specific members of the Sac7d family are disclosed herein.As shown in Figure 1, considering the sequence similarity of Sac7d family proteins and the fact that mutations can be carried from one protein to another, mutations can be drawn in the consensus sequence of Sac7d family.

[0024] In sequences SEQ ID NO:17 through SEQ ID NO:37, the amino acids designated as X or Xaa at positions 8-10, 22, 27, 30, 32, 42, 46, and 48 are as shown in Table 2. Other amino acids designated as X (or Xaa) are shown in Table 1 or Table 3 (where "-" designates no amino acid).

[0025] TIFF2024507780000001.tif97143 (Table 1) Description of amino acids in SEQ ID NO: 17 to SEQ ID NO: 37

[0026] The amino acids disclosed in Table 1 correspond to the variation observed in the Sac7d family of proteins, as also shown in FIG.

[0027] The amino acids disclosed in Table 2 correspond to the amino acids involved in albumin binding in the variants disclosed herein. Note that such variants further differ from the wild-type protein in the following amino acids: Position 23: the presence of W; Position 25: the presence of L; Position 34: the presence of K; Position 44: the presence of Y.

[0028] These amino acids have been shown by the inventors to be important for binding to albumin (altering the amino acids dramatically reduces the ability to bind to albumin or the affinity for albumin). In contrast, other amino acids from the optimized conjugates (SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 37) can be modified according to Table 2. In particular, it has been noted that replacement of the amino acids at positions 22, 27, 30, 32, 42, or 48 of these conjugates with different amino acids did not essentially alter the binding or its properties. As for the amino acids at positions 8 and 10 in these conjugates, they can be changed with hydrophobic / aromatic and polar / charged amino acids, respectively, as described in Table 2.

[0029] It has also been shown that D17 (aspartic acid) of the consensus sequence (not involved in binding) can be replaced by E (glutamic acid), as represented in the sequences above (SEQ ID NO: 20, SEQ ID NO: 23 carrying X at position 17; SEQ ID NO: 27 and SEQ ID NO: 30 carrying D17; SEQ ID NO: 34 and SEQ ID NO: 37 carrying E17).

[0030] TIFF2024507780000002.tif76145 (Table 2) Explanation of amino acids designated as Xaa in SEQ ID NO: 17 through SEQ ID NO: 37. Note that this table is used for the sequences listed above for the Xaa they carry. Some sequences have amino acids specified at some positions in Table 2, so the rows of the table do not apply.

[0031] As noted above, the methionine at position 1 of SEQ ID NOs: 17-37 can be omitted, as can amino acids 61-68 of SEQ ID NOs: 17-37.

[0032] In one embodiment, the polypeptide comprises amino acids 2-60 of SEQ ID NO:17 or one of the sequences from Table 3.

[0033] TIFF2024507780000003.tif48145TIFF2024507780000004.tif245145(Table 3) List of variant sequences based on consensus sequences of Sac7d family proteins. The nature of X is provided in Tables 1 and 2. X at position 17 can be E or D.

[0034] Description of Sac7d- and Aho7c-based variants Sac7d and Aho7c are proteins that share great similarity.

[0035] SEQ ID NO:87 corresponds to the consensus sequence after alignment of amino acids 2-66 of Sac7d (SEQ ID NO:1) and amino acids 3-60 of Aho7c (SEQ ID NO:14). The initiation amino acid has been omitted as it is not essential to the structure of the protein. TIFF2024507780000005.tif11136

[0036] In this consensus sequence, X (or Xaa) is as shown in Table 4.

[0037] TIFF2024507780000006.tif42148 (Table 4) Description of the amino acids represented as Xaa in SEQ ID NO: 87 and SEQ ID NO: 38-44

[0038] Such variants are represented by the following:

[0039] TIFF2024507780000007.tif102145 (Table 5) Sequences of variants based on the consensus sequence of Sac7d-Aho7c. Amino acids represented by X are further described in Tables 4, 6, and 7.

[0040] TIFF2024507780000008.tif76148 (Table 6) Explanation of amino acids designated as Xaa in SEQ ID NO: 38 through SEQ ID NO: 86. Note that this table is used for the sequences listed above for Xaa retained. Some sequences have amino acids designated at some positions in Table 6, so the rows of the table do not apply.

[0041] In these sequences, some amino acids that are not involved in binding can also be modified with variants (see in particular SEQ ID NO: 38 to SEQ ID NO: 86), which are shown in Table 7.

[0042] TIFF2024507780000009.tif28128 (Table 7) Amino acids represented as Xaa in SEQ ID NOs: 36-44, 45-51, and 66-72. Xaa at position 56 is only present in SEQ IDs 45-51.

[0043] Variant description based on Sac7d (SEQ ID NO: 1) Variants of Sac7d that bind human and mouse serum albumin are depicted by SEQ ID NO:45 to SEQ ID NO:65.

[0044] In these sequences, the initiating methionine (M) of the Sac7d protein is omitted. It has been shown by the applicant that the deletion of this amino acid does not alter the activity of the protein. Similarly, it is possible to omit the last 7 amino acids from the variants and still retain activity.

[0045] As a result, proteins represented by amino acids 1-57 of any of SEQ ID NO:45 to SEQ ID NO:65 are also variants according to the invention. Mention may also be made of proteins represented by amino acids 1-58, 1-59, 1-60, 1-61, 1-62, 1-63 of any of SEQ ID NO:45 to SEQ ID NO:65, which are also variants according to the invention.

[0046] Thus, the polypeptides include any of SEQ ID NO: 45 to SEQ ID NO: 65, or any truncated proteins based on these sequences, as disclosed above.

[0047] Such variants are represented by the following:

[0048] TIFF2024507780000010.tif123145TIFF2024507780000011.tif170145(Table 8) Sequences of variants based on Sac7d. The amino acids represented by X are further described in Tables 6 and 7.

[0049] As described above, it is possible to modify D16 of Sac7d (located at position 15 in SEQ ID NOs: 45-65 due to the omission of M1 present in Sac7d), N37 of Sac7d (located at position 36 herein) or M57 of Sac7d (located at position 56 herein) as disclosed in Table 7.

[0050] With respect to the consensus sequence of Sac7d / Aho7c, any combination of substitutions is predicted, not all of which are depicted in the sequence listing (numbering is based on SEQ ID NOs: 45-65): TIFF2024507780000012.tif50128.

[0051] As noted above, all sequences contain mutant amino acids W21, L23, K32, Y41 (corresponding to positions 22, 24, 33, and 42, respectively, of SEQ ID NO: 1, including an N-terminal methionine) that differ from the amino acids naturally occurring in Sac7d.

[0052] The applicant has indeed found that these amino acids are present in various variants that bind to serum albumin, and that such modifications result in reduced binding (loss of affinity or loss of binding). Other amino acids may be variable, subject to the conditions set forth in Table 6.

[0053] Very interesting variants are depicted by SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, and SEQ ID NO: 65.

[0054] In particular, highly interesting variants are depicted by SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 62, and SEQ ID NO: 65.

[0055] SEQ ID NO:62 (designated F06) is of particular interest, as is SEQ ID NO:55. Note that L56 in SEQ ID NO:62 can be replaced by M56.

[0056] SEQ ID NO:58 (designated B11) is also of particular interest, as is SEQ ID NO:65.

[0057] Protein description based on Aho7c (SEQ ID NO: 14) Variants of Aho7c that bind human and mouse serum albumin are depicted by SEQ ID NO: 66 to SEQ ID NO: 86. As noted above, such proteins are highly similar to Sac7d. It is also shown and contemplated herein that mutations in Sac7d can be transferred from Sac7d to other proteins (WO 2012 / 150314).

[0058] In these sequences, the initiating methionine and alanine (MA) of the Aho7c protein (SEQ ID NO: 14) are omitted. It has been shown by the applicant that the deletion of these amino acids does not alter the activity of the protein. Similarly, it is possible to omit the last four amino acids from the variants and still retain activity.

[0059] As a result, proteins represented by amino acids 1-55 of any of SEQ ID NO:66 to SEQ ID NO:86 are also variants according to the invention. Mention may also be made of proteins represented by amino acids 1-56, 1-57, 1-58 of any of SEQ ID NO:66 to SEQ ID NO:86, which are also variants according to the invention.

[0060] Thus, the polypeptides include any of SEQ ID NO: 66 to SEQ ID NO: 86, or any truncated proteins based on these sequences, as disclosed above.

[0061] Such variants are represented by the following:

[0062] TIFF2024507780000013.tif130145TIFF2024507780000014.tif164145 (Table 9) Sequences of variants based on Ao7c. The amino acids represented by X are further described in Tables 6 and 7.

[0063] As described above, it is possible to modify D17 of Aho7c (located at position 15 in SEQ ID NOs:66-86 due to the omission of M1A2 present in Aho7c), or N38 of Aho7c (located at position 36 herein), as disclosed in Table 7.

[0064] With respect to the consensus sequence of Sac7d / Aho7c, any combination of substitutions is predicted, not all of which are depicted in the sequence listing (numbering is based on SEQ ID NOs: 66-86): TIFF2024507780000015.tif24128.

[0065] As noted above, all sequences contain mutant amino acids W21, L23, K32, Y41 (corresponding to positions 23, 25, 34, and 43, respectively, of SEQ ID NO: 14 and containing a methionine and an alanine at the N-terminus) that differ from the amino acids naturally occurring in Aho7c.

[0066] The applicant has indeed found that these amino acids are present in various variants that bind to serum albumin, and that such modifications result in reduced binding (loss of affinity or loss of binding). Other amino acids may be variable, subject to the conditions set forth in Table 6.

[0067] Very interesting variants are depicted by SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 85, and SEQ ID NO: 86.

[0068] In particular, highly interesting variants are depicted by SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 83, and SEQ ID NO: 86.

[0069] SEQ ID NO: 83 (based on F06) is of particular interest.

[0070] SEQ ID NO: 86 (based on B11) is also of particular interest.

[0071] In certain embodiments, the polypeptide is comprised of a variant of the Sac7d family of proteins that binds human serum albumin (HSA).

[0072] In certain embodiments, a variant of a Sac7d family protein that binds HSA is linked or fused (also referred to herein as associated) to another protein or polypeptide.

[0073] The present invention also relates to a genetic construct comprising a DNA sequence encoding a polypeptide as described herein, a vector comprising such a genetic construct, and a host cell comprising the genetic construct (nucleic acid or vector) in its genome.

[0074] The present invention also provides a method for producing a polypeptide as disclosed herein, comprising the steps of: a. culturing a cell culture, the cells being transformed with the disclosed genetic construct; and b. Recovering the polypeptide The present invention relates to a method comprising the steps of:

[0075] The present invention also relates to the polypeptides disclosed herein as pharmaceutical agents.

[0076] The present invention also relates to fusion proteins comprising the variants disclosed herein fused to a pharmaceutical therapeutically active peptide or protein.

[0077] The present invention also relates to a method for treating a subject in need thereof, comprising administering to the subject a therapeutic amount of the polypeptide disclosed herein, particularly when the subject has cancer, metabolic disease (such as diabetes), neurological disease, inflammatory disease or autoimmune disease (such as rheumatoid arthritis, psoriasis, lupus or multiple sclerosis), or enzyme deficiency, particularly in the brain.The polypeptide administered to the subject comprises a variant of the protein of the Sac7d family that binds to human serum albumin, fused to a protein or peptide that has therapeutic activity against the disease to be treated.For example, a protein that neutralizes interleukin-17 for treating psoriasis, a protein that neutralizes tumor necrosis factor alpha for treating rheumatoid arthritis, a protein that blocks the interaction of programmed death ligand 1 with its receptor on the surface of cancer cells, a protein that neutralizes stress resistance factors such as heat shock proteins (particularly HSP 110).

[0078] Therapeutic amount (or effective amount) is an amount sufficient to produce beneficial or desired results, such as clinical results, and thus depends on the situation in which it is applied.In the context of administering the polypeptide comprising variants that bind to HSA and peptides or polypeptides with therapeutic activity to patients with diseases disclosed herein, the effective amount of the drug is, for example, an amount sufficient to achieve the cure of the patient, improve the condition of the patient, or accelerate the cure of the patient, compared to the response obtained without administering the drug.It can also be an amount that stops or slows the progression of the disease.It can also be an amount that allows the regression of the disease or the regression of the biological markers associated with a particular disease.

[0079] A therapeutically active agent (or therapeutic agent) is an agent that, when administered in an effective amount to a subject having a disease, improves the subject's condition or reduces a biomarker associated with the disease.

[0080] Note that the sequence of Sac7d is: TIFF2024507780000016.tif11143.

[0081] The HSA binding variants disclosed herein contain mutations at positions corresponding to positions 7, 8, 9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, and 46 of the Sac7 sequence (positions 6, 7, 8, 20, 21, 23, 25, 28, 30, 32, 39, 41, 43, and 45 of SEQ ID NOs: 38-86). The lysine at position 21 in SEQ ID NO: 1 may be maintained in some variants. The lysine at position 8 in SEQ ID NO: 1, the natural amino acid at positions 26, 29, 31, 40, or 46 in SEQ ID NO: 1, or the phenylalanine at position 43 in SEQ ID NO: 1 may also be maintained in some variants.

[0082] However, the variant contains at least four mutated amino acids, preferably at least six, more preferably at least nine, or at least or exactly twelve mutated amino acids compared to the native sequence, which are W22, L24, K33, and Y42 in SEQ ID NO: 1 (W21, L23, K32, and Y41 in SEQ ID NOs: 38-86).

[0083] It is preferred when the variant of a Sac7d family protein that binds HSA is linked or fused to another protein or polypeptide.

[0084] This may be conjugated or fused to another variant of a protein of the Sac7d family, a peptide, or an antibody (as described in WO2019096797).

[0085] In particular, other variants of the Sac7d family of proteins may bind to IL-17 (see WO2019096797), or TNF-α (see WO2020074402).

[0086] In another embodiment, the polypeptide is conjugated to an organic molecule.

[0087] It also makes it possible to obtain a genetic construct comprising a DNA sequence encoding a polypeptide as defined above, a vector comprising such a genetic construct, and a host cell comprising such a genetic construct in its genome, such vector also comprising elements (promoter, terminator, enhancer... etc.) allowing the transcription and translation of the polypeptide.

[0088] It is possible to carry out a method for producing a polypeptide comprising culturing a cell culture, the cells being transformed with a genetic construct, and recovering the polypeptide.

[0089] The disclosed polypeptides may also be embodied as pharmaceuticals, in particular polypeptides comprising variants that bind to HAS fused to another peptide or polypeptide having therapeutic activity.

[0090] It should be noted that the above sequences may contain a methionine at their N-terminus, but it is also possible to carry out the present invention when the N-terminus of each sequence does not contain such a methionine as described. Similarly, amino acids located at the termini of the protein may be omitted. In particular, the amino acids located after residue L58 ​​of Sac7d (residues located after L60 of SEQ ID NO: 16) may be omitted.

[0091] It should be noted that the present specification discloses variants of Sac7d as an example, but the teachings are applicable to other proteins of the Sac7d family. The teachings are also applicable to other OB-fold domains, as disclosed in WO2007139397. In particular, reference is made to WO2012150314, which demonstrates that it is possible to carry mutations from one sequence of the Sac7d family to another, using the alignment shown in FIG. 1. The present invention is also applicable to the SH3 domain, a small protein domain of about 60 amino acid residues, originally described as a conserved sequence in the viral adaptor protein v-Crk and described in the PFAM database under PF00018. The SH3 domain has a characteristic β-barrel fold consisting of five or six β-strands arranged as two closely packed antiparallel β-sheets. The linker region may contain a short helix. It should be noted that the OB-fold and SH3 domains share homology, and given knowledge of the sequences and structures of these domains, it is possible to determine which amino acids in either the OB-fold or SH3 domain correspond to those disclosed below for Sac7d.

[0092] Sac7d protein family The Sac7d family is defined as referring to the Sac7d protein, which corresponds to a family of 7 kDa DNA-binding proteins isolated from extremophilic bacteria. It is disclosed herein as a representative of the OB-fold domain, which is preferably used in the context of the present invention. Since SH3 domain shares homology with OB-fold domain, the teachings on Sac7d are also applicable to SH3 scaffolds.

[0093] These proteins and this family are described in particular in WO 2008 / 068637. Thus, within the context of the present invention, a protein belongs to the Sac7d family if it has one of the sequences SEQ ID NO: 1 to SEQ ID NO: 15 or if it has a sequence corresponding to the consensus sequence SEQ ID NO: 16 (derived from SEQ ID NO: 1 to SEQ ID NO: 9 and SEQ ID NO: 12 to SEQ ID NO: 15, in which the dash "-" indicates the absence of an amino acid and indicates that the proteins do not all have the same size). This Sac7d family is particularly represented by the Sac7d or Sac7e protein from Sulfolobus acidocaldarius, the Sso7d protein from Sulfolobus solfataricus, the DBP 7 also called Sto7 protein from Sulfolobus tokodaii, the Ssh7b protein from Sulfolobus shibatae, the Ssh7a protein from Sulfolobus shibatae, the Mse7 from Metallosphaera sedula, the Mcu7 from Metallosphaera cuprina, the Aho7a or Aho7b or Aho7c from Acidianus hospitalis, the Aho7b or Aho7c from Sulfolobus islandicus, the Aho7c from Sulfolobus sedula, the Aho7d protein from Sulfolobus solfataricus, the Aho7c from Sulfolobus tokodaii, the Aho7d protein from Sulfolobus solfataricus, the Aho7b protein from Sulfolobus shibatae, the Aho7c protein from Sulfolobus sedula, the Aho7c protein from Sulfolobus solfataricus, the Aho7d protein from Sulfolobus solfataricus ... These include the Sis7a or Sis7b from Sac7d family proteins, and the p7ss protein from Sulfolobus solfataricus. Given the extensive sequence similarity of the Sac7d family proteins, it is directly possible and easy to identify the amino acid in another protein than Sac7d that corresponds to a given amino acid in Sac7d. In particular, the teachings of WO 2008 / 068637 can be used, which shows (in the figures) and explains (in the specification) that such proteins are superimposable.

[0094] WO 2012 / 150314 shows that mutations from one protein of the Sac7d family can be carried into another protein of the same family. This transferability corresponds to starting from a mutant of one protein of the Sac7d family to generate a mutant of another protein of the Sac7d family. The initial mutant can in particular be obtained by carrying out the process of WO 2008 / 068637. As a result, it is possible to obtain a mutant of any protein of the Sac7d family starting from a mutant of any other protein of such family, in particular using the teachings of WO 2012 / 150314 and the teachings of FIG. 1. By way of illustration, in the case of a mutant of Sac7d, the sequence alignment of FIG. 1 can be used to introduce the mutated amino acids of the Sac7d mutant into another protein scaffold. By way of illustration, the variants of Aho7c obtained from the Sac7d variants disclosed herein are described as SEQ ID NO: 66 to SEQ ID NO: 86.

[0095] The number of mutated residues introduced into the wild-type protein sequence to obtain a variant is preferably between 4 and 25, or more specifically between 4 and 22. It is thus possible to obtain variants which preferably have at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7 or 8, even more preferably at least 10, but generally less than 25, more preferably less than 22, even more preferably less than 20, or less than 15 or 14 substituted amino acids compared to the wild-type OB-fold protein (or domain). It is noted that all and any ranges (such as 5-20 or 7-25, etc.) are contemplated herein. Particularly preferred ranges are 4-17 and 6-17, 4-11, 4-12, 5-12, 6-12, 4-14 and 6-14.

[0096] It is preferred if 7, 8, 9, 10, 11, 12, 13 or 14 amino acids are mutated in the binding site of the OB-fold domain compared to the wild-type OB-fold domain. These mutations are introduced at amino acids corresponding to V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50, and P51 of Sac7d (SEQ ID NO: 1), preferably at amino acids corresponding to K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46.

[0097] Of particular interest are cases where the number of mutated amino acids is between 7 and 14 (inclusive). In particular, the variants may also include amino acid insertions as described above.

[0098] As shown, the Sac7d family proteins include Sac7d or Sac7e from Sulfolobus acidocaldarius, Sso7d from Sulfolobus solfataricus, DBP 7 also called Sto7 from Sulfolobus tokodayi, Ssh7b from Sulfolobus shibatae, Ssh7a from Sulfolobus shibatae, Mse7 from Metallosphaera sedula, Mcu7 from Metallosphaera cuprina, Aho7a or Aho7b or Aho7c from Acidianus hospitalis, Sis7a or Sis7b from Sulfolobus islandicus, and p7ss from Sulfolobus solfataricus. The various sequences of Sac7d, Sso7d, Sac7e, Ssh7b, Ssh7a, DBP7, Sis7a (3 alleles), Mse7, Mcu7, Aho7a, Aho7b, Aho7c, and Sto7d proteins are represented by SEQ ID NO: 1 to SEQ ID NO: 15, respectively.

[0099] This variant of the Sac7d family protein may be called nanophytin. Thus, the present invention is preferentially carried out on variants of the proteins represented by SEQ ID NO: 1 to SEQ ID NO: 15 or the protein having the sequence SEQ ID NO: 16, in particular on variants of Sac7d.

[0100] Association of Sac7d with OB-fold proteins and SH3 domains OB-fold proteins are known in the art. They are described in particular in the documents cited above, and also in Arcus (Curr Opin Struct Biol. 2002 Dec; 12(6):794-801). The OB-fold is in the form of a cylinder with five beta (β) sheets. Most OB-fold proteins use the same binding interface with a natural ligand, which may be an oligosaccharide, an oligonucleotide, a protein, a metal ion, or a catalytic substrate. This binding interface mainly comprises residues located in the β-sheets. Certain residues located in the loops may also be involved in the binding of OB-fold proteins with their natural ligands. Thus, the applications WO 2007 / 139397 and WO 2008 / 068637 as well as the document of Arcus (2002, op. cit.) describe OB-fold protein domains for binding with their natural ligands.

[0101] In particular, the document WO 2008 / 068637 describes exactly how to identify the binding domain of OB-fold proteins.By superimposing several sequences and three-dimensional structures of proteins with OB-fold domains, it is possible to identify the location of the binding domain and in particular the amino acids that can be modified using the websites WU-Blast2 (http: / / www.ebi.ac.uk / blast2 / index.html) (Lopez et al., 2003, Nucleic Acids Res 31, 3795-3798), T-COFFEE (http: / / www.ch.embnet.org / software / TCoffee.html) (Notredame et al., 2000, J Mol Biol 302, 205-217) and DALI lite (http: / / www.ebi.ac.uk / DaliLite / ) (Holm and Park, 2000, Bioinformatics 16, 566-567). With reference to the sequence of Sac7d (SEQ ID NO: 1), these include residues V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D35, D36, N37, G38, K39, T40, G41, R42, A44, S46, E47, K48, D49, A50, and P51, preferably K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46. Using the alignment shown in Figure 1, it is possible to identify the amino acids in each protein that correspond to these amino acids in Sac7d.

[0102] WO 2008 / 068637 describes that it is possible to carry out a superposition of the three-dimensional structure of an OB-fold protein or domain (in the present application, 10 domains were used, including Sac7d) using the DALI website (http: / / www.ebi.ac.uk / dali / interactive.html) (Holm and Sander, 1998, Nucleic Acids Res 26, 316-319). Thus, for any OB-fold protein (or any OB-fold domain), it is easy to identify the amino acids that are involved in the binding site and that correspond to the Sac7d amino acids mentioned above. As a result, by providing amino acids that can be mutated in one of these proteins, it is possible to identify the corresponding amino acids for any other OB-fold domain.

[0103] It is also noted that OB-fold domains resemble SH3 domains, and it is possible to identify amino acid equivalents of Sac7d in SH3 domains.

[0104] Examples of OB-fold or SH3 domains Non-limiting examples of OB-fold proteins that can be used according to the invention include the N-terminal domains of Sac7d, Sso7d, SEB (Papageorgiou et al., 1998), chain A of Shiga-like toxin IIe (PDB 2bosa), human neutrophil activatin peptide-2 (NAP-2, PDB 1tvxA), molybdenum-binding protein (modg) of Azotobacter vinelandii (PDB 1h9j), the N-terminal domain of SPE-C (Roussel et al., 1997), the B5 subunit of E. coli Shiga-like toxin (Kitov et al., 2000), Cdc13 (Mitton-Fry et al., 2002), the cold shock DNA binding domain of the human Y-box protein YB-1 (Kloks et al., 2002), the E. coli inorganic pyrophosphatase EPPase (Samygina et al., 2001), or any of the proteins listed in Table 3 of the paper by (Arcus, 2002), such as 1krs (lysyl-tRNA synthetase LysS, E. coli), 1c0aA (Asp-tRNA synthetase, E. coli), 1b8aA (Asp-tRNA synthetase, P. kodakaraensis), 1lylA (lysyl-tRNA synthetase LysU, E. coli), 1quqA (replication protein A, 32 kDa subunit, human), 1quqB (replication protein A, 14 kDa subunit, human), 1jmcA (replication protein A, 70 kDa subunit (RPA70) fragment, human), 1otc (telomere end-binding protein, O. nova), 3ullA (mitochondrial ssDNA-binding protein, human), 1prtF (pertussis toxin S5 subunit, B. pertussis), 1bcpD (pertussis toxin S5 subunit (ATP-binding), B. pertussis), 3chbD (cholera toxin, V. cholerae),cholerae), 1tiiD (heat-labile toxin, E. coli), 2bosA (verotoxin-1 / shiga toxin, B-pentamer, E. coli), 1br9 (TIMP-2, human), 1an8 (superantigen SPE-C, S. pyogenes), 3seb (superantigen SPE, S. aureus), 1aw7A (toxic shock syndrome toxin, S. aureus), 1jmc (major cold shock protein, E. coli), 1bkb (initiation translation factor 5a, P. aerophylum), 1sro (S1 RNA-binding domain of PNPase, E. coli), 1d7qA (initiation translation factor 1, elF1a, human), 1ah9 (initiation translation factor 1, IF1, E. coli), 1b9mA (Mo-dependent transcriptional regulator ModE, coli), 1ckmA (RNA guanylyltransferase, Chlorella virus, PBCV-1), 1a0i (ATP-dependent DNA ligase, bacteriophage T7), 1snc (Staphylococcal nuclease, Staphylococcus aureus), 1hjp (DNA helicase RuvA subunit, N-terminal domain, E. coli), 1pfsA (gene V protein, Pseudomonas aeruginosa bacteriophage pf3), 1gvp (gene V protein, filamentous bacteriophage (f1, M13)), 1gpc (gene 32 protein (gp32) core, bacteriophage T4), 1wgjA (inorganic pyrophosphatase, S. cerevisiae), and 2prd (inorganic pyrophosphatase, T. thermophilus).

[0105] Non-exhaustive examples of proteins with SH3 domains are signaling adaptor proteins, CDC24, Cdc25, PI3 kinase, phospholipases, Ras GTPase activating proteins, Vav proto-oncogene, GRB2, p54 S6 kinase 2 (S6K2), SH3D21, C10orf76 (possible), STAC3, some myosins, SHANK1,2,3, ARHGAP12, C8orf46, TANGO1, integrase, focal adhesion kinase (FAK, PTK2), proline-rich tyrosine kinases (Pyk2, CADTK, PTK2beta), or TRIP10 (cip4).

[0106] Production of identified variants The sequences of the identified variants can be cloned into any suitable vector by any molecular genetic method known in the art.

[0107] These recombinant DNA constructs comprising a nucleotide sequence encoding a polypeptide comprising the variants described above are used in conjunction with a vector, such as a plasmid, phagemid, phage, or viral vector.

[0108] These recombinant acid molecules can be produced by the techniques described in Sambrook et al., 1989 (Sambrook J, Fritschi EF and Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York). Alternatively, the DNA sequences can be chemically synthesized, for example, using a synthesizer.

[0109] The recombinant construct of the present invention includes an expression vector that can express RNA and thus produce protein from the gene sequence.Therefore, the vector can further include a regulatory sequence, including a suitable promoter that is operably linked to the open reading frame (ORF) of the gene sequence disclosed herein.The vector can further include a selectable marker sequence, such as an antibiotic resistance gene.When bacteria are used as expression hosts, specific initiation signals and bacterial secretion signals may also be required for efficient translation of the coding sequence.

[0110] Molecular production Cells are transfected or transformed with vectors containing sequences encoding polypeptides comprising variants as disclosed above.

[0111] The cells are cultured under conditions such that the protein is expressed and secreted well. The cell culture conditions are generally those used for recombinant antibody production and are known in the art. Such conditions, known in the art, can also be optimized by the skilled artisan as necessary. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461) provide an overview of such methods and provide ample references thereto.

[0112] For one, bacterial, phage (Shukra et al, Eur J Microbiol Immunol (Bp). 2014; 4(2): 91-98), or eukaryotic production systems can be used.

[0113] For one thing, it may be preferable to use eukaryotic cells to obtain proper post-translational modifications such as glycosylation.

[0114] In particular, CHO (Chinese Hamster Ovary) cells, PER.C6 cells (human cell line, Pau et al, Vaccine. 2001 21;19(17-19):2716-21), HEK 293b cells (human embryonic kidney cells 293), NS0 cells (cell line derived from a non-secretory mouse myeloma) or EB66 cells (duck cell line Valneva, Lyons, France) can be used.

[0115] Also provided by the present disclosure is a host cell comprising at least one of the DNA constructs encoding the polypeptide comprising variants disclosed herein.Host cell can be any cell that expression vector is available for.As mentioned above, it can be higher eukaryotic host cell such as mammalian cell, lower eukaryotic host cell such as yeast cell, or prokaryotic cell such as bacterial cell.

[0116] The introduction of recombinant construct into host cell is carried out by any method known in the art (such as calcium phosphate transfection, lipofection, DEAE, dextran-mediated transfection, electroporation, or phage infection).Vector can be inserted into the genome of host cell or can be maintained as an extragenomic vector (such as bacterial artificial chromosome or yeast artificial chromosome).When introduced into cell genome, such introduction can be targeted or random using methods known in the art (such as homologous recombination).

[0117] Bacterial host and expression Expression vectors useful for bacteria are constructed by inserting a recombinant DNA sequence, with appropriate translation initiation and termination signals, in functional reading phase with a functional promoter. The vector may contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide for amplification within the host.

[0118] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0119] Eukaryotic hosts and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, the above mentioned cells can be used.

[0120] The transformed or transfected cells are cultured by methods known in the art and the polypeptide is recovered from the intracellular or extracellular fraction (depending on whether it is secreted or not).

[0121] Molecular Isolation The produced recombinant protein can be separated and purified from the intracellular or extracellular fraction by any of a variety of known separation methods that exploit the physical or chemical properties of the protein.

[0122] In particular, methods such as precipitation, tangential flow filtration, ultrafiltration, various liquid chromatography techniques such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and combinations thereof can be used.

[0123] Generally, any method known and used to purify recombinant polypeptides can be adapted to purify the molecules disclosed herein.

[0124] If a tag (such as a polyhistidine tag) has been introduced into the recombinant sequence, this tag can be used to purify the molecule, however it is preferred to purify the molecule using affinity.

[0125] In particular, the fact that the molecules produced herein bind to specific targets can be used and any affinity method (affinity columns, FACS, beads) can be used to isolate such molecules.

[0126] One particular advantage of the molecules disclosed herein is that they do not need to be glycosylated to be active and therefore may be produced in any type of cell, not necessarily a eukaryotic cell. They are particularly well produced in bacterial cells.

[0127] Modification of variants The above variants having the ability to bind to human serum albumin and mouse serum albumin can be modified by any method known in the art.

[0128] Preparation of polypeptides containing variants It is possible to prepare a DNA sequence that contains two coding sequences, one for the variant disclosed herein and the other for the protein or peptide of interest (especially having therapeutic activity).The resulting expressed protein is thus a polypeptide that contains both proteins.Vector can be constructed to contain a sequence that codes for a linker that is located between the two proteins in the expressed polypeptide.

[0129] Consequently, the present invention also encompasses polypeptides comprising a variant of an OB-fold protein (preferably of the Sac7d family) that binds to HSA, which is linked to another protein or polypeptide (preferably via an amine bond as disclosed above).

[0130] In certain embodiments, the other protein or polypeptide comprises another variant of a protein of the Sac7d family.

[0131] Of particular interest are the following: - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSA, fused to a variant of an OB-fold protein of the Sac7d family that binds (at the N-terminus or C-terminus) to interleukin-17 and neutralizes its interaction with the receptor for the treatment of an autoimmune disease such as psoriasis or psoriatic arthritis. - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSA, fused to a variant of an OB-fold protein of the Sac7d family that binds (at the N-terminus or C-terminus) to programmed death ligand 1 and neutralizes its interaction with its cognate receptor for the treatment of cancer, such as solid tumors (particularly breast, lung, kidney, pancreatic cancer). - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSA, fused to a variant of an OB-fold protein of the Sac7d family that binds to heat shock protein 110 (at the N-terminus or C-terminus) and neutralizes its ability to induce cellular resistance to stress and macrophage polarization, thereby alleviating resistance to anti-cancer drugs and / or eliciting anti-cancer activity of its own. - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSA, fused to a variant of an OB-fold protein of the Sac7d family that binds (at the N-terminus or C-terminus) to tumor necrosis factor alpha. Such polypeptides act as inhibitors of inflammatory signals mediated by TNFα and are useful for reducing inflammation in diseases such as rheumatoid arthritis, Crohn's disease, and more generally inflammatory bowel disease.

[0132] In another embodiment, the other protein or polypeptide is an antibody. In this embodiment, the OB-fold domain variant is fused to at least one of the heavy or light chains of the immunoglobulin monomer, preferably to the N-terminus or C-terminus of the light or heavy chain. In another embodiment, the variant may be fused to both the heavy or light chain.

[0133] To obtain such a compound, a genetic construct can be used that comprises a DNA sequence selected from the group consisting of: a. A sequence encoding an antibody heavy chain fused at its 3' end to a sequence encoding a variant of an OB-fold protein (potentially via a sequence encoding a linker), b. A sequence encoding an antibody heavy chain fused at its 5' end to a sequence encoding a variant of an OB-fold protein (potentially via a sequence encoding a linker). c. A sequence encoding an antibody light chain fused at its 3' end to a sequence encoding a variant of an OB-fold protein (potentially via a sequence encoding a linker). d. A sequence in which a sequence encoding the light chain of an antibody is fused at its 5' end to a sequence encoding a variant of an OB-fold protein (potentially via a sequence encoding a linker).

[0134] This fusion can be made at the N-terminus and / or C-terminus of the antibody chains (heavy and / or light). It should be noted that, especially when using small OB-fold domains (about 70 amino acids) such as proteins from the Sac7d family, it is possible to obtain molecules with the structure of an antibody (two light chains paired with two heavy chains, and such co-paired dimers) with an antibody region and an additional binding region consisting of a modified OB-fold domain.

[0135] In certain embodiments, the antibody portion of the proteins disclosed herein is an IgG molecule.

[0136] In another embodiment, the antibody portion of the proteins disclosed herein is an IgA molecule.

[0137] In another embodiment, the antibody portion of the proteins disclosed herein is an IgM molecule.

[0138] In another embodiment, the antibody portion of the proteins disclosed herein is an IgD molecule.

[0139] In another embodiment, the antibody portion of the proteins disclosed herein is an IgE molecule.

[0140] The antibody may be a human antibody, a rodent antibody (such as a mouse antibody or a rat antibody), a feline antibody, a dog antibody, a chicken antibody, a goat antibody, a camelid antibody (such as a camel antibody, a llama antibody, an alpaca antibody or a nanobody), a shark antibody, or an antibody from any other species. The antibody may be a chimeric antibody or a humanized antibody. As recalled in Wikipedia, a humanized antibody is an antibody from a non-human species whose protein sequence has been modified to increase similarity to antibody variants naturally produced in humans. A chimeric antibody contains sequences from a different species.

[0141] It is preferred if the antibody that is part of the molecules disclosed herein is an antibody that contains two identical heavy chains (about 400-500 amino acids, typically around 450 amino acids) and two identical light chains. As a result, the antibody contains the same Fab variable region. This antibody is therefore a monospecific antibody in that both parts of the antibody (combination of light and heavy chains) bind to the same epitope of an antigen.

[0142] However, the antibody may present different heavy and / or light chains. In particular, in some embodiments, the antibody is a bispecific antibody. Thus, the term "antibody" encompasses both "classical antibodies" as disclosed above, which have identical heavy and light chains, but also engineered antibodies, which have two or more specificities.

[0143] In certain embodiments, an antibody displays one heavy and light chain from one antibody and another heavy and light chain from another antibody.

[0144] The antibody may bind to a target selected from the group consisting of: Cell surface receptors: insulin receptor, low density lipoprotein receptor-related protein 1, transferrin receptor, epidermal growth factor receptor, epidermal growth factor receptor variant III, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, Her2, Her3, Her4, PMSA, IGF-1R, GITR, RAGE, CD28. Cell surface proteins: Mesothelin, EpCam, CD19, CD20, CD38, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, MadCam, a4b7, CD7, CD4, CD138; integrins such as αVβ6, α4β7, α4β1, αEβ7. Angiogenic and growth factors: VEGF, Angiopoietin 2, HGF, PDGF, EGF, GM-CSF, HB-EGF, TGF Immune checkpoint inhibitors or activators: PD-1, PD-L1, CTLA4, CD28, B7-1, B7-2, ICOS, ICOSL, B7-H3, B7-H4, LAG3, KIR, 4-1BB, OX40, CD27, CD40L, TIM3, A2aR Circulating proteins: TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Bace1, α-synuclein, Tau, Amyloid.

[0145] It is therefore specifically foreseen that: - a polypeptide comprising a variant as disclosed above that binds to HSA and an antibody that binds to IL-17 or IL-17R (such as ixekizumab or brodalumab) for the treatment of autoimmune diseases such as psoriasis. - Polypeptides comprising the variants disclosed above that bind to HSA and antibodies that bind to PD-L1 (such as atezolizumab, avelumab, or durvalumab) for the treatment of cancers such as kidney cancer, lung cancer, breast cancer, pancreatic cancer, and various types of blood cancers such as multiple myeloma, lymphoma, etc. - Polypeptides comprising the variants disclosed above that bind to HSA and antibodies that bind to EGFR for treating solid tumors (such as cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab).

[0146] Alternatively, the polypeptide may include the variants disclosed above and biologically active molecules such as erythropoietin, interferon, lysosomal disrupting enzymes, or etanercept.

[0147] In another embodiment, the variant of the OB-fold domain (particularly the variant of the Sac7d family protein) is conjugated to an organic molecule. This can be done by any method known in the art. In particular, the molecule can be chemically linked to the protein. The molecule can include anti-proliferative agents (cytotoxic and cytostatic agents), including cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors. Anti-inflammatory molecules can also be used.

[0148] In particular, DNA binding or alkylating drugs, such as anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and their analogs, alkylating agents, such as calicheamicin, dactinomycin, mithromycin, pyrrolobenzodiazepines, etc., may be mentioned. Cell cycle progression inhibitors, such as CDK inhibitors, Rho kinase inhibitors, checkpoint kinase inhibitors, aurora kinase inhibitors, PLK inhibitors, and KSP inhibitors, may also be mentioned. Thalidomide and its derivatives, lenalidomide and pomalidomide, may also be mentioned. Cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitors, quercetin, and / or resveratrol may also be used as molecules conjugated to the polypeptides comprising variants to treat inflammatory disorders.

[0149] It is therefore foreseen, inter alia: - Polypeptides, including variants as disclosed above, that bind to HSA, as well as Janus kinase inhibitors for the treatment of inflammatory diseases, in particular Crohn's disease. - Polypeptides, including the variants disclosed above that bind to HSA, as well as doxorubicin for the treatment of solid tumors, such as pancreatic, ovarian, lung, renal, liver, breast cancer.

[0150] Molecules of interest and preferred molecules are also disclosed above.

[0151] Using variants In particular the variants may be used in therapeutic methods, particularly for treating cancer, metabolic diseases (such as diabetes), neurological diseases, inflammatory diseases and autoimmune diseases (such as rheumatoid arthritis, psoriasis, lupus or multiple sclerosis), or enzyme deficiencies, particularly in the brain, using biomolecules having activity for the treatment of such diseases.

[0152] The present invention therefore relates to a method for the treatment of cancer, metabolic diseases (such as diabetes), neurological diseases, inflammatory diseases and autoimmune diseases (such as rheumatoid arthritis, psoriasis, lupus or multiple sclerosis), or enzyme deficiencies, particularly in the brain, comprising the administration to a subject in need thereof of a therapeutic amount of an OB-fold variant disclosed herein (in particular a variant of a protein of the Sac7d family) fused to a molecule (a polypeptide or other molecule having a therapeutic effect against the disease being treated).

[0153] The term "therapeutic amount" or "effective amount" as used herein refers to an amount sufficient to produce beneficial or desired results, such as clinical results, and "effective amount" depends on the context in which it is applied. An effective amount is an amount that produces therapeutic improvement while minimizing side effects or adverse effects. For example, therapeutic improvement can be regression of tumor size of solid tumors, improvement of quality of life of a subject, improvement of efficacy of combination therapy.

[0154] The variants can be administered by any method known in the art.

[0155] In particular, the variant can be injected. In another embodiment, the variant can be applied topically (on the patient's skin or eye) as disclosed in WO 2014 / 173899. In another embodiment, the variant can be administered orally as disclosed in WO 2016 / 062874.

[0156] The variants or polypeptides containing the variants may also be used in diagnostic methods, in particular such variants or polypeptides may be linked to any marker known in the art and used in imaging methods. [Brief description of the drawings]

[0157] [Figure 1] Alignment of Sac7d family proteins. [Diagram 2]Binding kinetic profiles and binding kinetic values ​​of B11 (panel A) and F06 (panel B) to human serum albumin (HSA) obtained by biolayer interferometry. B11 and F06 were run on a streptavidin biosensor loaded with biotinylated HSA over a concentration range of 15.6-1000 nM. Association and dissociation were performed for 180 s each. [Diagram 3] Percentage of fluorescein remaining in serum after a single intravenous (iv) injection in mice either in the form of anti-albumin nanophytin conjugate (blue, n=3) or not (red). Data for fluorescein alone are extracted from (Chahalet et al., Invest. Ophthalmol. Vis. Sci. 26(1985)764-768) and are included here for reference. [Figure 4] Concentration measurements as a function of time of anti-albumin variants fused to protein partners. [Diagram 5] ELISA signals of nanophytin B11 (panel A) and F06 (panel B) on different domains of human serum albumin. EXAMPLES

[0158] Example 1. Characterization of Sac7d variants that bind albumin The affinity of nanophytins B11 (SEQ ID NO: 58) and F06 (SEQ ID NO: 65 with L56M) towards human serum albumin (HSA) was investigated by biolayer interferometry (octet red96) and is shown in Figure 2. Biotinylated HSA was immobilized on a streptavidin biosensor and the association (180 sec) and dissociation (180 sec) of nanophytins were studied over a concentration range from 15.6 nM to 1000 nM. In these conditions, B11 and F06 were found to have affinities of 0.1 and 8 nM, respectively. Similar affinities were obtained at pH 7.4 and pH 6.

[0159] TIFF2024507780000017.tif21146 (Table 10) Affinity and binding kinetic parameters of B11 and F06 determined by biolayer interferometry experiments.

[0160] Using similar conditions, the ability of the two nanophytins to bind mouse (MSA), rat (RSA), dog (DSA), cynomolgus monkey (CSA), pig (PSA), bovine (BSA), and rabbit (RabSA) serum albumins was also investigated. Cross-reactivity was observed with all albumins from the various species investigated, except for rabbit serum albumin.

[0161] These results were repeated with point mutations, namely D16E, N37Q, and M57L, either individually or simultaneously, for both nanophytins, without any significant changes.

[0162] TIFF2024507780000018.tif21148 (Table 11) A table showing the cross-reactivity patterns of B11 and F06 against albumins of different species. ND stands for not done. Increasing "3" symbols refer to increasing strength of binding. Symbol "-" refers to no detectable binding.

[0163] Example 2. Variants increase the half-life of small molecules Serum half-life (t 1 / 2 To illustrate the ability of anti-albumin nanophytin to extend the t , we used fluorescein as a model. Fluorescein is a small molecule (332 Da) that is widely used as a fluorescent tracer. Its biodisponibility in mouse serum after a single intravenous injection has already been studied and reported. Analysis of data from the literature (Chahalet et al., Invest. Ophthalmol. Vis. Sci. 26(1985)764-768) showed a t of approximately 8 minutes. 1 / 2It has been suggested that fluorescein is conjugated to anti-albumin nanophytin under its isothiocyanate derivative (FITC). After a single bolus intravenous injection of anti-albumin nanophytin-fluorescein conjugate in mice (20 mg / kg, 3 mice per time point), the presence of fluorescein in serum could be followed by monitoring the fluorescence (excitation: 488 nm and emission: 520 nm) of serum samples taken at time intervals (0, 1, 4, 6, and 24 hours) as shown in Figure 3. The resulting t in mouse serum of fluorescein upon conjugation to anti-albumin 1 / 2 was calculated to be 174 minutes.

[0164] Example 3. Increasing the half-life of a protein partner The serum half-life (t 1 / 2 To illustrate the ability of anti-albumin nanophytin to prolong the t , we used another nanophytin targeting TNFα as a model. Nanophytin is a small protein (7 kDa) that is naturally and rapidly excreted from the systemic circulation because its size is below the glomerular filtration cutoff of the kidney (50 kDa) and it is essentially not recycled. Anti-TNFα nanophytin and a chimeric construct consisting of a genetic fusion of anti-TNFα nanophytin with anti-albumin nanophytin were injected into mice as a single bolus injection. Blood sampling was performed at different time intervals (5 min, 0.5, 4, 24, and 48 h) and monitoring of the presence of nanophytin products in the serum was performed by Western blot using anti-His tag antibody to reveal. Data were plotted as a function of concentration measurement against time (Figure 4). Anti-TNFα nanophytin had a t of about 10-15 min. 1 / 2 showed rapid clearance at t = 1.0, whereas fusion with anti-albumin nanophytin showed a t = 1.0 in mice of >16 h. 1 / 2 This resulted in a long retention time in serum.

[0165] Example 4. Domains targeted by anti-albumin nanophytin Albumin has been described as having three domains, designated domain I, II, and III. The interaction of albumin with the FcRn receptor involves domain I and III. Domain I (aa1-197), domain II (aa189-385), and domain III (aa381-585) as well as domain I-II (aa1-385) were obtained from Albumin Bioscience. The interaction of anti-albumin nanophytins with the different domains was investigated by biolayer interferometry according to the following procedure. His-tagged nanophytins B11 and F06 were immobilized at 1 nm on a Ni:NTA biosensor. The sensor was then equilibrated for 60 s in protein-free blocking buffer (Thermo Scientific) supplemented with 0.002% tween20. The binding of different albumin domains (250 nM in protein-free blocking buffer supplemented with 0.002% tween 20) was monitored for 180 seconds. The response at 180 seconds was then recorded and used to reflect the binding capacity of B11 and F06 nanophytins to different albumin domains.

[0166] Both nanophytins were found to interact exclusively with domains I-II and II, demonstrating that they target domain II of albumin ( Figure 5 ).

[0167] This was further demonstrated by competition experiments with the albumin binding domain G148-GA3 (He et al.; Protein Science; 16(2007)1490-1494). The interaction of the two anti-albumin nanophytins is inhibited by the presence of G148-GA3. G148-GA3 has been shown to bind to domain II of HSA (Lejon et al.; J. Biol. Chem.; 279(2004)42924-42928). Domain II of HSA is distant from the interaction site of albumin with FcRn. By targeting domain II of HSA, anti-albumin nanophytins can bind to albumin without compromising its binding with FcRn and nanophytins can benefit from its recycling mechanism to extend their serum half-life. This is even more plausible considering that the affinity of anti-albumin nanophytin remains similar at both pH 7.4 and pH 6, allowing it to remain bound to its target during the recycling process.

[0168] Example 5. Anti-albumin nanophytin improves the pharmacokinetics of active related peptides As disclosed, a genetic fusion of exenatide peptide (a glucagon-like peptide-1 receptor agonist) with albumin-bound nanophytin (ABNF) was performed. The specific activity of exenatide-ABNF was measured and was not affected by the fusion. In vivo mouse results provide compelling data with sustained pharmacological activity over 3 days (t of exenatide peptide compared to 20 hours of exenatide-ABNF). 1 / 2 provided a 35-minute (8 min) time window (Michot et al, Peptides. 2022 Feb 9; 170760).

Claims

1. A polypeptide comprising a variant of a Sac7d family member that binds to human and mouse serum albumin, the variant comprising between 4 and 20 mutated residues in the binding interface of the Sac7d family member to its natural ligand, and the variant comprising K22W, W24L, T33K, R42Y mutations numbered corresponding to positions in the Sac7d sequence SEQ ID NO:

1.

2. 2. The polypeptide of claim 1, further comprising at least one mutation selected from D16E, N37Q, and M57L, numbered corresponding to positions in the Sac7d sequence SEQ ID NO:

1.

3. 3. The polypeptide of claim 1 or 2, wherein the mutated residues in the binding interface of a member of the Sac7d family to its natural ligand are selected from the group consisting of V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, A44, S46, E47, K48, D49, A50, and P51 of Sac7d.

4. 4. The polypeptide of any one of claims 1 to 3, further comprising at least one mutation selected from Y8F, Y8P, A44F, A44V, numbered corresponding to positions in the Sac7d sequence SEQ ID NO:

1.

5. Sac7d from Sulfolobus acidocaldarius, Sac7e from Sulfolobus acidocaldarius, SSo7d from Sulfolobus solfataricus, Ssh7b from Sulfolobus shibatae, Ssh7a from Sulfolobus shibatae, DBP7 from Sulfolobus tokodaii, Sis7a from Sulfolobus islandicus, Mse7 from Metallosphaera sedula, Mcu7 from Metallosphaera cuprina, Acidianus hospitalis The polypeptide of any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of Aho7a from Acidianus hospitalis, Aho7b from Acidianus hospitalis, Aho7c from Acidianus hospitalis, and Sto7 from Sulfurisphaera tokodaii.

6. 6. The polypeptide of any one of claims 1 to 5, comprising SEQ ID NO: 38 or amino acids 1 to 54 of SEQ ID NO:

38.

7. 7. The polypeptide of claim 6, comprising a sequence selected from SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, and amino acids 1 to 57 of these sequences.

8. 7. The polypeptide of claim 6, comprising a sequence selected from SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 86, and amino acids 1 to 55 of these sequences.

9. 9. A polypeptide according to any one of claims 1 to 8, which is composed of a variant of a member of the Sac7d family that binds to human and mouse serum albumin.

10. 9. The polypeptide of any one of claims 1 to 8, wherein the variant of a member of the Sac7d family that binds human and mouse serum albumin is conjugated to an organic molecule.

11. 9. The polypeptide of any one of claims 1 to 8, wherein the variant of a member of the Sac7d family that binds to human and mouse serum albumin is conjugated to another polypeptide, in particular another variant of a protein of the Sac7d family.

12. The other polypeptide is selected from the group consisting of insulin receptor, low density lipoprotein receptor-related protein 1, transferrin receptor, epidermal growth factor receptor, epidermal growth factor receptor variant III, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, Her2, Her3, Her4, PMSA, IGF-1R, GITR, RAGE, CD28, mesothelin, EpCam, CD19, CD20, CD38, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, MadCam, a4b7, CD7, CD4, CD138; integrins such as αVβ6, α4β7, α4β1, αEβ7; V 12. The polypeptide of claim 11, which binds to a target selected from EGF, Angiopoietin 2, HGF, PDGF, EGF, EGFR, GM-CSF, HB-EGF, TGF, PD-1, PD-L1, CTLA4, CD28, B7-1, B7-2, ICOS, ICOSL, B7-H3, B7-H4, LAG3, KIR, 4-1BB, OX40, CD27, CD40L, TIM3, A2aR, TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Bace1, alpha-synuclein, Tau, and amyloid.

13. The polypeptide of claim 11, wherein the other polypeptide is another variant of a protein of the Sac7d family selected from a variant that binds to interleukin-17 and neutralizes its interaction with its receptor, a variant that binds to PD-L1, a variant that binds to heat shock protein 110, and a variant that binds to tumor necrosis factor alpha.

14. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 9 and 11 to 13.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 13 or a nucleic acid according to claim 14, and a pharma- ceutically acceptable carrier.

16. A method for producing a polypeptide according to any one of claims 1 to 9 and 11 to 13, comprising the steps of: (a) culturing a cell culture, the cells being transformed with a nucleic acid according to claim 14; and (b) recovering said polypeptide. A method comprising the steps of:

17. Use of a polypeptide according to any one of claims 1 to 13 in the manufacture of a medicament.

18. The method of claim 17, wherein the polypeptide is a variant that binds to interleukin-17 and neutralizes its interaction with its receptor, and the medicament is for the treatment of an autoimmune disease, such as psoriasis or psoriatic arthritis. the other polypeptide is a variant that binds to PD-L1 and the medicament is for the treatment of cancer, such as solid tumors (particularly breast cancer, lung cancer, kidney cancer, pancreatic cancer); The other polypeptide is a variant that binds to heat shock protein 110, and the medicament is for the treatment of cancer by alleviating resistance to anti-cancer drugs and / or eliciting its own anti-cancer activity, or Another polypeptide is a variant that binds to tumor necrosis factor alpha and the medicament is for reducing inflammation in diseases such as rheumatoid arthritis, Crohn's disease and more generally inflammatory bowel disease.

18. The use according to claim 17.

19. A pharmaceutical composition for the treatment of an autoimmune disease comprising the polypeptide of claim 11, wherein another polypeptide is another variant of a protein of the Sac7d family that binds to interleukin-17.

20. A pharmaceutical composition for the treatment of cancer comprising the polypeptide of claim 11, wherein another polypeptide is another variant of a Sac7d family protein that binds to PD-L1.

21. A pharmaceutical composition for the treatment of inflammation in diseases such as rheumatoid arthritis, Crohn's disease and more generally inflammatory bowel disease comprising a polypeptide as described in claim 11, wherein another polypeptide is another variant of a protein of the Sac7d family that binds to tumor necrosis factor alpha.

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