Polypeptides as carriers for crossing the intestinal barrier

Abstract

The present invention relates to polypeptides, including variants of proteins of the Sac7d family, that specifically bind to the human leptin receptor and are capable of inducing crossing of the intestinal barrier, in particular the crossing of biological agents associated therewith.

Description

[Background technology]

[0001] Introduction Increasing understanding of disease mechanisms and their pathogenesis at the molecular level has driven the development of targeted therapies, where biopharmaceuticals are fully integrated into clinical practice and are expected to account for an increasing proportion of therapeutic development due to their potential to provide greater specificity compared to traditional small molecules.

[0002] There remain two main challenges that need to be addressed to close the gap between small molecule and biopharmaceutical development: i) the development costs of biopharmaceuticals are several times higher, and ii) the administration of biopharmaceuticals is almost exclusively restricted to parenteral routes.

[0003] Patient feedback ranks oral administration as the most preferred route of administration, which correlates with higher compliance with oral therapy compared to parenteral drugs. Despite the preference for oral therapy, the development of oral biopharmaceuticals has been limited by the lack of stability of biopharmaceuticals in the gastric environment and the poor passive diffusion of large molecules through biological barriers, such as the intestinal barrier. Receptor-mediated active transport mechanisms have been designed by nature for the transfer of large biomolecules through biological barriers. Hijacking these transport mechanisms has been explored for the transport of drugs through the blood-brain barrier using ligands that bind to receptors that naturally mediate the transport of biomolecules in the brain, such as TfR, LRP1, and insulin receptor, as Trojan horse delivery systems.

[0004] The development of a similar strategy for transport across the intestinal barrier would allow for systemic delivery of cargo molecules upon oral administration. This would require technologies that could maintain stability within the intestinal lumen. Nanofitins are small alternative scaffolding proteins derived from the natural hyperthermostable protein sac7d that can be engineered to be highly specific and highly affinitive for a given target while retaining the extraordinary stability of the parent protein, including resistance to protease degradation.

[0005] Leptin is a peptide hormone secreted mainly by tissues such as adipocytes and stomach, and has many physiological effects.Leptin mainly acts on the central nervous system to regulate energy homeostasis and neuroendocrine function, and plays a role in various functions in the gastrointestinal tract, such as tissue healing or glucose metabolism.Therefore, this multipotent hormone requires interaction with its cell surface receptor, human leptin receptor (human LepR or hLepR, UniProtKB-P48357), which is present in various barriers, such as the blood-brain barrier or the intestinal barrier.Therefore, leptin receptor is responsible for the receptor-mediated active transfer of its natural ligand, leptin, as described in the blood-brain barrier and the intestinal barrier.

[0006] EP 3 632 924 A1 discloses the production of variants of members of the Sac7d family that are capable of binding to subunits of multimeric proteins and inhibiting multimer formation.

[0007] WO 2008 / 068637 (Patent Document 2) discloses the ability to obtain variants of the Sac7d protein against various targets.

[0008] US 20190113512 (Patent Document 3) discloses proteins comprising a target-binding domain for detecting a target of interest, including a cellulose-binding domain (CBD) or a carbohydrate-binding module (CBM) and an engineered charge-reduced Sso7d (rcSso7d) antigen-binding protein, and methods, compositions, and kits thereof.

[0009] D4 Loussouarn et al (Scientific Reports, vol. 10, no. 1, 1 2020) disclose the stability of Sac7d variants under simulated gastric and intestinal digestive conditions and the design to enhance their properties to acquire resistance to proteolytic digestion while maintaining or increasing the critical pH and temperature stability of these proteins. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] EP 3 632 924 A1 [Patent Document 2] WO 2008 / 068637 [Patent Document 3] US 20190113512 [Non-patent literature]

[0011] [Non-Patent Document 1] D4 Loussouarn et al (Scientific Reports, vol. 10, no. 1, 1 2020) Summary of the Invention

[0012] In a first aspect, the invention relates to a polypeptide comprising a variant of a member of the Sac7d family that binds to the human leptin receptor (as well as to the mouse and porcine leptin receptors), the variant comprising 4-20 mutated residues at the interface of the binding of the Sac7d family member to its natural ligand, and the variant comprising 5-15 mutations according to the numbering corresponding to the Sac7d (SEQ ID NO: 1) residues. In particular, the variant comprises 5-14 or 5-13 mutated residues at the interface of the binding of the Sac7d family member to its natural ligand. In other embodiments, the variant comprises 6-15, or 6-14, or 6-13 mutated residues. In some embodiments, the variant contains exactly 15 mutated residues (not considering deletions at the N- or C-terminus). In some embodiments, the variant contains exactly 14 mutated residues (not considering deletions at the N- or C-terminus). In some embodiments, the variants contain exactly 13 mutated residues (not considering deletions at the N- or C-terminus). In some embodiments, the variants contain exactly 12 mutated residues (not considering deletions at the N- or C-terminus).

[0013] In particular, the polypeptide further comprises W24Y, T33W, and A44H mutations, with numbering corresponding to the Sac7d (SEQ ID NO: 1) residues.

[0014] As shown in the Examples, these mutations are present in all members of Cluster A, which is a rearrangement of identified binders that exhibit homology (the same amino acid, or the same type (in terms of size, hydrophobicity, polarity, etc.) of amino acids) in the mutations present in the binding site. Analysis of the other mutated amino acids indicates that there may be variation in these other amino acids (as shown in Tables 3, 8, and 10), which gives weight to the presence of the W24Y, T33W, and A44H mutations present in all members of Cluster A.

[0015] Moreover, the examples show that these variants are very suitable for pharmacological development, since they bind to human, mouse and porcine leptin receptors. The fact that variants belonging to cluster A bind to leptin receptor and do not compete with leptin is also advantageous for use in pharmacological contexts, since it can reduce side effects that may be observed when leptin's binding to its receptor is eliminated or reduced. It should also be noted that, although it is known that variants of the Sac7d family can cross the intestinal barrier (as shown in WO2016062874 and US 20180085427, and in the examples that confirm that a small portion of non-specific variants cross the barrier), the amount of specific variants disclosed herein that cross the intestinal barrier is much greater than the amount of non-specific variants or the amount of leptin receptor binding bodies belonging to other clusters. This strongly suggests the active passage of cluster A binding bodies, as opposed to the passive passage of other Sac7d variants. Indeed, it has been shown that binding to leptin receptors on the surface of intestinal cells induces transport of the variant (alone or bound to another polypeptide) to the opposite surface of the intestinal tract.

[0016] In particular, the mutated residues in the binding interface of a Sac7d family member 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 (SEQ ID NO: 1).

[0017] In some embodiments, the mutated residues are on K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46 of Sac7d (SEQ ID NO: 1).

[0018] In some embodiments, the mutated residues are on K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, R42, and A44 of Sac7d (SEQ ID NO: 1).

[0019] In some embodiments, the polypeptide further comprises at least one mutation selected from D16E, N37Q, and M57L, numbered according to the corresponding Sac7d (SEQ ID NO: 1) residues.

[0020] In particular, the members of the Sac7d family include 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, and Acidianus hospitalis. The host cell is selected from the group consisting of Aho7a from Acidianus hospitalis, Aho7b from Acidianus hospitalis, Aho7c from Acidianus hospitalis, and Sto7 from Sulfurisphaera tokodaii.

[0021] Based on the Sac7d family consensus sequence (SEQ ID NO:16), variants include SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29, or amino acids 5-56, 5-57, 5-58, 5-59, 5-60, or 5-61 of these sequences.

[0022] In some embodiments, the polypeptide is based on Sac7d and includes SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49, or amino acids 1-54 of these sequences.

[0023] These sequences differ according to (1) the preferred mutation specifications and (2) the presence or absence of certain mutations that lie outside the binding site and that favor stability and / or industrial production.

[0024] In some embodiments, the polypeptide is based on Aho7c and includes 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, or SEQ ID NO:64, or amino acids 1-54 of these sequences.

[0025] Variants based on the consensus sequence of Sac7d and Aho7c (represented by SEQ ID NO:65) include SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or amino acids 1 to 54 of these sequences.

[0026] In some embodiments, the polypeptide is based on Sso7d and comprises SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70, or amino acids 1-53 or 1-54 of these sequences.

[0027] In some embodiments, the polypeptide can include amino acids 1-55, 1-56, 1-57, 1-58, or 1-59 of the preceding sequences.

[0028] In some embodiments, the polypeptide is within a variant of a member of the Sac7d family that binds to the human leptin receptor.

[0029] In some embodiments, the variant of a Sac7d family member that binds to the human leptin receptor is conjugated to an organic molecule.

[0030] In some embodiments, the variant of a Sac7d family member that binds to the human leptin receptor is conjugated to another polypeptide, particularly another polypeptide that includes another variant of a protein of the Sac7d family. In some embodiments, the variant of a Sac7d family member that binds to the human leptin receptor is conjugated to another variant of a protein of the Sac7d family.

[0031] The present invention also relates to nucleic acid molecules encoding the described polypeptides, expression vectors comprising such nucleic acid molecules (including elements enabling transcription in a host cell), and host cells comprising said nucleic acid molecules or said expression vectors.

[0032] The invention also relates to a pharmaceutical composition comprising a disclosed polypeptide, a disclosed nucleic acid, a disclosed expression vector, or a disclosed host cell and a pharma- ceutically acceptable carrier.

[0033] The present invention also provides a method for producing the disclosed polypeptides, comprising: a. culturing a cell culture in which the cells are transformed with a disclosed expression vector; and b. Recovering the polypeptide The present invention relates to a method comprising the steps of:

[0034] The invention also relates to the disclosed polypeptides or nucleic acids for use as a medicament.

[0035] The present invention also relates to the disclosed leptin receptor binding variants fused to an active molecule, particularly another polypeptide, or a nucleic acid encoding same, an expression vector, or a cell, for use in the treatment or prevention of a disease, wherein the variant is associated with a substance active for the treatment or prevention of said disease, which substance may be designated as a therapeutically active substance.

[0036] The present invention also relates to a method of treating a subject having or at risk of developing a given disease, comprising administering to the subject a composition comprising an effective amount of a LepR binding variant associated or bound to a therapeutically active substance for treating or preventing the given disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention In particular, the present invention relates to a polypeptide comprising a variant of Sac7d protein or a variant of a protein of the Sac7d family, which specifically binds to the human leptin receptor (referred to as P48357 in the UNIPROT database). In particular, the variant binds to human, porcine and mouse leptin receptors. Also, the variant preferably allows crossing of the intestinal barrier when bound to another polypeptide, in particular another variant of a protein of the Sac7d family. Such variants are useful in fusing, binding or associating with therapeutically active substances to allow such substances to be administered orally or rectally, to cross the intestinal barrier and to act, in particular locally in the intestine or in other organs of the body after passing through the systemic circulation.

[0038] In preferred embodiments, such variants include SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29. These sequences are based on consensus sequences for the Sac7d family and were obtained according to the disclosure of WO 2012 / 150314, which shows that mutations can be transferred from one protein of the Sac7d family to another, which proteins exhibit similar structures and binding sites, as well as similar amino acid sequences. SEQ ID NOs:22-25 are consensus sequences having a D at position 17, while SEQ ID NOs:26-29 exhibit an E at position 17, which corresponds to position 16 of SEQ ID NO:1 as seen in the sequence alignment of Figure 1. The first one, two or three amino acids of these sequences may be omitted, as may some or all of the last twelve.

[0039] In some embodiments, the variant is based on the Sac7d protein and comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 18, or SEQ ID NO: 49, or amino acids 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, or 1-63 of these sequences. SEQ ID NOs:41-44 represent variants having the natural amino acids D, N, and M at positions 16, 37, and 57 of SEQ ID NO:1 (these sequences omit the initial M of SEQ ID NO:1), while SEQ ID NOs:45-49 represent variants having the modified amino acids E, Q, and L at these positions. SEQ ID NOs:35-39 represent sequences which are consensus for these positions.

[0040] In some embodiments, the variant is based on the Sso7d protein (SEQ ID NO:2) and includes SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70, or amino acids 1-53, 1-54, 1-55, 1-56 of these sequences, in which the first three amino acids of SEQ ID NO:2 are omitted.

[0041] In some embodiments, the variant is based on the Aho7c protein (SEQ ID NO:14) and includes 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, or SEQ ID NO:64, or amino acids 1-54, 1-55, 1-56, or 1-57 of these sequences. SEQ ID NOs:55-59 represent variants of SEQ ID NO:14 having the natural amino acids D and N at positions 17 and 38 (these sequences omit the two initial amino acids MA of SEQ ID NO:14), while SEQ ID NOs:60-64 represent variants having the modified amino acids E and Q at these positions. SEQ ID NOs:50-54 represent the consensus sequences for these positions.

[0042] Variants based on the consensus sequences of Sac7d and Aho7c are represented by SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

[0043] It should be noted that although the sequences shown above do not contain a methionine at their N-terminus, the invention can be practiced with each sequence having a methionine at its N-terminus.

[0044] Such variants can be fused to biomolecules and / or conjugated to small chemical entities for use by oral or rectal administration, allowing the molecule to cross the intestinal barrier, thereby increasing efficacy or bioavailability, or allowing the use of smaller doses. Exemplary molecules that can be conjugated include small chemical molecules (e.g., cytotoxic agents for cancer, such as auristatins or doxorubicin), peptides (insulin analogs, such as GLP-1), small protein scaffolds, such as derivatives of Sac7d (and analogs, such as Aho7c or Sso7d) from Sulfolobus acidocaldarius, 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 receptors, ankyrin repeat motifs, and the like. motive), SH3 domain of Fyn, Kunits domain of protease inhibitor, 10th type III domain of fibronectin, 3- or 4-helix bundle protein, armadillo repeat domain, leucine-rich repeat domain, PDZ domain, SUMO or SUMO-like domain, immunoglobulin-like domain, phosphotyrosine binding domain, pleckstrin homology domain, src homology 2 domain, or synthetic peptide ligands, antibody fragments (including Fab, VHH, ScFv), enzymes, antisense oligonucleotides, siRNA, shRNA, and other therapeutic nucleic acids. Larger polypeptides can also be fused to the variants disclosed herein.

[0045] All these sequences describe specific variants based on proteins of the Sac7d family, and it is possible to design variants of other proteins of the family using the alignment in Figure 1, as explained below.

[0046] The consensus sequence for the proteins Sac7d and Aho7c is SEQ ID NO: 65. SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 34 are variants based on this consensus sequence.

[0047] In the event of any discrepancy between the present specification and the sequence listing, the sequences referred to in the present specification shall prevail.

[0048] As indicated above, the present specification specifically discloses variants of Sac7d (SEQ ID NO: 1), Aho7c (SEQ ID NO: 14), Sso7d (SEQ ID NO: 2), but the disclosure is applicable to other proteins of the Sac7d family, especially Sto7 (SEQ ID NO: 15), which is highly similar to Sac7d and Aho7c. The disclosure is 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 listed under PF00018 in the PFAM database. The SH3 domain has a characteristic beta-barrel fold consisting of five or six beta strands arranged as two closely packed antiparallel beta sheets. The linker region may contain a short helix. It should be noted that, in light of the homology shared between the OB-fold and SH3 domains, and 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.

[0049] In certain embodiments, the polypeptide is within a variant of the Sac7d family of proteins that bind to the human leptin receptor.

[0050] In certain embodiments, the variant of the protein of the Sac7d family that binds to human leptin receptor is linked or fused to another protein or polypeptide.In particular, the other protein or polypeptide can be the same variant or another variant of the protein of the Sac7d family that binds to another target.In this embodiment, it is preferable that the other variant of the Sac7d family binds to a target related to a disease, so that the other variant is useful for treating or preventing the disease.In some embodiments, the other variant of the Sac7d family binds to human serum albumin.In some embodiments, the leptin receptor binding variant is bound to (at least) two other variants of the Sac7d family.

[0051] In certain embodiments, the variants are present within a polypeptide and are thus covalently linked to other proteins or polypeptides of biological interest via amine bonds.

[0052] In one embodiment, the polypeptide is conjugated to an organic molecule that exhibits some therapeutic activity.

[0053] The present invention also relates to genetic constructs comprising DNA sequences encoding the polypeptides described herein, vectors comprising such genetic constructs, and host cells comprising the genetic constructs within their genome.

[0054] The present invention also provides 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 a disclosed genetic construct; and b. Recovering the polypeptide The present invention relates to a method comprising the steps of:

[0055] The present invention also relates to the polypeptides disclosed herein for use as a medicament.

[0056] The present invention also relates to a method for treating a subject in need thereof, comprising administering to the subject a therapeutic amount of a leptin receptor binding variant disclosed herein, wherein the variant is fused to a peptide or protein having therapeutic activity against the disease as a pharmaceutical.

[0057] The present invention also relates to a composition containing a leptin receptor binding variant disclosed herein for a subject, wherein the variant is fused to a peptide or protein having therapeutic activity against a given disease and to another agent for simultaneous, separate or sequential (distributed) use in the treatment of said disease, said other agent being selected from among known agents for the treatment of the given disease.

[0058] It is particularly applicable when the disease is as disclosed below.

[0059] The present invention also relates to these variants in therapeutic, diagnostic or purification uses. The present invention also relates to compositions, particularly oral compositions, containing the polypeptides or variants. Indeed, and even in the case of the treatment of systemic diseases (not localized to the gastrointestinal system), oral administration is preferred whenever possible over other modes of administration (particularly injections), which may require the involvement of a medical staff member. In this embodiment, the composition also preferably contains pharma- ceutically acceptable excipients (dyes, sweeteners, texturizing agents, etc.) that can be used for such oral administration.

[0060] The sequence of Sac7d is Please note that the file is TIFF2024542807000001.tif11143.

[0061] The sequence of Sso7d is The file is TIFF2024542807000002.tif11143.

[0062] The sequence of Sso7d is The file is TIFF2024542807000003.tif11143.

[0063] The human leptin receptor 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 / or 46 of the Sac7 sequence. Note, however, that the threonine at position 40 may be maintained in the variant, and / or within the serine at position 46. However, the human leptin receptor binding variants disclosed herein contain at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12 variant amino acids compared to SEQ ID NO: 1 (i.e., amino acids that differ from the corresponding amino acids in SEQ ID NO: 1 or compared to the sequence of the Sac7d family protein from which the variant is derived). Generally, there are up to 20 mutated amino acids compared to SEQ ID NO: 1 (or the sequence of the Sac7d family protein from which it is derived), more preferably up to 18, more preferably up to 16, 15, 14 or 13 mutated amino acids.

[0064] Sac7d protein family The Sac7d family is defined as Sac7d protein, which corresponds to a family of 7kDa DNA binding proteins isolated from extremophilic bacteria.The Sac7d family is disclosed herein as a representative of the OB-fold domain, which is preferably used in the context of the present invention.Because SH3 domain shares homology with OB-fold domain, the disclosure related to Sac7d is also applicable to SH3 scaffold.

[0065] 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 that corresponds 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 that the proteins do not all have the same size). This Sac7d family includes, among others, the Sac7d or Sac7e protein from Sulfolobus acidocaldarius, the Sso7d protein from Sulfolobus solfataricus, the DBP 7 also called Sto7 protein from Sulfolobus tokodayi, 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 Sis7a or Sis7b from Sulfolobus islandicus, and the p7ss protein from Sulfolobus solfataricus. In view of the extensive sequence similarity of the proteins of the Sac7d family, it is directly possible and easy to identify the amino acid of another protein other than Sac7d that corresponds to a given amino acid of Sac7d. In particular, the disclosure of WO 2008 / 068637 can be used, which shows (in the figures) and explains (in the specification) that such proteins are superimposable. The contents of this document are incorporated herein by reference, in particular with respect to the description of methods for aligning and superimposing various OB-fold proteins. As indicated above, it is useful to use the numeration of amino acids from the Sac7d protein to designate particular amino acids, and equivalent amino acids can be obtained from FIG. 1.

[0066] 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 leads to the creation of a mutant of one protein of the Sac7d family, starting from a mutant of another protein of the family. The initial mutant can in particular be obtained by carrying out the process of WO 2008 / 068637. Thus, in particular using the disclosure of WO 2012 / 150314 and the disclosure of FIG. 1, 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. As an example, in the case of a mutant of Sac7d, the sequence alignment of FIG. 1 can be used to introduce the mutant amino acid of the Sac7d mutant into the scaffold of another protein. By way of example, variants of Sso7d derived from the Sac7d variants disclosed herein are set forth as SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70.

[0067] Using the consensus sequence for the Sac7d family, the variants are designated by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 28, or SEQ ID NO: 28.

[0068] The number of mutated residues introduced into the wild-type protein sequence to obtain a variant is preferably 4-25, or more specifically 4-22 or 4-20. 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 in the present specification all and any ranges (e.g. 5-20 or 7-25, etc.) are contemplated. Particularly preferred ranges are 4-20, 4-17 and 6-17, 4-14 and 6-14.

[0069] 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 therefore preferably 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), more specifically In the variant, the amino acids corresponding to K7, Y8, K9, K21, K22, W24, V26, G27, K28, M29, S31, T33, T40, R42, A44, and S46, or K7, Y8, K9, K21, K22, W24, V26, G27, K28, M29, S31, T33, R42, and A44 are introduced. As shown, the variant contains the mutations W24Y, T33W, and A44H. Other mutations (D16E, N37Q, and M57L) can also be made. All amino acid numbers refer to SEQ ID NO: 1.

[0070] Of particular interest are cases where the number of mutated amino acids is between 7 and 14 (inclusive). In particular, variants may also include amino acid insertions as shown in WO 2008 / 068637.

[0071] As shown, the proteins of the Sac7d family are 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 Sto7 proteins are represented by SEQ ID NO: 1 to SEQ ID NO: 15, respectively.

[0072] Variants of this Sac7d family of proteins may be called nanophytins. The present invention is therefore preferentially practiced with respect to variants of proteins represented by SEQ ID NO: 1 to SEQ ID NO: 15 or having a sequence that reads on SEQ ID NO: 16 (consensus sequence), in particular with respect to variants of Sac7d.

[0073] Relationship of Sac7d to 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 of their natural ligands, which can be oligosaccharides, oligonucleotides, proteins, metal ions or catalytic substrates. This binding interface is mainly composed of residues located in the beta 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, supra) describe OB-fold protein domains for binding with their natural ligands.

[0074] In particular, document WO 2008 / 068637 elaborates how to identify binding domains of OB-fold proteins. By superimposing several sequences and 3D structures of proteins with OB-fold domains using WU-Blast2 (Lopez et al., 2003, Nucleic Acids Res 31, 3795-3798), T-COFFEE (Notredame et al., 2000, J Mol Biol 302, 205-217) or DALI lite (Holm and Park, 2000, Bioinformatics 16, 566-567), it is possible to identify the location of the binding domain and in particular the amino acids that can be modified. With reference to the sequence of Sac7d (SEQ ID NO:1), these are 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.

[0075] WO 2008 / 068637 states that it is possible to carry out a superposition of the 3D structure of an OB-fold protein or domain (in this application ten domains including Sac7d were used) 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 aforementioned Sac7d amino acids. Thus, 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.

[0076] It is also noted that OB-fold domains resemble SH3 domains, and it is also possible to identify amino acid equivalents of Sac7d in SH3 domains.

[0077] Examples of OB-fold or SH3 domains Non-limiting examples of OB-fold proteins that may be used in accordance with the present 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 (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), 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 of PNPase), RNA-binding domain, E. coli), 1d7qA (initiation translation factor 1, elF1a, human), 1ah9 (initiation translation factor 1, IF1, E. coli), 1b9mA (Mo-dependent transcription factor ModE, E. 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 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).

[0078] 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 (possibly), STAC3, certain myosins, SHANK1, 2, 3, ARHGAP12, C8orf46, TANGO1, integrase, focal adhesion kinase (FAK, PTK2), proline-rich tyrosine kinase (Pyk2, CADTK, PTK2beta), or TRIP10 (cip4).

[0079] Description of leptin receptor (LepR) binding variants based on Sac7d and Aho7c Sac7d and Aho7c are proteins with great similarity. It can also be noted that Sto7 (SEQ ID NO: 15) also exhibits similarity to these proteins.

[0080] SEQ ID NO:654 corresponds to the consensus sequence after alignment of amino acids 2-66 of Sac7d (SEQ ID NO:1) with amino acids 3-60 of Aho7c (SEQ ID NO:14). The initiation amino acid has been omitted because it is not essential to the structure of the protein. TIFF2024542807000004.tif11143

[0081] In this consensus sequence, X is as shown in Table 1.

[0082] Table 1: Description of some of the amino acids represented as X in SEQ ID NOs: 59-73. TIFF2024542807000005.tif69148

[0083] The consensus sequence for variants that bind to the human leptin receptor is represented by the following:

[0084] (Table 2) Sequences of variants based on the consensus sequence of Sac7d-Aho7c. Amino acids represented by X are further described in Tables 1, 3, and 4. TIFF2024542807000006.tif75145

[0085] Table 3: Description of the amino acids designated as X in SEQ ID NOs: 30-64. Please note that this table is used for the aforementioned sequences with respect to the X they possess. For some sequences, the amino acids are specified at some positions in Table 3, so that row of this table does not apply. The listed sequences are indicated with the X in the second column of this table. At position 39, it can be noted that there are three possibilities: X is any amino acid, preferably X is T, Y, L, or A, or more preferably X is T or Y. TIFF2024542807000007.tif164145

[0086] In these sequences, some amino acids that are not involved in binding can also be modified on the variants without modifying the binding and biological properties of the protein. They are listed in Table 4.

[0087] (Table 4) Amino acids represented as X in SEQ ID NOs: 30-64. X at position 56 is only present in SEQ ID NOs: 30-49. TIFF2024542807000008.tif28128

[0088] Description of leptin receptor binding variants based on Sac7d (SEQ ID NO: 1) Specific variants based on Sac7d that bind to the human leptin receptor are represented by SEQ ID NO:35 to SEQ ID NO:49.

[0089] In these sequences, the initiation methionine (M) of the Sac7d protein is omitted. It has been shown by the applicant that the activity of the protein is not altered when this amino acid is deleted. Similarly, all or part of the last seven amino acids can be omitted from the variants and still retain activity.

[0090] Thus, the protein represented by amino acids 1-57 of any of SEQ ID NOs:35 to 49 is also a variant according to the invention. Mention may also be made of the proteins represented by amino acids 1-58, 1-59, 1-60, 1-61, 1-62, 1-63 of any of SEQ ID NOs:35 to 49, which are also variants according to the invention.

[0091] Thus, the polypeptides include any of SEQ ID NO: 35 to SEQ ID NO: 49, or any truncated proteins based on these sequences, and those disclosed above.

[0092] Such variants are represented by the following:

[0093] Table 5: Sequences of variants based on Sac7d. The amino acids represented by X are further described in Tables 3 and 4. TIFF2024542807000009.tif211145

[0094] As noted above, D16 of Sac7d (which is located at position 15 of SEQ ID NO: 35 to SEQ ID NO: 49 since M1 present in Sac7d has been omitted), N37 of Sac7d (located at position 36 herein), or M57 of Sac7d (located at position 56 herein) can be modified as disclosed in Table 4.

[0095] Regarding the consensus sequence of Sac7d / Aho7c, any combination of substitutions is foreseen, although not all are shown in the sequence listing (numbering refers to SEQ ID NO: 35 to SEQ ID NO: 49): TIFF2024542807000010.tif50128

[0096] As shown above, all sequences contain mutant amino acids Y23, W32, and H43 (corresponding to positions 24, 33, and 44, respectively, of SEQ ID NO: 1, which contain an N-terminal methionine) that differ from the amino acids naturally occurring in Sac7d.

[0097] The applicant has indeed found that these amino acids are present in various variants that bind to the human leptin receptor, and that such modifications lead to reduced binding (loss of affinity or loss of binding). Other amino acids may be variable under the conditions mentioned in Table 3.

[0098] Preferred is when the variant contains M7 (corresponding to position 8 of SEQ ID NO:1), T39 (corresponding to position 40 of SEQ ID NO:1), and / or S45 (corresponding to position 46 of SEQ ID NO:1).

[0099] Very interesting variants are represented by SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 48 and SEQ ID NO: 49.

[0100] Protein description based on Aho7c (SEQ ID NO: 14) Variants of Aho7c that bind to human leptin receptor are represented by SEQ ID NO: 50 to SEQ ID NO: 64. As shown above, Aho7c is very similar to Sac7d. It is also shown and recalled herein that mutations of Sac7d can be carried from Sac7d to other proteins (WO 2012 / 150314).

[0101] 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 activity of the protein is not altered when these amino acids are deleted. Similarly, all or part of the last four amino acids can be omitted from the variants while retaining activity.

[0102] Thus, the protein represented by amino acids 1-55 of any of SEQ ID NO:50 to SEQ ID NO:64 is also a variant according to the invention. Mention may also be made of the proteins represented by amino acids 1-56, 1-57, 1-58 of any of SEQ ID NO:50 to SEQ ID NO:64, which are also variants according to the invention.

[0103] Thus, the polypeptides include any of SEQ ID NO:50 to SEQ ID NO:64, or any truncated proteins based on these sequences, and those disclosed above.

[0104] Such variants are represented by the following:

[0105] Table 6: Sequences of Aho7c-based variants. The amino acids represented by X are further described in Tables 3 and 4. TIFF2024542807000011.tif211145

[0106] As noted above, D17 of Aho7c (which is located at position 15 of SEQ ID NO:50 to SEQ ID NO:64 due to the omission of M1A2 present in Aho7c), or N38 of Aho7c (located at position 36 herein) can be modified as disclosed in Table 4.

[0107] Regarding the consensus sequence of Sac7d / Aho7c, any combination of substitutions is foreseen, although not all are shown in the sequence listing (numbering refers to SEQ ID NO: 50 to SEQ ID NO: 64): TIFF2024542807000012.tif24128

[0108] As shown above, all sequences contain mutant amino acids Y23, W32, and H43 (corresponding to positions 25, 34, and 45, respectively, of SEQ ID NO: 14, which contain an N-terminal methionine and alanine) that differ from the amino acids naturally occurring in Sac7d.

[0109] The applicant has indeed found that these amino acids are present in various variants that bind to the human leptin receptor, and that such modifications lead to reduced binding (loss of affinity or loss of binding). Other amino acids may be variable under the conditions mentioned in Table 3.

[0110] Preferred is when the variant contains M7 (corresponding to position 9 of SEQ ID NO:14), T39 (corresponding to position 41 of SEQ ID NO:14), and / or S45 (corresponding to position 47 of SEQ ID NO:14).

[0111] Very interesting variants are represented by SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 63 and SEQ ID NO: 64.

[0112] Protein description based on Sso7d (SEQ ID NO: 2) Variants based on Sso7d that bind to the human leptin receptor are represented by SEQ ID NO: 66 to SEQ ID NO: 70. As shown above, Sso7d is very similar to Sac7d. It has also been shown that mutations in Sac7d can be carried into Sso7d (WO 2012 / 150314).

[0113] In these sequences, the initiating methionine, alanine, and threonine (MAT) of the Sso7d protein (SEQ ID NO: 2) are omitted. It has been shown by the applicant that the activity of the protein is not altered when these amino acids are deleted. Similarly, all or part of the last six amino acids can be omitted from the variants while retaining activity.

[0114] Thus, proteins represented by amino acids 1-53 of any of SEQ ID NOs:66 to 70 are also variants according to the invention. Also included are proteins represented by amino acids 1-54, 1-55, 1-56, 1-57, or 1-58 of any of SEQ ID NOs:66 to 70, which are also variants according to the invention.

[0115] Thus, the polypeptides include any of SEQ ID NO:66 to SEQ ID NO:70, or any truncated proteins based on these sequences, and those disclosed above.

[0116] Such variants are represented by the following:

[0117] Table 7: Sequences of variants based on Sso7d. The amino acids represented by X are further described in Table 8. TIFF2024542807000013.tif75145

[0118] Table 8: Description of the amino acids designated as X in SEQ ID NOs: 66-70. Please note that this table is used for the aforementioned sequences for the X they possess. For some sequences, the amino acids are specified at some positions in Table 8, so that row of this table does not apply. The listed sequences are indicated with the X in the second column of this table. At position 38, it can be noted that there are three possibilities: X is any amino acid, preferably X is T, Y, L, or A, or more preferably X is T or Y. TIFF2024542807000014.tif164145

[0119] As noted above, it is possible to modify D18 of Sso7d (which is located at position 13 of SEQ ID NO:66 to SEQ ID NO:68, due to the omission of M1A2T3 present in Sso7d).

[0120] As shown above, all sequences contain mutant amino acids Y21, W30, and H42 (corresponding to positions 24, 33, and 45, respectively, of SEQ ID NO:14, which contain an N-terminal methionine, alanine, and threonine) that differ from the amino acids naturally occurring in Sso7d.

[0121] The applicant has indeed found that these amino acids are present in various variants that bind to the human leptin receptor, and that such modifications lead to reduced binding (loss of affinity or loss of binding). Other amino acids may be variable under the conditions mentioned in Table 3.

[0122] Preferred is when the variant contains M5 (corresponding to position 8 of SEQ ID NO:2), T38 (corresponding to position 41 of SEQ ID NO:2), and / or S44 (corresponding to position 47 of SEQ ID NO:2).

[0123] Very interesting variants are represented by SEQ ID NO:69 and SEQ ID NO:70.

[0124] Description of variants based on the Sac7d family consensus sequence (SEQ ID NO: 16) Variants of specific members of the Sac7d family are disclosed above. In view of the sequence similarity of the Sac7d family proteins shown in Figure 1 and the fact that mutations may be carried from one protein to another, mutations may be depicted in the consensus sequence of the Sac7d family.

[0125] In the sequences of SEQ ID NO:17 to SEQ ID NO:29, the amino acids designated as X, or X at positions 8-10, 22, 23, 27, 30, 32, 42, 44, and 48 are as set forth in Table 9. Other amino acids designated as X (or X) are set forth in Table 10 or Table 11.

[0126] Table 9: Amino acid description for SEQ ID NO: 17 to SEQ ID NO: 29 TIFF2024542807000015.tif110143

[0127] The amino acids disclosed in Table 1 correspond to the variability observed in the Sac7d family of proteins, as also shown in the consensus sequence in FIG.

[0128] The amino acids disclosed in Table 10 correspond to the amino acids involved in binding to the human leptin receptor in the variants disclosed herein. Note that all such variants differ from the wild-type protein in the following amino acids: position 25: presence of Y; position 34: presence of W; position 46: presence of H.

[0129] These amino acids have been shown by the inventors to be important for binding to the human leptin receptor (altering the amino acids significantly reduces the ability to bind to albumin or the affinity for albumin). In contrast, the disclosure of Table 10 allows for modification of other amino acids from the optimized conjugates (SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 29). In particular, it has been noted that replacement of the amino acids at positions 8, 22, or 23 of these conjugates with other amino acids did not essentially change the binding or their properties. According to the inventors' analysis, the amino acid at position 9 also appears to tolerate any amino acid. The amino acids at positions 10 and 32 are preferably very small, and the amino acid at position 10 is preferably small. The amino acids at positions 30 and 42 are preferably hydrophobic, and the amino acid at position 48 is preferably polar. All of the above information can be readily converted to the specific members of the Sac7d family disclosed above and the corresponding amino acids in Tables 3 and 8.

[0130] It has also been shown that D17 (aspartic acid) of the consensus sequence, which is not involved in binding, can be replaced by E (glutamic acid), as shown in the sequences above (SEQ ID NO: 17 to SEQ ID NO: 21 carrying X at position 17; SEQ ID NO: 22 to SEQ ID NO: 25 carrying D17; SEQ ID NO: 26 to SEQ ID NO: 29 carrying E17).

[0131] Table 10: Description of the amino acids designated as X in SEQ ID NO: 17 to SEQ ID NO: 29. Note that this table is used for the sequences mentioned above for the X they possess. For some sequences, the amino acid is specified at some positions in Table 10, so that row of the table does not apply. At position 42, it can be noted that there are three possibilities: X is any amino acid, preferably X is T, Y, L, or A, or more preferably X is T or Y. TIFF2024542807000016.tif137145

[0132] As noted above, the methionine at position 1 of SEQ ID NOs: 17-29 may be omitted. Similarly, amino acids 61-68 of SEQ ID NOs: 17-29 may be omitted.

[0133] In one embodiment, the polypeptide comprises amino acids 2-60 of SEQ ID NO: 17 or one of the sequences in Table 11.

[0134] Table 11: List of variant sequences based on the consensus sequence of Sac7d family proteins. The nature of X is provided in Tables 9 and 10. X at position 17 can be E or D. TIFF2024542807000017.tif184145

[0135] Generation of identified variants The sequences of the identified variants may be cloned into any suitable vector by any molecular genetic method known in the art.

[0136] These recombinant DNA constructs comprising a nucleotide sequence encoding a polypeptide comprising the variants described above are used in conjunction with a vector, for example a plasmid, phagemid, phage, or viral vector.

[0137] These recombinant acid molecules can be produced by the techniques described in Sambrook et al., 1989 (Sambrook J, Fritsch 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.

[0138] The recombinant construct of the present invention comprises an expression vector, which can express RNA, thus resulting in the production of protein from the gene sequence.Therefore, the vector can further comprise 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 comprise a selection 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 coding sequences.

[0139] Molecular Creation Cells are transfected or transformed with vectors containing sequences encoding the polypeptides, including the variants disclosed above.

[0140] The cells are cultured under conditions such that the protein is expressed and secreted well. The cell culture conditions are those commonly 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) provides an overview of such methods and provides extensive references thereto.

[0141] Bacterial, phage (Shukra et al, Eur J Microbiol Immunol (Bp). 2014; 4(2): 91-98) or eukaryotic production systems can be used.

[0142] It is preferable to use eukaryotic cells in order to obtain appropriate post-translational modifications such as glycosylation.

[0143] 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.

[0144] Also provided by the present disclosure is a host cell that contains at least one of the DNA constructs that code for the polypeptide comprising variants disclosed herein.Host cell can be any cell that can utilize expression vector.As shown above, host cell 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.

[0145] The introduction of recombinant constructs into host cells is carried out by any method known in the art, such as calcium phosphate transfection, lipofection, DEAE, dextran-mediated transfection, electroporation, or phage infection. Vectors can be inserted into the genome of host cells or maintained as extragenomic vectors, such as bacterial artificial chromosomes or yeast artificial chromosomes. When introduced into cell genome, such introduction can be random or targeted, using methods known in the art, such as homologous recombination.

[0146] Bacterial host and expression Useful expression vectors for use in bacteria are constructed by inserting a recombinant DNA sequence in reading phase with a functional promoter, with appropriate translation initiation and termination signals. The vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide amplification within the host.

[0147] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0148] Eukaryotic hosts and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, the aforementioned cells can be used.

[0149] 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).

[0150] 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.

[0151] In particular, methods such as precipitation, 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.

[0152] Generally, any method known and used for purifying recombinant polypeptides can be adapted to purify the molecules disclosed herein.

[0153] 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, in certain embodiments it is preferred to purify the molecule using affinity.

[0154] 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.

[0155] 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.

[0156] Modification of variants The above variants, and variants that have the ability to bind to the human leptin receptor, can be modified by any method known in the art.

[0157] Preparation of polypeptides containing variants It is possible to prepare a DNA sequence that contains two coding sequences in frame, one for the variant disclosed herein and the other for the protein or peptide of interest.Therefore, the resulting expressed protein is a polypeptide that contains both proteins.Vector can be constructed in such a way that it contains a sequence that codes for a linker located between the two proteins in the expressed polypeptide.

[0158] Thus, the present invention also encompasses polypeptides comprising a protein variant of an OB-fold protein (preferably of the Sac7d family) that binds to the human leptin receptor, the protein variant being fused or linked (preferably via an amine bond as disclosed above) to another protein or polypeptide.

[0159] In certain embodiments, the other protein or polypeptide comprises another variant of the Sac7d family of proteins, particularly as disclosed herein.In other embodiments, the other protein or polypeptide comprises at least two other variants of the Sac7d family of proteins.In some embodiments, the variant of the Sac7d family of proteins that binds to human leptin receptor is bound to another variant of the Sac7d family at N-terminus and C-terminus.Variants can bind to targets as described below for antibodies.

[0160] Of particular interest are the following: - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to human leptin receptor fused to a variant of an OB-fold protein of the Sac7d family that binds to albumin and a variant of an OB-fold protein of the Sac7d family that binds to PD-L1 (particularly as disclosed in WO2021180823), such polypeptide being particularly useful for the treatment of cancer, in particular non-small cell lung cancer, metastatic Merkel cell carcinoma, urothelial carcinoma, solid tumors, hematological cancers, and in particular lymphomas (particularly classical Hodgkin's lymphoma), squamous cell carcinoma of the skin, squamous cell carcinoma of the lung, renal cell carcinoma, or melanoma. - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to the human leptin receptor fused to a variant of an OB-fold protein of the Sac7d family that binds to albumin and to a variant of an OB-fold protein of the Sac7d family that binds to tumor necrosis factor alpha (TNF-alpha) or RANK-L (in particular those disclosed in WO2020074402). Such polypeptides are particularly useful for the treatment of immune or inflammatory diseases (such as rheumatoid arthritis, ankylosing spondylitis, psoriasis and psoriatic arthritis, Crohn's disease, ulcerative colitis, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease), diseases of the central nervous system (particularly demyelinating diseases such as multiple sclerosis, optic neuritis, or non-demyelinating diseases such as meningitis, meningoencephalitis, encephalitis, neurosarcoidosis, or CNS vasculitis), respiratory diseases (such as idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), pulmonary Langerhans cell histiocytosis, emphysema, and pulmonary fibrosis), osteoporosis, or for the treatment of COVID-19. - a polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to the human leptin receptor fused to a variant of an OB-fold protein of the Sac7d family that binds to albumin and a variant of an OB-fold protein of the Sac7d family that binds to IL-17 (in particular as disclosed in WO2019096797), for the treatment of the same diseases as mentioned for TNF-alpha.

[0161] 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.

[0162] To obtain such a compound, a genetic construct can be used which comprises a DNA sequence selected from the group consisting of: A sequence encoding the heavy chain of an antibody fused at its 3' end to a sequence encoding a variant of an OB-fold protein (optionally with a sequence encoding a linker). b. A sequence encoding the heavy chain of an antibody fused at its 5' end to a sequence encoding a variant of an OB-fold protein (optionally with a sequence encoding a linker). c. A sequence encoding the light chain of an antibody fused at its 3' end to a sequence encoding a variant of an OB-fold protein (optionally with a sequence encoding a linker). d. A sequence encoding the light chain of an antibody fused at its 5' end to a sequence encoding a variant of an OB-fold protein (optionally with a sequence encoding a linker).

[0163] This fusion can be made at the N-terminus and / or C-terminus of the antibody chains (heavy and / or light chains). 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 dimers paired together) with an antibody region and an additional binding region consisting of a modified OB-fold domain.

[0164] In certain embodiments, the antibody portion of the proteins disclosed herein is an IgG molecule.

[0165] In another embodiment, the antibody portion of the proteins disclosed herein is an IgA molecule.

[0166] In another embodiment, the antibody portion of the proteins disclosed herein is an IgM molecule.

[0167] In another embodiment, the antibody portion of the proteins disclosed herein is an IgD molecule.

[0168] In another embodiment, the antibody portion of the proteins disclosed herein is an IgE molecule.

[0169] 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 with the antibody variants naturally generated in humans. A chimeric antibody contains sequences from a different species.

[0170] 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, generally around 450 amino acids) and two identical light chains. Thus, the antibody contains the same Fab variable region. Therefore, this antibody is a monospecific antibody in that both parts of the antibody (combination of light and heavy chains) bind to the same epitope of the antigen.

[0171] However, antibodies may exhibit different heavy and / or light chains. In particular, in some embodiments, the antibodies are bispecific antibodies. Thus, the term "antibody" encompasses both "classical antibodies" as disclosed above, which have identical heavy and light chains, and in addition, engineered antibodies with two or more specificities.

[0172] In certain embodiments, an antibody displays one heavy and light chain from one antibody and another heavy and light chain from another antibody.

[0173] 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, PSMA, IGF-1R, GITR, RAGE, CD28, FcRn, platelet-derived growth factor receptor. Cell surface proteins: CD19, CD20, CD22, CD30, CD38, CD40, CD248, CD47, CD73, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, MadCam, a4b7, CD7, CD4, CD223, CD138. 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, complement component 5. Circulating proteins: HSA, TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IL1, IL22, IL36, IFNg, IL17, RANKL, Bace1, alpha-synuclein, tau, amyloid, coagulation factor XI, coagulation factor XII, IgE, LPAM.

[0174] In another embodiment, the variant of the OB-fold domain (particularly the variant of the Sac7d family of proteins) 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.

[0175] In particular, DNA binding or alkylating agents, such as anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and analogs thereof, alkylating agents, such as calicheamicins, dactinomycins, mithromycins, 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.

[0176] Interesting and preferred molecules are also disclosed above.

[0177] Using variants The variants may be used in particular in therapeutic methods when fused to another polypeptide having therapeutic activity to treat a given disease.

[0178] Biological agents have been developed for the treatment of numerous diseases and can be fused to the LepR binding variants disclosed herein for the treatment of cancer, autoimmune diseases, inflammatory diseases, or infectious diseases. An example can be trastuzumab for the treatment of Her2 / neu breast cancer. Various other therapeutic antibodies are listed in WO2019096797 (pages 16-18, incorporated herein by reference) and can also be used with the LepR binding variants disclosed herein.

[0179] Thus, the present invention relates to a method for the treatment of a particular disease comprising administering to a subject in need thereof a therapeutic amount of a LepR-binding variant of the OB-fold disclosed herein (in particular a variant of a protein of the Sac7d family) fused to a therapeutic agent active against that disease.

[0180] 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. Therapeutic improvement can be disease regression, improvement in the quality of life of a subject, or improvement in the effectiveness of a combination treatment.

[0181] In the context of administering a polypeptide comprising a variant that binds to the leptin receptor and a therapeutic peptide or polypeptide to a patient with a disease targeted by the therapeutic peptide or polypeptide, an effective amount is an amount sufficient to achieve a cure of the patient, an improvement in the patient's condition, or an acceleration of the cure of the patient, for example, compared to the response obtained without administration. An effective amount can also be an amount that stops or slows the progression of a disease. It can be an amount that allows for regression of the disease or a diminution of a biological marker associated with a particular disease. It is obtained by allowing passage through the intestinal barrier of an amount of the variant fused to the therapeutic peptide or polypeptide such that the concentration / amount at the site of action of the therapeutic peptide or polypeptide is sufficient to produce a therapeutic effect.

[0182] 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.

[0183] The variants can be administered by any method known in the art.

[0184] However, as disclosed in WO 2016 / 062874, it is preferred that the variant is administered by oral route. In this embodiment, the variant and any other elements attached thereto are formulated for oral administration with a pharma- ceutically acceptable excipient. Such formulations are known in the art.

[0185] In another embodiment, the variant is administered by the rectal route. In this embodiment, the variant and any other elements attached thereto are formulated with a pharma- ceutically acceptable excipient for rectal administration. Such formulations are known in the art.

[0186] In some embodiments, the polypeptide or variant (or the nucleotide encoding the polypeptide or variant) can be associated (e.g., physically associated) with a delivery agent, such as a nanoparticle (e.g., lipid nanoparticle), a dendrimer, a polymer, a liposome, or a cationic delivery system. It is particularly interesting when other polypeptides or organic molecules bound to the LepR-binding variant are protected from gastric degradation through a suitable coating or by inclusion in a suitable vector. In this case, the LepR-binding variant is bound to this protective element. Indeed, WO2016062874 shows that variants of the Sac7d family are resistant to gastric degradation (enzymatic or pH-induced degradation). Thus, the variant does not need to be protected. However, in some embodiments, both the LepR-binding variant bound to the therapeutically active molecule are encapsulated / protected from gastric degradation. In this case, the protection allows the release of the LepR-binding variant bound to the therapeutically active molecule in the intestinal tract.

[0187] 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.

[0188] The present invention therefore also relates to a method for detecting the presence of or quantifying the human leptin receptor in a sample, comprising the steps of: a. exposing the sample to a disclosed variant that binds to the human leptin receptor under conditions that allow such binding. b. Recovering the variant and / or detecting or measuring the amount of human leptin receptor bound to the variant. The present invention relates to a method comprising the steps of:

[0189] The recovery of b) can be carried out by various washing or methods common in the art. Detection or quantification can be carried out by any method such as ELISA, chromatography, fluorescence, or other methods in the art.

[0190] Diseases Treated with Variants As noted above, the variants disclosed herein are essentially used as a means to transport biological agents through the intestinal barrier. Thus, the diseases treated using the variants disclosed herein are related to the biological activity of the molecule associated with the variant. [Brief description of the drawings]

[0191] [Figure 1] Alignment of Sac7d family proteins. [Diagram 2] Verification of binding to hLepR. [Diagram 3] Competition assay between human leptin (hLep) and nanophytin for binding to hLepR. [Figure 4] Cross-reactivity to the mouse leptin receptor (mLepR) by assessing the binding signal in an ELISA. [Diagram 5] Validation of binding on a cell model expressing hLepR and porcine LepR (pLepR). [Figure 6] Validation of pLepR expression in porcine jejunum. [Figure 7] Improved intestinal barrier crossing in an ex vivo assay for nanophytins from cluster A compared to a nanophytin (NF) identified in the screen not belonging to the cluster (F02) and a nanophytin that binds to a different target (unrelated NF). [Figure 8] Preservation of binding properties of nanophytin alone (F08) or nanophytin fused to different protein sequences (GFP, F08-GFP), or nanophytin fused to another nanophytin (F08-NF). [Figure 9]Improved intestinal barrier crossing in ex vivo assays for nanophytin from cluster A alone (F08) or fused to another nanophytin (F08-NF) and nanophytin binding to another target (unrelated NF). EXAMPLES

[0192] Example 1. Identification of nanophytin variants that bind to recombinant human leptin receptor Screening of 95 leptin receptor binding variants (such variants based on the Sac7d sequence are called nanophytins) from crude bacterial supernatants using immobilized hLepR as target by ELISA showed a high percentage of positive binders (Figure 2). Subsequent sequencing showed strong sequence diversity with few repetitive sequences. Fractionation of nanophytins into clusters was performed based on the homology of their binding mutations. One cluster of nanophytins (cluster "A"

[0193] Example 2. Nanophytins of Cluster A bind to LepR on an epitope that does not overlap with its native leptin ligand Leptin is a 16KDa hormone that interacts with the leptin receptor, whose main function is to regulate satiety and energy storage at the hypothalamic location. Leptin interacts more specifically at the second cytokine receptor homology domain, CRH2, of the complete leptin receptor. In addition, interactions with two or more LepRs through Ig-like domain interactions can stabilize the binding in these species.

[0194] Overlap between nanophytin- or leptin-bound epitopes was identified through competitive ELISA assays using at least one anti-hLepR nanophytin from each of the different sequence clusters (cluster A and others). Results are shown as binding ratios, where the ELISA signal of the various nanophytins with hLepR measured in the presence of hLeptin is divided by their binding to hLepR measured in the absence of hLep (Figure 3). The binding response of nanophytins from cluster A was not affected by the presence of hLep (ratio approx. 1), suggesting that they bind to hLepR on an epitope distinct from the hLep binding site. Nanophytins not included in cluster A showed reduced binding response in the presence of hLep (ratio <0.5), suggesting that hLep overlaps with their epitopes.

[0195] Example 3: Nanophytins of Cluster A cross-react with mouse leptin receptor and share similar binding patterns For the nanophytins found to be specific for hLepR in cluster A and other clusters, cross-reactivity between mouse and human LepR was evaluated by ELISA. Cross-reactivity was assessed by the ratio of the binding response measured at mLepR divided by the binding response measured at hLepR (Figure 4). Nanophytins from cluster A bind equally to receptors from both species (ratio approx. 1), whereas other nanophytins gave increased ELISA signals at mLepR (ratio >1.5), suggesting that nanophytins from cluster A bind to a different epitope than nanophytins from other clusters.

[0196] Example 4. Binding to human and porcine leptin receptors on cell models Cells were modified to express either the human leptin receptor (hLepR) or the porcine leptin receptor (pLepR) and used as models for cell surface LepR. The human (NM_002303.5) and porcine (NM_001024587.1) long isoforms of the leptin receptor gene were inserted into the plasmid pCNDA3.1+C-eGFP by Genscript (Piscataway, New Jersey, US), which contains a reporter GFP gene. The plasmids were transfected into HEK293 cells (Manassas, VA, USA). Positively transfected cells were identified by measuring GFP fluorescence. Receptor expression was confirmed by flow cytometry using an anti-LepR antibody (FAB867R, Bio techne). These cell models were then used to evaluate the ability of anti-hLepR nanophytins of cluster A to bind both human and porcine leptin receptors expressed on the cell surface, using clone F08 as a representative of cluster A. It was found that F08 could specifically bind on hLepR- and pLepR-expressing cells, but not on the mock cell line transfected with empty vector (Fig. 5), demonstrating that nanophytins of cluster A can bind to receptors in the cellular context and that they can cross-react with pLepR.

[0197] Example 5. Expression of porcine LepR in the porcine jejunum The functional evaluation of anti-LepR nanophytin in the porcine intestinal model implies that the receptor is effectively expressed in the tissue. The expression of pLepR in the porcine intestine was examined by Western blot (Figure 6). Cells were collected from the porcine jejunum intestine and compared with hLepR expressing cells (HepG2) and h / pLepR negative cells (HEK293). The supernatant of the cell lysate was run on an SDS page gel and h / pLepR was identified by Western blot using anti-LepR antibody followed by visualization by chemiluminescence. The results shown in Figure 6 show that the bands in the lysates from HepG2 cells and the porcine jejunum are at the expected molecular weight of LepR (132.5 kDa). As expected, no bands could be observed in the conditions using HEK293 lysates, demonstrating the specificity of anti-LepR staining. Taken together, these data confirmed the expression of LepR in the porcine jejunum.

[0198] Example 6. Active transport of anti-LepR nanophytins through the intestinal barrier is observed for anti-LepR nanophytins of cluster A but not for other nanophytins, including other anti-LepR nanophytins and unrelated nanophytins The active transfer of anti-LepR nanophytin through the intestinal barrier was assessed ex vivo using porcine intestinal tissue introduced into an Ussing chamber. Fresh porcine intestines were prepared and cultured to a volume of 1.26 cm2 with an exposed surface area of ​​1.26 cm2. 2The donor (mucosa) and acceptor (serosa) compartments were created by inserting the donor (mucosa) and acceptor (serosa) compartments into Ussing chamber sliders at 38°C in KBR buffer. Tissue viability and integrity were followed by electrical resistance (TEER). Fluorescently labeled nanophytin was introduced into the mucosal compartment and after 120 min, samples from each compartment were analyzed to quantify the presence of nanophytin. The percentage of fluorescently labeled nanophytin found in the acceptor compartment was calculated relative to the amount of material applied to the donor compartment and used as a measure of transport efficiency across the intestinal barrier (Figure 7). In this experiment, an irrelevant nanophytin targeting GFP was used as a negative control. The passage rate in the serosal compartment was found to be similar (≦0.05%) for the irrelevant nanophytin and the anti-LepR nanophytin F02, suggesting that such levels of passage could be due to passive diffusion. Anti-LepR nanophytins of cluster A (F08 and D07) were found to benefit from a higher passage (>0.05%) through the intestinal barrier than unrelated nanophytins and other anti-LepR nanophytins, suggesting that an active mechanism is employed for their transport, highlighted by their significantly higher percentage measured in the serosal compartment, demonstrating that targeting LepR alone is not sufficient to provide active delivery through the intestinal barrier and that nanophytins of cluster A share a common epitope that provides a selective advantage for this property.

[0199] Example 7: Tolerance of fusions at both the N- and C-terminus The binding sites of nanophytins and their N- and C-terminal parts are located on opposite faces. As a result, nanophytins can be conjugated at their ends with cargo molecules without altering the target engagement, which may include genetic fusion or conjugation to peptides or proteins. This was demonstrated for the anti-LepR nanophytin F08. F08 nanophytin retains its functionality regardless of the protein cargo fused to its C-terminus. This was demonstrated using proteins of different sequence and length. It is noted here that similar properties have been observed in fusion to a full-length antibody (WO2019096797) or to another nanophytin. Furthermore, nanophytins can be expressed either recombinantly in a wide range of expression hosts (prokaryotic or eukaryotic), examples of which include E. coli and CHO, or by full chemical synthesis. In all cases investigated with F08 nanophytin, fully functional F08 nanophytin is recovered, as demonstrated in ELISA experiments (Figure 8).

[0200] Example 8. Improved barrier crossing of protein cargo fused to nanophytin of cluster A Nanophytins can be fused to other proteins while preserving their binding properties. As a result, nanophytins of cluster A can be conjugated to cargo proteins and used as vehicles or Trojan horses to effect active transport of cargo through the intestinal barrier. This was demonstrated using the ex vivo experimental setup described in Example 6 with F08 fused to a second nanophytin as cargo (construct F08-NF). The percentage of fluorescently labeled nanophytin fusion found in the acceptor compartment was calculated relative to the amount of material applied to the donor compartment and used as a measure of transport efficiency across the intestinal barrier (Figure 9). Active transport, highlighted by a percentage in the serosal compartment above 0.05%, was observed both for nanophytin F08 alone and its counterpart fused to a cargo protein. Furthermore, the crossing rates in the serosal compartment were very similar for both nanophytin F08 alone and its conjugated counterpart, demonstrating that anti-LepR nanophytin F08 fully retains the active translocation properties through the intestinal barrier when conjugated to cargo proteins, demonstrating that anti-LepR nanophytins of cluster A can be used for active transport of cargo molecules through the intestinal barrier.

Claims

1. A polypeptide comprising a variant of a Sac7d family member that binds to a human leptin receptor, The variant comprises 4 to 20 mutated residues in the binding interface of the member of the Sac7d family to its natural ligand, wherein the mutated residues in the binding interface of the 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 as shown in SEQ ID NO: 1, and wherein the member of the Sac7d family is Sulfolobus acidocaldarius. 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 Aho7a from L. acidianus hospitalis, Aho7b from L. acidianus hospitalis, Aho7c from L. acidianus hospitalis, and Sto7 from Sulfurisphaera tokodaii, wherein the variant comprises W24Y, T33W, and A44H mutations according to the numbering corresponding to the numbering of the Sac7d residues shown in SEQ ID NO:

1. The polypeptide.

2. 2. The polypeptide of claim 1, further comprising Y8M, K9G, S31G, and R42G mutations, numbered according to the numbering of the Sac7d residues shown in SEQ ID NO:

1.

3. 2. The polypeptide of claim 1, further comprising at least one mutation selected from D16E, N37Q, and M57L, with numbering corresponding to the numbering of the Sac7d residues shown in SEQ ID NO:

1.

4. SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID 2. The polypeptide of claim 1, comprising SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO:

29.

5. 2. The polypeptide of claim 1, comprising SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, or amino acids 1 to 54 of these sequences.

6. The polypeptide of claim 1, which is within a variant of a member of the Sac7d family that binds to the human leptin receptor.

7. The polypeptide of claim 1, wherein the variant of a member of the Sac7d family that binds to the human leptin receptor is conjugated to another polypeptide, particularly to another variant of a protein of the Sac7d family.

8. The polypeptide of claim 1, wherein the variant of a Sac7d family member that binds to the human leptin receptor is conjugated to an organic molecule.

9. A nucleic acid molecule encoding the polypeptide of claim 1.

10. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 8 or the nucleic acid of claim 9 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the polypeptide is conjugated to a substance having therapeutic activity in a specific disease.

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