Variants of Sac7d and its use in cancer treatment

Abstract

The present invention relates to variants of OB-fold proteins, particularly of the Sac7d family, that bind to PD-L1 or HSP110 and can be used alone or in combination for the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to the field of cancer treatment, and more particularly to the development of a novel binder for PDL-1 that can be used for such purposes. [Background technology]

[0002] Introduction Cancer immunotherapy has become the optimal treatment for some cancers. Its essence lies in using the patient's immune system to fight cancer cells. In particular, cell-based immunotherapy utilizes immune effector cells such as lymphocytes (especially T lymphocytes) and non-specialized cells such as macrophages, dendritic cells, or natural killer cells (NK cells) to target tumor antigens expressed on the surface of tumor cells.

[0003] However, it has been observed that tumor cells can evade the immune response by stimulating immune checkpoint targets. Immune checkpoints are regulators of the immune system that, when stimulated, can reduce or inhibit the immune response.

[0004] Immune checkpoints include CTLA4, PD-1, and PD-L1. PD-1 is a transmembrane programmed cell death 1 protein (also known as PDCD1 and CD279) that interacts with PD-L1 (PD-1 ligand 1, or CD274). Upregulation of PD-L1 on the cell surface can inhibit T cells. By blocking the interaction between PD-1 and PD-L1, T cells may be able to counter tumor cells. This protein is present in the UniProt database under accession number Q9NZQ7 (human protein) or Q9EP73 (mouse protein). RefSeq accession numbers are NP_001254635, NP_001300958, and NP_054862 (human protein), as well as NP_068693 (mouse protein).

[0005] HSP110, also known as Hsp110, HSPH1, or Hsp105 (Hsp105α or Hsp105β), is a mammalian member of the HSP105 / 110 family, a subgroup of the HSP70 family. Hsp105α is expressed constitutively and in response to various forms of stress, while Hsp105β is an alternative splicing of Hsp105α, specifically expressed during mild heat shock, and was cloned by Yasuda et al. (J Biol Chem 270, 29718-29723 (Non-Patent Literature 1)) and Ishihara et al. (Biochim Biophys Acta 1444, 138-142 (Non-Patent Literature 2)). HSP110 has been shown to be a major determinant of both prognosis and treatment response in colorectal cancer (CRC), particularly by Dorard et al. (Nat Med. 2011 Sep 25;17(10):1283-9 (Non-Patent Literature 3)). In particular, binding between Hsp110 and STAT3 may enable control of cancer, especially CRC cancer (Gozzi et al, Cell Death Differ. 2020 Jan;27(1):117-129 (Non-Patent Literature 4)) and improve the response to chemotherapy (Dubrez et al, Oncogene. 2020 Jan;39(3):516-529 (Non-Patent Literature 5)). This protein is located in the UniProt database under accession number Q92598 (human protein) or Q61699 (mouse protein). Therefore, identifying inhibitors of HSP110-STAT3 binding is of interest.

[0006] The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. EGFR overexpression is associated with several cancers, including lung adenocarcinoma (40% of cases), anal cancer, glioblastoma (50%), and epithelial tumors of the head and neck (80–100%). Mutations, amplifications, or misregistrations of EGFR or its family members are involved in approximately 30% of all epithelial cancers. Anticancer drugs targeting EGFR have been developed, including gefitinib, erlotinib, afatinib, brigatinib, and icotinib for lung cancer, and cetuximab for colon cancer. Other drugs include panitumumab and osimertinib. These drugs include monoclonal antibodies as well as tyrosine kinase inhibitors. This protein is located in the UniProt database under accession number P00533 (human protein) or Q01279 (mouse protein).

[0007] EP1930342 (Patent Document 1) discloses that it is possible to screen for variants of the Sac7d family of proteins.

[0008] WO2019 / 096797 (Patent Document 2) discloses the creation of multispecific molecules using the Sac7d variant, and several variants that bind to IL-17.

[0009] Goux et al. (Bioconjug Chem. 2017 Sep 20;28(9):2361-2371 (Non-Patent Literature 6)) have disclosed that anti-EGFR nanophytin (a variant of Sac7d) can be used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumors.

[0010] Gocha et al. (Sci Rep 7, 12021; 2017 (Non-Patent Literature 7)) disclose the identification and characterization of a novel Sso7d scaffold-based binder for Notch1.

[0011] Marchetti et al. (J Thorac Dis. 2017 Dec; 9(12): 4863-4866 (Non-Patent Literature 8)) discuss the roles of PD-L1 and PD1 in cancer treatment.

[0012] Overall, none of these documents, individually or in combination, disclose or suggest that it is possible to obtain variants of Sac7d family proteins that can inhibit PD-L1 / PD1 binding or bind to both human and mouse PD-L1. In fact, all of these documents rely on the ability to bind to the protein, while biological activity (inhibition of effect) also requires appropriate affinity and binding at a strategic location. As will be explained below, human PDL1 and mouse PDL1 have structural homology but a low degree of identity, and some drugs may not be able to target both proteins. Furthermore, the surface area of ​​the binding site between PD-L1 / PD1 is small, and inhibiting binding may require steric hindrance that cannot be achieved with small molecules (Sac7d family proteins are 60-70 amino acids long). [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] EP1930342 [Patent Document 2] WO2019 / 096797 [Non-patent literature]

[0014] [Non-Patent Document 1] Yasuda et al, J Biol Chem 270, 29718-29723 [Non-Patent Document 2] Ishihara et al, Biochim Biophys Acta 1444, 138-142

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0015] The present invention relates to a polypeptide comprising a variant of a Sac7d protein or a Sac7d family protein that binds to PD-L1. In some embodiments, the variant binds to human PD-L1. In other embodiments, the variant binds to PD-L1 of both human and mouse.

[0016]

[0017] Considering that the interaction region between PDL1 and PD1 is relatively narrow, it may be difficult to create a neutralizing ligand for PDL1.

[0018] ​This is especially true for small antibody substitutes such as nanophytin (a variant of the Sac7d family of proteins). Given its extremely compact structure, nanophytin needs to bind to PDL1 very close to its interaction region with PD1 in order to exert its direct neutralizing ability. In fact, the small size of such variants prevents neutralization by steric hindrance alone, which may be possible with larger proteins such as antibodies.

[0019] The extracellular domain of PDL1 consists of 220 amino acids (aa) and has two immunoglobulin-like domains: Ig-like V type (aa19-aa127) and Ig-like C2 type (aa133-aa225). Only the Ig-like V type domain is involved in the interaction with PD1. Therapeutic antibodies such as atezolizumab, durvalumab, and avelumab all target the Ig-like V type domain, and their epitopes overlap with the interaction region between PDL1 and PD1 (Figure 14).

[0020] Interestingly, the extracellular domains of human PDL1 and mouse PDL1 share high structural homology but a relatively low percentage of identity (72%), yet they have been found to have different drug potential profiles (Magiera-Mularz et al., iScience. 2021 Jan;24(1):101960). For example, both atezolizumab and avelumab were demonstrated to be cross-reactive to human and mouse PDL1, while durvalumab was found to be specific only to human PDL1.

[0021] Much development has been done to improve the predictability of mouse tumor models, particularly regarding the evaluation of immune checkpoint inhibitor combinations (Sanmamed et al, Annals of Oncology, 27 (7), 2016, 1190-1198; Chulpanova et al. Int J Mol Sci. 2020;21(11):4118). All these efforts reflect the continued importance of mouse models in the development scheme of oncology drugs, given their low cost, short breeding cycle, high tumor growth rate, and ease of genetic modification. In this context, the development of novel immune checkpoint inhibitors can be made more efficient by using cross-reactive ligands that can target both mouse and human targets. Therefore, mouse models can be used not only for evaluating efficacy but also, for example, for evaluating target engagement, in vivo distribution, and toxicity.

[0022] Accordingly, the present invention comprises polypeptides comprising variants of members of the Sac7d family that bind to human PD-L1 and inhibit liaison between PD-L1 and PD1. In certain embodiments, the polypeptide consists of such variants. In certain embodiments, the variant comprises 4 to 20 mutated residues at the interface of binding of the Sac7d family member to its native ligand. In certain embodiments, the variant comprises 8 to 14 mutated residues at the interface of binding of the Sac7d family member to its native ligand.

[0023] Polypeptides containing variants of members of the Sac7d family that bind to human PD-L1 contain 4 to 20 (particularly 8 to 14) mutated residues at the interface of binding of the Sac7d family member to its native ligand, and the variant may contain the Y8M, V26L, S31L, R42L, and A44F mutations, or the Y8I, V26I, S31L, R42M, and A44L mutations (the numbering corresponds to the position in Sac7d sequence SEQ ID NO: 1). Variants containing the Y8M, V26L, S31L, R42L, and A44F mutations correspond to SEQ ID NO: 51 to 71. Variants containing the Y8I, V26I, S31L, R42M, and A44L mutations correspond to SEQ ID NO: 15 to 23 and SEQ ID NO: 30 to 50.

[0024] In certain embodiments, the polypeptide also binds to mouse PD-L1. In this embodiment, the polypeptide may contain Y8M, W24T, V26L, M29A, S31L, T33R, R42L, and A44F mutations, or Y8I, W24R, V26I, M29Y, S31L, T33K, R42M, and A44L mutations, with the numbering corresponding to the position in Sac7d sequence SEQ ID NO: 1. Variants containing the Y8M, W24T, V26L, M29A, S31L, T33R, R42L, and A44F mutations correspond to SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 71. Variants containing the Y8I, W24R, V26I, M29Y, S31L, T33K, R42M, and A44L mutations correspond to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 18, and SEQ ID NO: 17.

[0025] In certain embodiments, the variant further comprises at least one mutation selected from D16E, N37Q, and M57L (the numbering corresponds to the position in Sac7d sequence SEQ ID NO: 1).

[0026] Such variants can be used in cancer treatment because they can reduce tumor weight, as shown in the examples. Such variants include SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. In particular, the variant is a variant of the Sac7d protein and includes SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In another embodiment, the variant is a variant of the Sso7d protein and includes SEQ ID NO: 22, or SEQ ID NO: 23.

[0027] In particular, in some embodiments, the variants include SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60 (all binding to human PD-L1), SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 62 (all binding to human and mouse PD-L1) based on the Sac7d protein.

[0028] In some embodiments, the variants include SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69 (all binding to human PD-L1), SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 71 (all binding to both human and mouse PD-L1).

[0029] In some embodiments, the variant is based on the Sso7d protein and includes SEQ ID NO: 22 or SEQ ID NO: 23.

[0030] All of these sequences describe specific variants based on Sac7d family proteins. As described below, the alignment in Figure 1 can be used to design variants of other proteins in the family.

[0031] Sequences associated with the consensus variants of proteins Sac7d and Aho7c that bind to PD-L1 may also include SEQ ID NO: 30, SEQ ID NO: 51 (all bind to human PD-L1), SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 52, or SEQ ID NO: 53 (all bind to human and mouse PD-L1).

[0032] In the event of any discrepancy between the specification and the sequence listing, the sequence listed in the specification shall prevail.

[0033] The present invention also relates to polypeptides comprising variants of the Sac7d protein or Sac7d family proteins that bind to HSP110. Such variants can be used in cancer treatment because they are capable of reducing tumor weight, as shown in the examples. Such variants include SEQ ID NO: 24 or SEQ ID NO: 25. In particular, the variant is a variant of the Sac7d protein and comprises SEQ ID NO: 26 or SEQ ID NO: 27. In another embodiment, the variant is a variant of the Sso7d protein and comprises SEQ ID NO: 28 or SEQ ID NO: 29.

[0034] In particular, in some embodiments, the variant is based on the Sac7d protein and includes 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, or SEQ ID NO: 83. In some embodiments, the variant is based on the Aho7c protein and includes SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, or SEQ ID NO: 92.

[0035] The consensus sequences for proteins Sac7d and Aho7c, and the sequences associated with variants that bind to HSP110, may also include SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0036] Such variants inhibit the binding of HSP110 to Stat3.

[0037] While some of the sequences described above contain methionine at the N-terminus, please note that the present invention can also be implemented even if the sequences do not contain such methionine at the N-terminus.

[0038] The present invention also relates to nucleic acids encoding variants, polypeptides containing variants and active substances, methods for producing variants alone or in combination with active substances, and methods for using them.

[0039] In particular, the present invention relates to the following: - Polypeptides disclosed herein, comprising variants of members of the Sac7d family that bind to human and / or mouse PD-L1. - A polypeptide disclosed herein, wherein a variant of a member of the Sac7d family that binds to human and / or mouse PD-L1 is conjugated to an organic molecule. - A polypeptide disclosed herein, wherein a variant of a member of the Sac7d family that binds to human and / or mouse PD-L1 is conjugated into another polypeptide. - Polypeptides disclosed herein, wherein other polypeptides are alternative variants of Sac7d family proteins. - A nucleic acid molecule encoding a polypeptide disclosed herein. - An expression vector comprising a nucleic acid molecule disclosed herein. - A host cell containing a nucleic acid molecule disclosed herein, or an expression vector disclosed herein. - A pharmaceutical composition comprising a polypeptide, a nucleic acid, an expression vector, or a host cell disclosed herein, and a pharmaceutically acceptable carrier. - A method for producing the polypeptides disclosed herein, (a) The step of culturing a cell culture in which cells have been transformed with an expression vector disclosed herein, and (b) Steps to produce polypeptides Methods that include... - Polypeptides disclosed herein as pharmaceuticals. - Polypeptides disclosed herein for use in the treatment or prevention of a disease, wherein a variant is associated with a substance active for the treatment or prevention of a disease. [Invention 1001] A polypeptide comprising a variant of a member of the Sac7d family that binds to human PD-L1 and inhibits liaison between PD-L1 and PD1, wherein the variant comprises 4 to 20 mutated residues at the interface of the binding of the Sac7d family member to its native ligand. [Invention 1002] The polypeptide of the present invention 1001, wherein the variant comprises Y8M, V26L, S31L, R42L, and A44F mutations, or Y8I, V26I, S31L, R42M, and A44L mutations, the numbering corresponding to the position in Sac7d sequence SEQ ID NO: 1. [Invention 1003] The polypeptide of the present invention 1001 or 1002, wherein the variant comprises Y8M, W24T, V26L, M29A, S31L, T33R, R42L and A44F mutations, or Y8I, W24R, V26I, M29Y, S31L, T33K, R42M and A44L mutations, the numbering corresponds to a position in Sac7d sequence SEQ ID NO: 1, and the variant also binds to mouse PD-L1. [Invention 1004] A polypeptide according to any of invention 1001 to 1003, wherein the variant further comprises at least one mutation selected from D16E, N37Q, and M57L, and the numbering corresponds to a position in Sac7d sequence SEQ ID NO: 1. [Invention 1005] A polypeptide according to any of the invention 1001 to 1004, wherein the mutated residue at the interface of binding of the member of the Sac7d family to its native ligand is selected from the group consisting of Sac7d 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. [Invention 1006] 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 Polypeptides of any of the present invention 1001 to 1005, selected from the group consisting of Aho7a derived from *Sulfurisphaera hospitalis*, Aho7b derived from *Sulfurisphaera hospitalis*, Aho7c derived from *Sulfurisphaera hospitalis*, and Sto7 derived from *Sulfurisphaera tokodaii*. [Invention 1007] A polypeptide according to any of the Invention 1001 to 1006, comprising SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and a sequence selected from amino acids 1 to 57 of these sequences. [Invention 1008] The polypeptide of Invention 1007, comprising SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and a sequence selected from amino acids 1 to 57 of these sequences. [Invention 1009] The polypeptide of the present invention 1007, comprising SEQ ID NO: 62, SEQ ID NO: 59, SEQ ID NO: 41, SEQ ID NO: 38, and a sequence selected from amino acids 1 to 57 of these sequences. [Invention 1010] A polypeptide according to any of the Invention 1001 to 1006, comprising SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23, or comprising amino acids 2 to 54 of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23. [Invention 1011] A polypeptide according to any of the Invention 1001 to 1006, comprising SEQ ID NO: 21 or amino acids 2 to 54 of SEQ ID NO: 21. [Invention 1012] A polypeptide according to any of the Invention 1001 to 1006, comprising SEQ ID NO: 71, SEQ ID NO: 68, SEQ ID NO: 50, SEQ ID NO: 47, and a sequence selected from amino acids 1 to 54 of these sequences. [Invention 1013] A polypeptide according to any of the inventions 1001 to 1012, comprising variants of members of the Sac7d family that bind to human and mouse PD-L1. [Invention 1014] A polypeptide according to any one of the invention 1001 to 1013, wherein the variant of the member of the Sac7d family that binds to human PD-L1 is conjugated to an organic molecule. [Invention 1015] A polypeptide according to any of the invention 1001 to 1013, wherein the variant of the member of the Sac7d family that binds to human PD-L1 is conjugated to another polypeptide, in particular another variant of a protein of the Sac7d family. [Invention 1016] The polypeptide of the present invention 1015, wherein the other protein is a variant of the Sac7d family that binds to HSP110 or EGFR. [Invention 1017] The polypeptide of Invention 1016, wherein the other variant comprises a polypeptide selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 80, SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, 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, and amino acids 1 to 57 of these sequences. [Invention 1018] The polypeptide of the present invention 1017, wherein the other variant comprises sequence SEQ ID NO: 83 or SEQ ID NO: 80, or amino acids 1 to 57 of these sequences. [Invention 1019] A nucleic acid molecule encoding any of the polypeptides 1001-1013 and 1015-1018 of the present invention. [Invention 1020] A pharmaceutical composition comprising a polypeptide according to any of Invention 1001 to 1018 or a nucleic acid according to Invention 1019, and a pharmaceutically acceptable carrier. [Invention 1021] A method for producing any polypeptide of the present invention 1001-1013 and 1015-1018, a. A step of culturing a cell culture, wherein the cells are transformed with the nucleic acid of the present invention 1019, and b. The step of recovering the polypeptide. A method comprising the steps of: [Invention 1022] A polypeptide according to any of invention 1001 to 1018, as a pharmaceutical. [Invention 1023] Polypeptides of any of 1001 to 1018 of the present invention for use in the treatment of cancer, particularly non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high-frequency microsatellite instability cancer, bladder cancer, breast cancer, small cell lung cancer, colorectal cancer, pancreatic cancer, or prostate cancer. [Invention 1024] Polypeptides for use in combination with chemotherapy or CAR-T cell therapy. [Invention 1025] A composition comprising any polypeptide from Invention 1001 to 1018 and a chemotherapeutic agent or CAR-T cells for use simultaneously, separately, or sequentially (gradually expanding) in the treatment of cancer, particularly non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high microsatellite instability cancer, bladder cancer, breast cancer, small cell lung cancer, colorectal cancer, pancreatic cancer, or prostate cancer. [Modes for carrying out the invention]

[0040] Detailed description of the invention This specification discloses variants of Sac7d (SEQ ID NO: 1), Aho7c (SEQ ID NO: 14), and Sso7d (SEQ ID NO: 2), but it should be noted that this instruction is applicable to other proteins in the Sac7d family, particularly Sto7 (SEQ ID NO: 94), which is very similar to Sac7d and Aho7c. The amino acids to be mutated are identified using sequence alignment with the Sac7d sequence, as disclosed in Figure 1 in particular. This instruction 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, which was originally described as a conserved sequence in the viral adapter protein v-Crk and is described under PF00018 in the PFAM database. The SH3 domain has a characteristic β-barrel fold consisting of 5 or 6 β-chains arranged as two densely packed antiparallel β-sheets. The linker region may contain a short helix. Note that the OB-fold domain and the SH3 domain share homology, and given the knowledge regarding the sequence and structure of these domains, it is possible to determine which amino acids correspond to the amino acids disclosed below for Sac7d in either the OB-fold domain or the SH3 domain.

[0041] In certain embodiments, the polypeptide consists of variants of Sac7d family proteins that bind to PD-L1. In certain embodiments, the polypeptide consists of variants of Sac7d family proteins that bind to HSP110.

[0042] In certain embodiments, a variant of the Sac7d family protein that binds to PD-L1 is ligated or fused to another protein or polypeptide. In particular, the other protein or polypeptide comprises another variant of the Sac7d family protein. In this embodiment, it is preferable that the other variant of the Sac7d family binds to HSP110. Such other variants may include any of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 72-92.

[0043] SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, or SEQ ID NO: 92 are particularly preferred. SEQ ID NO: 80 and SEQ ID NO: 83 are particularly preferred. In another embodiment, other variants of the Sac7d family bind to EGFR.

[0044] In certain embodiments, the variant resides within a polypeptide and is therefore covalently bonded via amine links to other proteins that present a biological object of interest.

[0045] In one embodiment, the polypeptide is conjugated to an organic molecule exhibiting some functionality, particularly a tyrosine kinase inhibitor used as an EGFR inhibitor.

[0046] The present invention also relates to a gene construct comprising a DNA sequence encoding a polypeptide described herein, a vector comprising such a gene construct, and a host cell containing such gene construct in its genome.

[0047] The present invention also relates to a method for producing polypeptides disclosed herein, a. The step of culturing cell cultures in which cells have been transformed by the disclosed gene construct, and b. The stage of collecting polypeptides. The present invention relates to a method that includes a stage consisting of the following steps.

[0048] The present invention also relates to polypeptides disclosed herein as pharmaceuticals.

[0049] The present invention also relates to polypeptides disclosed herein for use in the treatment of cancer, either alone or in combination with chemotherapy or CAR-T cell therapy.

[0050] The present invention also relates to a method for treating a subject in need, particularly when the subject has cancer, comprising the step of administering a therapeutic dose of the polypeptide disclosed herein to the subject.

[0051] The present invention also relates to a composition containing a polypeptide and a chemotherapeutic agent or CAR-T cells according to any one of claims 1 to 12 for use simultaneously, separately, or sequentially (gradually) in the treatment of cancer.

[0052] This is particularly applicable when cancer is disclosed as follows:

[0053] The present invention also relates to these variants for use in therapeutic, diagnostic, or purifying applications. The present invention also relates to compositions containing polypeptides or variants, particularly oral or topical (skin) compositions.

[0054] The sequence of Sac7d is Please note that TIFF0007879809000001.tif11143 exists.

[0055] The PD-L1-binding variants disclosed herein contain mutations at positions 7, 8, 9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, and 46 of the Sac7 sequence. However, it should be noted that lysine at position 21 and threonine at position 40, or serine at position 46, may be maintained in the variants. It should also be noted that methionine at position 1, as well as some amino acids 59-66, may be deleted. Therefore, the variants may contain amino acids 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, or 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65 of Sac7d. This corresponds to amino acids 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, or 1-63 in any of the following sequences: SEQ ID NO: 33-41, SEQ ID NO: 54-62, or SEQ ID NO: 75-83.

[0056] In a preferred embodiment, the polypeptide sequence is as follows: Includes TIFF0007879809000002.tif11144.

[0057] In this sequence, the amino acids designated as X at positions 8, 10, 22-23, 25, 30, 34, 42, and 48 can be any amino acid. Other amino acids designated as X are shown in Table 1 (where "-" means no amino acid).

[0058] (Table 1) Explanation of amino acids TIFF0007879809000003.tif90143

[0059] The amino acids in Table 1 are those that exhibit variability among different sequences of Sac7d family proteins. As shown in this table, there is little variability among these proteins.

[0060] In another embodiment, polypeptides are arranged Includes TIFF0007879809000004.tif11141.

[0061] In this sequence, the amino acid designated as X at positions 8, 10, 22-23, 42, and 48 can be any amino acid. Other amino acids designated as X are shown in Table 1.

[0062] In another embodiment, polypeptides are arranged Includes TIFF0007879809000005.tif11141.

[0063] In this sequence, the amino acid designated as X is shown in Table 1.

[0064] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000006.tif11143.

[0065] In this sequence, the amino acid designated as X at positions 7, 9, 21-22, 24, 29, 33, 40, and 46 can be any amino acid.

[0066] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000007.tif11141.

[0067] In this sequence, the amino acid designated as X at positions 7, 9, 21-22, 40, and 46 can be any amino acid.

[0068] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000008.tif11141. Such sequences are particularly preferred.

[0069] In another embodiment, polypeptides are arranged This includes TIFF0007879809000009.tif11141. In this sequence, the amino acid designated as X at positions 7, 9, 21-22, 24, 29, 33, 41, and 47 can be any amino acid.

[0070] In another embodiment, polypeptides are arranged Includes TIFF0007879809000010.tif11141.

[0071] The polypeptide may also contain SEQ ID NO: 21, in which 1 to 10, more preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 2, and more preferably 1 amino acid, selected from the group consisting of V7, G9, K21, T22, T40, and S46, are replaced by any other amino acids.

[0072] The HSP110-binding variants disclosed herein include 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. However, it should be noted that tryptophan at position 24 may be retained in the variants.

[0073] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000011.tif11141.

[0074] In this sequence, the amino acid designated as X at positions 8, 28, 30, 44, and 48 can be any amino acid. Other amino acids designated as X are shown in Table 1 (where "-" means no amino acid).

[0075] In another embodiment, polypeptides are arranged Includes TIFF0007879809000012.tif11142.

[0076] In this sequence, the amino acid designated as X is shown in Table 1.

[0077] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000013.tif11141.

[0078] In this sequence, the amino acid designated as X at positions 7, 26, 29, 42, and 46 can be any amino acid.

[0079] In a preferred embodiment, the polypeptide is sequence Includes TIFF0007879809000014.tif11142. Such sequences are particularly preferred.

[0080] In another embodiment, polypeptides are arranged This includes TIFF0007879809000015.tif11141. In this sequence, the amino acid designated as X at positions 7, 26, 29, 43, and 47 can be any amino acid.

[0081] In another embodiment, polypeptides are arranged Includes TIFF0007879809000016.tif11142.

[0082] The polypeptide may contain SEQ ID NO: 29, in which 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 2, and more preferably 1 amino acid selected from the group consisting of M7, S26, T29, Q43, and H47 are replaced by any other amino acid.

[0083] Description of PD-L1 binding variants based on Sac7d and Aho7c Sac7d and Aho7c are proteins with significant similarities. It is also worth noting that Sto (SEQ ID NO: 94) exhibits similarities to these proteins.

[0084] SEQ ID NO: 93 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 starting amino acid is omitted because it is not essential to the protein structure. TIFF0007879809000017.tif11143

[0085] In this consensus sequence, X (or Xaa) is as shown in Table 2.

[0086] (Table 2) Explanation of some amino acids represented as Xaa in SEQ ID NO: 87 and SEQ ID NO: 30-32, 51-53, or 72-74. TIFF0007879809000018.tif42148

[0087] The consensus sequence for variants that bind to PD-L1 is represented as follows:

[0088] (Table 3) Variant sequences based on the consensus sequence of Sac7d-Aho7c. The amino acids represented by TIFF0007879809000019.tif89145X are further described in Tables 2, 4, and 5.

[0089] (Table 4) Explanation of the amino acid shown as Xaa in SEQ ID NO: 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66, 67, 68, 69. TIFF0007879809000020.tif124147 Please note that this table is used for the Xaa that the above sequences possess. For some sequences, the amino acids are specified at some positions in Table 4, so the corresponding rows in this table do not apply.

[0090] In these sequences, several amino acids that are not involved in binding can also be modified in variants without altering the protein's binding properties or biological characteristics. These are shown in Table 5.

[0091] (Table 5) Amino acids represented as Xaa in SEQ ID NO: 30-32, SEQ ID NO: 33-35, SEQ ID NO: 42-44, SEQ ID NO: 51-53, SEQ ID NO: 54-56, SEQ ID NO: 63-65, SEQ ID NO: 72-74, SEQ ID NO: 75-77, and SEQ ID NO: 84-85. Xaa at position 56 in TIFF0007879809000021.tif28128 exists only in SEQ ID NO: 33~35, SEQ ID NO: 54~56, and SEQ ID NO: 75~77.

[0092] Description of PD-L1 binding variants based on Sac7d (SEQ ID NO: 1) Specific variants based on Sac7d that bind to human and / or mouse PD-L1 are indicated by SEQ ID NO: 33–SEQ ID NO: 41 and SEQ ID NO: 54–SEQ ID NO: 62.

[0093] In these sequences, the initiating methionine (M) of the Sac7d protein is omitted. The applicant has demonstrated that the protein's activity remains unchanged even with the deletion of this amino acid. Similarly, it is possible to retain activity by omitting the last seven amino acids from the variant.

[0094] Therefore, proteins represented by amino acids 1-57 of any of SEQ ID NO: 33-41 and SEQ ID NO: 54-62 are also variants of the present invention. Proteins represented by amino acids 1-58, 1-59, 1-60, 1-61, 1-62, and 1-63 of any of SEQ ID NO: 33-41 and SEQ ID NO: 54-62 can also be listed, and these are also variants of the present invention.

[0095] Therefore, the polypeptide comprises any of the sequences SEQ ID NO: 33 to SEQ ID NO: 41 and SEQ ID NO: 54 to SEQ ID NO: 62, or any truncated protein based on these sequences, and those disclosed above.

[0096] Such variants are represented as follows:

[0097] (Table 6) Sequence of variants based on Sac7d. The amino acids represented by TIFF0007879809000022.tif252145X are further described in Tables 4 and 5.

[0098] SEQ IDs 36-38 (and 39-41 respectively) correspond to SEQ IDs 33-35, where amino acids not involved in binding or biological properties are specified. SEQ IDs 36-38 also correspond to SEQ IDs 19-21.

[0099] SEQ ID NOs 57-59 (60-62 respectively) correspond to SEQ ID NOs 54-56, where amino acids that are not involved in binding or biological properties are specified.

[0100] As described above, as disclosed in Table 5, it is possible to modify D16 of Sac7d (which is located at position 15 in SEQ ID NO: 33 to SEQ ID NO: 41 and SEQ ID NO: 54 to SEQ ID NO: 62, as M1 present in Sac7d is omitted), N37 of Sac7d (located at position 36 in this specification), or M57 of Sac7d (located at position 56 in this specification).

[0101] Regarding the consensus sequence for Sac7d / Aho7c, any combination of substitutions can be foreseen even if the sequence listing does not show all of them (the numbering is for SEQ ID NO: 33~SEQ ID NO: 41 and SEQ ID NO: 54~SEQ ID NO: 62): D15 N36 M56 SEQ ID NO: 36~38 and SEQ ID NO: 57~59 D15 N36 L56 D15 Q36 M56 D15 Q36 L56 E15 N36 M56 E15 N36 L56 E15 Q36 M56 E15 Q36 L56 SEQ ID NO: 39~41 and SEQ ID NO: 60~62

[0102] As described above, all sequences contain five mutated amino acids I7, I25, L30, M41, and L43, or M7, L25, L30, L41, and F43 (corresponding to positions 8, 26, 31, 42, and 44 of SEQ ID NO: 1, respectively, which are different from the amino acids naturally present in Sac7d).

[0103] The applicant has found that these amino acids are present in various variants that bind to PD-L1, and that such modifications result in a decrease in binding (loss of affinity or loss of binding). Other amino acids can be varied under the conditions described in Table 4.

[0104] Furthermore, if the variant includes R23, Y28, and K32, they will bind to both human and mouse PD-L1.

[0105] The particularly interesting variants are indicated by SEQ ID NO: 21, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 62. These variants bind to both human and mouse PD-L1. SEQ ID NO: 21 corresponds to SEQ ID NO: 38.

[0106] SEQ ID NO: 41 (which I've named B11) is particularly interesting, just like SEQ ID NO: 38.

[0107] SEQ ID NO: 62 (which I've named G02) is also particularly interesting, just like SEQ ID NO: 59.

[0108] Protein description based on Aho7c (SEQ ID NO: 14) Variants of Aho7c that bind to human and / or mouse PD-L1 are indicated by SEQ ID NO: 63–SEQ ID NO: 71 and SEQ ID NO: 42–SEQ ID NO: 50. As described above, such proteins are very similar to Sac7d. It is also noted herein, as shown and recalled, that mutations in Sac7d can be introduced from Sac7d into other proteins (WO 2012 / 150314).

[0109] In these sequences, the initiating methionine and alanine (MA) of the Aho7c protein (SEQ ID NO: 14) are omitted. The applicant has demonstrated that the protein's activity remains unchanged even with the deletion of these amino acids. Similarly, activity can be maintained even when the last four amino acids are omitted from the variant.

[0110] Therefore, proteins represented by amino acids 1-55 of any of SEQ ID NO: 63-71 and SEQ ID NO: 42-50 are also variants of the present invention. Proteins represented by amino acids 1-56, 1-57, and 1-58 of any of SEQ ID NO: 63-71 and SEQ ID NO: 42-50 can also be cited, and these are also variants of the present invention.

[0111] Therefore, the polypeptide comprises any of the sequences SEQ ID NO: 63 to SEQ ID NO: 71 and SEQ ID NO: 42 to SEQ ID NO: 50, or any truncated protein based on these sequences, and those disclosed above.

[0112] Such variants are represented as follows:

[0113] (Table 7) Variant sequences based on Ao7c. The amino acids represented by TIFF0007879809000023.tif42145TIFF0007879809000024.tif211145X are further described in Tables 4 and 5.

[0114] As described above, as disclosed in Table 5, it is possible to modify D17 of Aho7c (which is located at position 15 in SEQ ID NO: 63~SEQ ID NO: 71 and SEQ ID NO: 42~SEQ ID NO: 50, as M1A2 present in Aho7c is omitted), or N38 of Aho7c (located at position 36 in this specification).

[0115] Regarding the consensus sequence for Sac7d / Aho7c, any combination of substitutions can be foreseen even if the sequence listing does not show all of them (the numbering is for SEQ ID NO: 63~SEQ ID NO: 71 and SEQ ID NO: 42~SEQ ID NO: 50): D15 N36 SEQ ID NO: 66~68 and 45~47 D15 Q36 E15 N36 E15 Q36 SEQ ID NO: 69-71 and 48-50

[0116] As described above, all sequences contain mutated amino acids I7, I25, L30, M41, and L43, or M7, L25, L30, L41, and F43 (corresponding to positions 9, 26, 32, 43, and 45 of SEQ ID NO: 14, respectively, which are different from the amino acids naturally present in Aho7c).

[0117] The applicant has found that these amino acids are present in various variants that bind to PD-L1, and that such modifications result in a decrease in binding (loss of affinity or loss of binding). Other amino acids can be varied under the conditions described in Tables 4 and 5.

[0118] Furthermore, if the variant includes R23, Y28, and K32, they will bind to both human and mouse PD-L1.

[0119] The particularly interesting variants are indicated by SEQ ID NO: 66, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 50.

[0120] SEQ ID NO: 70 (based on G02) is particularly interesting.

[0121] SEQ ID NO: 50 (based on B11) is also particularly interesting.

[0122] In some embodiments, the polypeptide is such that a variant of a Sac7d family protein that binds to PDL-1 is ligated to or fused to another protein or polypeptide, in particular another variant of a Sac7d family protein or antibody, in particular those that bind to HSP-110 (in particular those disclosed by SEQ ID NO: 24 to SEQ ID NO: 29, and in particular SEQ ID NO: 27) or EGFR.

[0123] In some embodiments, the polypeptide is such that a variant of a Sac7d family protein that binds to HSP-110 is ligated to or fused to another protein or polypeptide, in particular another variant of a Sac7d family protein or antibody, in particular those that bind to PDL-1 (in particular those disclosed by SEQ ID NO: 16 to SEQ ID NO: 23, and in particular those disclosed by SEQ ID NO: 21) or EGFR.

[0124] Variant that binds to Hsp-110 Description of Hsp-110 variants based on Sac7d and Aho7c Such variants are represented as follows:

[0125] (Table 8) Variant sequences based on the consensus sequence of Sac7d-Aho7c. The amino acids represented by TIFF0007879809000025.tif48145X are further described in Tables 2, 5, and 9.

[0126] (Table 9) Explanation of the amino acid shown as Xaa in SEQ ID NO: 72 to SEQ ID NO: 92. TIFF0007879809000026.tif104145 Please note that this table is used for the Xaa that the above sequences possess. For some sequences, the amino acids are specified at some positions in Table 6, so the corresponding rows in this table do not apply.

[0127] In these sequences, several amino acids that are not involved in binding can also be modified with variants (see SEQ ID NO: 72-77, 84-86 in particular). These are shown in Table 5.

[0128] Description of variants based on Sac7d (SEQ ID NO: 1) Sac7d-based variants that bind to human Hsp110 are indicated by SEQ ID NO: 75 to SEQ ID NO: 83.

[0129] In these sequences, the initiating methionine (M) of the Sac7d protein is omitted. The applicant has demonstrated that the protein's activity remains unchanged even with the deletion of this amino acid. Similarly, it is possible to retain activity by omitting the last seven amino acids from the variant.

[0130] Therefore, proteins represented by amino acids 1-57 of any of SEQ ID NO: 75 to SEQ ID NO: 83 are also variants of the present invention. Proteins represented by amino acids 1-58, 1-59, 1-60, 1-61, 1-62, and 1-63 of any of SEQ ID NO: 75 to SEQ ID NO: 83 can also be listed, and these are also variants of the present invention.

[0131] Therefore, the polypeptide comprises any of SEQ ID NO: 75 to SEQ ID NO: 83, or any truncated protein based on these sequences, and those disclosed above.

[0132] Such variants are represented as follows:

[0133] (Table 10) Sequence of hsp110-binding variant based on Sac7d. The amino acids represented by TIFF0007879809000027.tif130145X are further described in Tables 5 and 9.

[0134] As described above, as disclosed in Table 7, it is possible to modify D16 of Sac7d (which is located at position 15 in SEQ ID NO: 45~65 because M1 present in Sac7d is omitted), N37 of Sac7d (located at position 36 in this specification), or M57 of Sac7d (located at position 56 in this specification).

[0135] Regarding the consensus sequence of Sac7d / Aho7c, any combination of substitutions can be foreseen even if the sequence listing does not show all of them (the numbering corresponds to SEQ ID NO: 45-65): D15 N36 M56 SEQ ID NO: 78~80 D15 N36 L56 D15 Q36 M56 D15 Q36 L56 E15 N36 M56 E15 N36 L56 E15 Q36 M56 E15 Q36 L56 SEQ ID NO: 81~83

[0136] As described above, all sequences contain mutated amino acids W7, D21; W23, H32, and K43 (corresponding to positions 8, 22, 24, 33, and 44 of SEQ ID NO: 1, respectively, which are different from the amino acids naturally present in Sac7d).

[0137] The applicant has found that these amino acids are present in various variants that bind to Hsp110, and that such modifications result in a decrease in binding (loss of affinity or loss of binding). Other amino acids can be varied under the conditions described in Table 9.

[0138] The particularly interesting variants are indicated by SEQ ID NO: 27, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 82, and SEQ ID NO: 83.

[0139] SEQ ID NO: 83 is particularly interesting, as is SEQ ID NO: 80. SEQ ID NO: 80 corresponds to SEQ ID NO: 27.

[0140] Protein description based on Aho7c (SEQ ID NO: 14) Variants of Aho7c that bind to human and / or mouse PD-L1 are indicated by SEQ ID NO: 84 to SEQ ID NO: 92. As described above, such proteins are very similar to Sac7d. It is also noted herein that mutations in Sac7d can be introduced into other proteins (WO 2012 / 150314).

[0141] In these sequences, the initiating methionine and alanine (MA) of the Aho7c protein (SEQ ID NO: 14) are omitted. The applicant has demonstrated that the protein's activity remains unchanged even with the deletion of these amino acids. Similarly, activity can be maintained even when the last four amino acids are omitted from the variant.

[0142] Therefore, proteins represented by amino acids 1-55 of any of SEQ ID NO: 84 to SEQ ID NO: 92 are also variants of the present invention. Proteins represented by amino acids 1-56, 1-57, and 1-58 of any of SEQ ID NO: 84 to SEQ ID NO: 92 can also be listed, and these are also variants of the present invention.

[0143] Therefore, the polypeptide comprises any of SEQ ID NO: 84 to SEQ ID NO: 92, or any truncated protein based on these sequences, and those disclosed above.

[0144] Such variants are represented as follows:

[0145] (Table 11) Variant sequences based on Ao7c. The amino acids represented by TIFF0007879809000028.tif130145X are further described in Tables 5 and 9.

[0146] As described above, as disclosed in Table 5, D17 of Aho7c (which is located at position 15 in SEQ ID NO: 84~92 because M1A2 present in Aho7c is omitted) and N38 of Aho7c (located at position 36 in this specification) can be modified.

[0147] Regarding the consensus sequence of Sac7d / Aho7c, any combination of substitutions can be foreseen even if the sequence listing does not show all of them (the numbering corresponds to SEQ ID NO: 84~92): D15 N36 SEQ ID NO: 87~89 D15 Q36 E15 N36 E15 Q36 SEQ ID NO: 90~92

[0148] As described above, all sequences contain mutated amino acids W7, D21; W23, H32, and K43 (corresponding to positions 9, 22, 24, 35, and 45 of SEQ ID NO: 14, respectively, which are different from the amino acids naturally present in Aho7c).

[0149] The particularly interesting variants are indicated by SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 92.

[0150] SEQ ID NO: 92 is particularly interesting, as is SEQ ID NO: 89.

[0151] Accordingly, the present invention also relates to a polypeptide comprising a variant of a member of the Sac7d family that binds to human Hsp110, wherein the variant comprises 4 to 20 (preferably 8 to 14) mutated residues at the interface of binding of the Sac7d family member to its native ligand. In particular, this variant also binds to mouse HSP110. In a preferred embodiment, the variant comprises the Y8W, K22D, W24W, T33H, and A44K mutations, the numbering corresponding to the position in Seq ID NO: 1 of the Sac7d sequence. This corresponds particularly to SEQ ID NO: 72 to 92.

[0152] Preferably, polypeptides containing variants of members of the Sac7d family that bind to human Hsp110 include Y8W, K21W, K22D, W24W, S31Y, T33H, T39M, and A44K mutations, the numbering corresponding to the position in Sac7d sequence SEQ ID NO: 1. This corresponds in particular to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 92. Such variants may be modified, manufactured, and used as disclosed herein, in the same way that variants bound to PD-L1 are modified, manufactured, and used.

[0153] Variant modification In another embodiment, polypeptides are conjugated to organic molecules, particularly tyrosine kinase inhibitors.

[0154] This also makes it possible to obtain a gene construct containing a DNA sequence encoding the polypeptide defined above, a vector containing such a gene construct, and a host cell containing such a gene construct in its genome.

[0155] A method for producing polypeptides can be carried out, which includes the steps of culturing a cell culture in which cells have been transformed by a gene construct, and recovering the polypeptide.

[0156] The disclosed polypeptides can also be materialized as pharmaceuticals.

[0157] The present invention also relates to polypeptides disclosed herein for use in the treatment of cancer, particularly solid tumors and lymphomas, which bind to PDL-1, HSP-110, or EGFR, either alone or in combination, such as as a multimer polypeptide.

[0158] Such cancers include, in particular, non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high-frequency microsatellite instability cancer, bladder cancer, breast cancer, small cell lung cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

[0159] Anti-PD-L1 variants are useful in a wide range of cancers as disclosed above, particularly in non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high-frequency microsatellite instability cancer, bladder cancer, breast cancer, and small cell lung cancer, which have already been treated with anti-PD-L1 agents.

[0160] Anti-HSP110 variants are particularly useful in treating colorectal cancer, pancreatic cancer, prostate cancer, or breast cancer.

[0161] The present invention also relates to polypeptides disclosed herein that bind to PDL-1, HSP-110, and / or EGFR for use in the treatment of cancer in combination with chemotherapy or CAR-T cell therapy, particularly for use in the treatment of the cancers listed above.

[0162] Sac7d protein family The Sac7d family is defined as relating to the Sac7d protein and corresponds to a family of 7 kDa DNA-binding proteins isolated from extremophilic bacteria. In this specification, it is disclosed as a representative species of the OB-fold domain, which is preferably used in the context of the present invention. Since the SH3 domain shares homology with the OB-fold domain, teachings relating to Sac7d are also applicable to the SH3 scaffold.

[0163] These proteins and this family are described in particular in WO 2008 / 068637. Therefore, in 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: 14, or a sequence corresponding to the consensus sequence SEQ ID NO: 15 (derived from SEQ ID NO: 1 to SEQ ID NO: 9 and SEQ ID NO: 12 to SEQ ID NO: 14, as well as SEQ ID NO: 94, in which the dash indicates the absence of an amino acid, indicating that not all proteins are the same size). The Sac7d family includes, in particular, the Sac7d or Sac7e protein derived from Sulfolobus acidocaldarius, the Sso7d protein derived from Sulfolobus solfataricus, DBP 7 (also called Sto7 protein) derived from Sulfolobus tokodaii, the Ssh7b protein derived from Sulfolobus shibatae, the Ssh7a protein derived from Sulfolobus shibatae, the Mse7 derived from Metallosphaera sedula, the Mcu7 derived from Metallosphaera cuprina, the Aho7a, Aho7b, or Aho7c derived from Acidianus hospitalis, and Sulfolobus islandis This includes Sis7a or Sis7b from *Islandicus* and the p7ss protein from *Sulfolobus solfataricus*. Given the extensive sequence similarities of the Sac7d family proteins, it is directly and easily possible to identify the amino acids of a protein other than Sac7d that correspond to a given amino acid in Sac7d.In particular, the teachings of WO 2008 / 068637, which show (in the figure) and explain (in the specification) that such proteins can be superimposed, can also be used.

[0164] WO 2012 / 150314 demonstrates that mutations from one protein in the Sac7d family can be introduced into another protein in the same family. This transferability corresponds to creating a variant of another protein in the Sac7d family starting from a variant of one protein in the Sac7d family. The initial variant may be obtained by performing the process in particular of WO 2008 / 068637. As a result, it is possible to obtain a variant of any protein in the Sac7d family, starting from a variant of any other protein in that family, particularly using the teachings in WO 2012 / 150314 and Figure 1. As an example, in the case of a Sac7d variant, the mutated amino acids of the Sac7d variant can be introduced into another protein scaffold using the sequence alignment in Figure 1. As an example, variants of Sso7d obtained from the Sac7d variants disclosed herein are described as SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, or higher.

[0165] The number of mutant residues introduced into the wild-type protein sequence to obtain a variant is preferably 4 to 25, or more specifically 4 to 22. Therefore, it is possible to obtain a variant that preferably has 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 folded protein (or domain). It should be noted that all and any range (such as 5 to 20 or 7 to 25) are considered herein. Particularly preferred ranges are 4 to 17 and 6 to 17, and 4 to 14 and 6 to 14.

[0166] It is preferable that 7, 8, 9, 10, 11, 12, 13, or 14 amino acids are mutated at the binding site of the OB fold domain compared to the wild-type OB fold domain. These mutations are introduced into the 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).

[0167] The case where the number of mutated amino acids is between 7 and 14 (including the values ​​at both ends) is particularly interesting. In particular, variants can also include amino acid insertions, as described above.

[0168] As shown, the Sac7d family proteins include Sac7d or Sac7e from Sulfolobus acidocardarius, Sso7d from Sulfolobus solfataricus, DBP 7, also known as Sto7, from Sulfolobus tokodaii, Ssh7b from Sulfolobus sivatae, Ssh7a from Sulfolobus sivatae, Mse7 from Metalosphaera sedula, Mcu7 from Metalosphaera cuprina, Aho7a or Aho7b or Aho7c from Acidianus hospitalis, Sis7a or Sis7b from Sulfolobus islandicus, and p7ss from Sulfolobus solfataricus. The various sequences of the Sac7d, Sso7d, Sac7e, Ssh7b, Ssh7a, DBP7, Sis7a (3 alleles), Mse7, Mcu7, Aho7a, Aho7b, and Aho7c proteins are represented by SEQ ID NO: 1 to SEQ ID NO: 14, respectively. SEQ ID NO: 94 represents the Sto7 protein from Sulfurisphaera tokodaii.

[0169] This variant of the Sac7d family protein may be called nanophytin. Therefore, the present invention is selectively carried out for variants of proteins represented by SEQ ID NO: 1 to SEQ ID NO: 14 and SEQ ID NO: 94, or for proteins having sequence SEQ ID NO: 15, and in particular for variants of Sac7d.

[0170] Relationship between Sac7d protein, OB folded protein, and SH3 domain OB-fold proteins are known in the art. They are described in particular in the documents cited above and in Arcus (Curr Opin Struct Biol. 2002 Dec; 12(6):794-801). OB-folds are cylindrical in form, having five beta (β) sheets. Most OB-fold proteins use the same binding interface as their native ligands, which can be oligosaccharides, oligonucleotides, proteins, metal ions, or catalytic substrates. This binding interface mainly involves residues located in the β sheets. Certain residues located in the loop can also be involved in the binding of OB-fold proteins to their native ligands. Accordingly, the OB-fold protein domains for binding to their native ligands are described in the applications WO 2007 / 139397 and WO 2008 / 068637 and in the Arcus document (2002, op. cit.).

[0171] In particular, document WO 2008 / 068637 accurately describes a method for identifying the binding domain of OB-fold proteins. By superimposing several sequences and 3D structures of proteins containing the OB-fold domain, 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). Referring 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.

[0172] WO 2008 / 068637 describes that it is possible to perform three-dimensional superposition of OB folded proteins or domains (in this application, 10 domains, including Sac7d) using the DALI website (http: / / www.ebi.ac.uk / dali / interactive.html) (Holm and Sander, 1998, Nucleic Acids Res 26, 316-319). Therefore, it is easy to identify the amino acids involved in the binding site and corresponding to the above-mentioned Sac7d amino acid for any OB folded protein (or any OB folded domain). As a result, by providing an amino acid that can be mutated in one of these proteins, it becomes possible to identify the corresponding amino acid for any other OB folded domain.

[0173] It should also be noted that the OB fold domain is similar to the SH3 domain, and that it is possible to identify amino acid equivalents of Sac7d in the SH3 domain.

[0174] Examples of OB fold domains or SH3 domains Non-limiting examples of OB-fold proteins that can be used by the present invention include Sac7d, Sso7d, the N-terminal domain of 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 of Azotobacter vinelandii (modg) (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), and Cdc13 (Mitton-Fry et al., 2002), human Y-box protein YB-1 cold shock DNA binding domain (Kloks et al., 2002), Escherichia coli inorganic pyrophosphatase EPPase (Samygina et al., 2001), or any of the proteins listed in Table 3 of the paper by (Arcus, 2002), for example, 1krs (lysyl-tRNA synthetase LysS, Escherichia coli), 1c0aA (Asp-tRNA synthetase, Escherichia coli), 1b8aA (Asp-tRNA synthetase, P. kodakaraensis), 1lylA (lysyl-tRNA synthetase LysU, Escherichia coli), 1quqA (replica protein A, 32 kDa subunit, human), 1quqB (replica protein A, 14 kDa subunit, human), 1jmcA (replica 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, pertussis (B. pertussis)), 1bcpD (pertussis toxin S5 subunit (ATP binding), pertussis), 3chbD (cholera toxin, V. cholera (V.cholerae), 1tiiD (thermolabitive toxin, Escherichia coli), 2bosA (verotoxin-1 / Shiga toxin, B-pentamer, Escherichia coli), 1br9 (TIMP-2, human), 1an8 (superantigen SPE-C, Streptococcus pyogenes), 3seb (superantigen SPE, Staphylococcus aureus), 1aw7A (toxic shock syndrome toxin, Staphylococcus aureus), 1jmc (major cold shock protein, Escherichia coli), 1bkb (initiator translation factor 5a, P. aerophylum), 1sro (S1 RNA binding domain of PNPase, Escherichia coli), 1d7qA (initiator translation factor 1, elF1a, human), 1ah9 (initiator translation factor 1, IF1, Escherichia coli), 1b9mA (Mo-dependent transcription regulator ModE, These include E. coli, 1ckmA (RNA guanylyl transferase, 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, budding yeast (S. cerevisiae)), and 2prd (inorganic pyrophosphatase, hyperthermophilic bacterium (T. thermophilus)).

[0175] Non-exclusive examples of proteins containing the SH3 domain include signal transduction adapter proteins, CDC24, Cdc25, PI3 kinases, phospholipases, Ras GTPase-activating proteins, Vav proto-oncogenes, GRB2, p54 S6 kinase 2 (S6K2), SH3D21, C10orf76 (possibly), STAC3, several myosins, SHANK1,2,3, ARHGAP12, C8orf46, TANGO1, integrases, local adhesion kinases (FAK, PTK2), proline-rich tyrosine kinases (Pyk2, CADTK, PTK2beta), or TRIP10 (cip4).

[0176] Manufacturing of identified variants The identified variant sequences can be cloned into any suitable vector by any molecular genetic method known in the art.

[0177] These recombinant DNA constructs, which contain nucleotide sequences encoding polypeptides containing the above variants, are used in conjunction with vectors such as plasmids, phagemids, phages, or viral vectors.

[0178] These recombinant acid molecules can be prepared 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 sequence may be chemically synthesized, for example, using a synthesizer.

[0179] The recombinant construct of the present invention comprises an expression vector capable of expressing RNA and thus resulting in the production of a protein from the gene sequence. Accordingly, the vector may further include a regulatory sequence, including a suitable promoter functionally linked to a gene sequence reading frame (ORF) disclosed herein. The vector may further include a selectable marker sequence, such as an antibiotic resistance gene. When bacteria are used as the expression host, specific initiation signals and bacterial secretion signals may also be required for efficient translation of the coding sequence.

[0180] Molecular production Cells are transfected or transformed with a vector containing a sequence encoding a polypeptide, such as the variants disclosed above.

[0181] Cells are cultured under conditions that allow for the expression and successful secretion of proteins. These cell culture conditions are generally those used for recombinant antibody production and are well known in the art. Such conditions, known in the art, can be optimized as needed by those skilled in the art. Such methods are outlined in Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461), and ample references are provided thereto.

[0182] One possible approach is to use bacterial, phage (Shukra et al, Eur J Microbiol Immunol (Bp). 2014; 4(2): 91-98) or eukaryotic production systems.

[0183] For one reason, it would be preferable to use eukaryotic cells to obtain appropriate post-translational modifications such as glycosylation.

[0184] 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 fetal kidney cells 293), NS0 cells (cell line derived from non-secretory mouse myeloma), or EB66 cells (duck cell line Valneva, Lyons, France) can be used.

[0185] Furthermore, the present disclosure provides a host cell comprising at least one DNA construct encoding a polypeptide containing the variant disclosed herein. The host cell can be any cell for which an expression vector is available. As described above, it may be a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell.

[0186] The introduction of the recombinant construct 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). The vector can be inserted into the genome of the host cell or maintained as an extragenomic vector (such as a bacterial or yeast artificial chromosome). If introduced into the cellular genome, such introduction may be targeted using methods known in the art (such as homologous recombination) or random.

[0187] Bacterial host and expression Expression vectors useful for bacteria are constructed by inserting recombinant DNA sequences, along with appropriate translation start and termination signals, into a functional read-through phase with a functional promoter. The vector includes one or more phenotypic selection markers and origins of replication to ensure vector retention and, if desired, provide amplification in the host.

[0188] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0189] Eukaryotic host and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, the above-mentioned cells can be used.

[0190] Transformed or transfected cells are cultured by methods known in the art, and polypeptides are recovered from intracellular or extracellular fractions (depending on whether they are secreted or not).

[0191] Molecular isolation The recombinant proteins produced can be separated and purified from intracellular or extracellular fractions by any of the known separation methods that utilize the physical or chemical properties of the proteins.

[0192] In particular, various liquid chromatography methods such as precipitation, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, as well as dialysis and combinations thereof, can be used.

[0193] In general, any method known and used for purifying recombinant polypeptides is applicable to the purification of the molecules disclosed herein.

[0194] If a tag (such as a polyhistidine tag) is introduced into the recombinant sequence, the molecule can be purified using this tag. However, it is preferable to purify the molecule using affinity.

[0195] In particular, the fact that the molecules produced herein bind to specific targets can be used to isolate such molecules using any affinity method (affinity column, FACS, beads).

[0196] One particular advantage of the molecules disclosed herein is that they do not need to be glycosylated to be active and can therefore be produced in any type of cell, not necessarily eukaryotic cells. They are produced particularly well in bacterial cells.

[0197] Variant modification The above variants, and variants having the ability to bind to PD-L1 or HSP110, can be modified by any method known in the art.

[0198] Preparation of polypeptides containing variants It is possible to prepare a DNA sequence containing two coding sequences: one for the variant disclosed herein and the other for the protein or peptide of interest. The resulting expressed protein will therefore be a polypeptide containing both proteins. The vector can be constructed such that it includes a sequence encoding a linker located between the two proteins in the expressed polypeptide.

[0199] Accordingly, the present invention also encompasses protein variants of OB folded proteins (preferably of the Sac7d family) that bind to PDL-1, or polypeptides comprising HSP110 linked to another protein or polypeptide (preferably through a bound amine as disclosed above).

[0200] In certain embodiments, other proteins or polypeptides include other variants of the Sac7d family proteins, particularly as disclosed herein.

[0201] The following are particularly interesting: - A polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSP-110, fused with a variant of an OB-fold protein of the Sac7d family that binds to PD-L1 (at the N-terminus or C-terminus). - A polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSP-110, fused with a variant of an OB-fold protein of the Sac7d family that binds to EGFR (at the N-terminus or C-terminus). - A polypeptide comprising a variant of an OB-fold protein of the Sac7d family that binds to HSP-110, fused with the same or another variant of the OB-fold protein of the Sac7d family that binds to HSP110 (at the N-terminus or C-terminus). - A polypeptide comprising a variant of a PD-L1-binding Sac7d family OB-fold protein, which is fused (at the N-terminus or C-terminus) with the same or another variant of a PD-L1-binding Sac7d family OB-fold protein. A polypeptide comprising a variant of a PD-L1-binding Sac7d family OB-fold protein, which is fused (at the N-terminus or C-terminus) with a variant of an EGFR-binding Sac7d family OB-fold protein.

[0202] In another embodiment, the other protein or polypeptide is an antibody. In this embodiment, the variant of the OB folded domain is fused to at least one of the heavy chain or light chain of the immunoglobulin monomer, preferably to the N-terminus or C-terminus of the light chain or heavy chain. In another embodiment, the variant may be fused to both the heavy chain and the light chain.

[0203] To obtain such compounds, a gene construct containing a DNA sequence selected from the group consisting of the following can be used. a. The sequence encoding the antibody's heavy chain is fused at its 3' end with the sequence encoding the variant of the OB-fold protein (potentially via the linker-encoding sequence). b. A sequence in which the sequence encoding the antibody's heavy chain is fused at its 5' end with the sequence encoding the variant of the OB-fold protein (potentially via the linker-coding sequence). c. The sequence encoding the antibody light chain is fused at its 3' end with the sequence encoding the variant of the OB folded protein (potentially via the linker-encoding sequence). d. A sequence in which the sequence encoding the antibody light chain is fused at its 5' end with the sequence encoding the variant of the OB folded protein (potentially via the linker-coding sequence).

[0204] This fusion can be performed at the N-terminal and / or C-terminal positions of the antibody chain (heavy and / or light chain). In particular, when using small OB-fold domains (approximately 70 amino acids), such as those derived from the Sac7d family, it is possible to obtain molecules with an antibody structure (two light chains paired with two heavy chains, and such a paired dimer) having an antibody region and a further binding region consisting of modified OB-fold domains.

[0205] In certain embodiments, the antibody portion of the protein disclosed herein is an IgG molecule.

[0206] In another embodiment, the antibody portion of the protein disclosed herein is an IgA molecule.

[0207] In another embodiment, the antibody portion of the protein disclosed herein is an IgM molecule.

[0208] In another embodiment, the antibody portion of the protein disclosed herein is an IgD molecule.

[0209] In another aspect, the antibody portion of the protein disclosed herein is an IgE molecule.

[0210] The antibody can be a human antibody, a rodent antibody (such as a mouse or rat antibody), a cat 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 can 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 its similarity to antibodies naturally produced in humans. A chimeric antibody contains sequences from different species.

[0211] Preferably, the antibody that is part of the molecule disclosed herein is an antibody that comprises two identical heavy chains (about 400 - 500 amino acids, generally around 450 amino acids) and two identical light chains. As a result, the antibody contains the same Fab variable region. This antibody is thus a monospecific antibody in which both parts of the antibody (the combination of the light and heavy chains) bind to the same epitope of the antigen.

[0212] However, the antibody can present different heavy and / or light chains. In particular, in some aspects, the antibody is a bispecific antibody. Thus, the term "antibody" encompasses both the "classical antibody" as disclosed above having the same heavy and light chains, but also engineered antibodies having two or more specificities.

[0213] In certain aspects, the antibody presents one heavy and one light chain from one antibody and another heavy and another light chain from another antibody.

[0214] The antibody can bind to a target selected from the group consisting of: Cell surface receptors: insulin receptor, low-density lipoprotein receptor-associated 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. Angiogenic factors 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: TNFα, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Bace1, α-synuclein, tau, amyloid.

[0215] This is particularly relevant when antibodies target IL17 or cell surface receptors specifically involved in cancer.

[0216] Therefore, the following is particularly foreseen: - A polypeptide comprising the variant disclosed above that binds to PD-L1 and an antibody that binds to HSP110. - A polypeptide comprising the variant disclosed above that binds to HSP110 and an antibody that binds to PD-L1 (such as atezolizumab, avelumab, or durvalumab). - A polypeptide comprising the variant disclosed above that binds to PD-L1 and an antibody that binds to EGFR (such as cetuximab, panitumumab, zaltumumab, nimotuzumab, and matuzumab). A polypeptide comprising the above-disclosed variant that binds to HSP110 and an antibody that binds to EGFR.

[0217] Alternatively, the polypeptide may comprise the variant disclosed above and a biologically active molecule such as erythropoietin, interferon, or etanercept.

[0218] In another embodiment, variants of the OB folded domain (particularly variants of the Sac7d family of proteins) are conjugated to organic molecules. This can be done by any method known in the art. In particular, molecules can be chemically linked to proteins. Examples of molecules include antiproliferative agents (cytotoxic agents and cell proliferation inhibitors) 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. Tubulin polymerization inhibitors such as auristatin or maytansine can also be used.

[0219] In particular, examples include DNA-binding drugs or alkylating drugs, such as anthracycline drugs (doxorubicin, epirubicin, idarubicin, daunorubicin) and their analogues, and alkylating agents, such as calicheamicin, dactinomycin, mitromycin, and pyrrolobenzodiazepines. Cell cycle progression inhibitors such as CDK inhibitors, Rho kinase inhibitors, checkpoint kinase inhibitors, aurora kinase inhibitors, PLK inhibitors, and KSP inhibitors can also be mentioned. Thalidomide and its derivatives, lenalidomide and pomalidomide, can also be mentioned. To treat inflammatory disorders, cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitors, quercetin, and / or resveratrol can be used as molecules to conjugate to polypeptides containing variants.

[0220] Interesting and desirable molecules are also disclosed above.

[0221] Use of variants The variant can be used in therapeutic applications, particularly in the treatment of cancer.

[0222] Accordingly, the present invention relates to a method for treating cancer, comprising administering a therapeutic dose of a variant of the OB fold disclosed herein (particularly a variant of the Sac7d family protein) to a subject in need thereof.

[0223] As used herein, the terms “therapeutic dose” or “effective dose” refer to a quantity sufficient to produce a beneficial or desirable outcome, such as a clinical result, and the “effective dose” depends on the context in which it is applied. An effective dose is a quantity that produces a therapeutic improvement while minimizing side effects or adverse effects. Therapeutic improvement may include regression of tumor size in solid tumors, inhibition of metastatic spread, improvement of the patient’s quality of life, or improvement of the efficacy of combination therapy.

[0224] The variant can be administered by any method in the art.

[0225] In particular, the variant can be injected. In another embodiment, the variant can be applied topically (on the patient's skin or on the eye) as disclosed in WO 2014 / 173899. In another embodiment, the variant can be administered orally as disclosed in WO 2016 / 062874.

[0226] The variant or a polypeptide containing the variant can also be used in a diagnostic method. In particular, such a variant or polypeptide can be linked to any marker used in imaging methods known in the art. In fact, PD-L1, like HSP110 which can be found on the cell surface, is a marker for cancer cells. This is particularly interesting for detecting metastases or especially for tracking the efficacy of cancer treatment in vivo. Such a polypeptide of the invention bound to any image-detectable marker can be administered, and the presence and binding of the administered molecule can be tracked by imaging methods.

[0227] Thus, the invention also relates to a method for detecting the presence of or quantifying a subunit of a multimeric protein in a sample, comprising the following steps: a. exposing the sample to the disclosed variant under conditions such that the variant binds to the subunit but not to the fully formed multimeric protein; b. recovering the variant and / or detecting the subunit or measuring the amount of the subunit, by ELISA, fluorescence, column quantification.

[0228] The recovery in b) can be carried out by various washing or methods common in the art. The detection or quantification can be carried out by any method such as ELISA or other methods in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0229] [Figure 1]Alignment of Sac7d family proteins. [Figure 2] B11 binding to proteins in both human (upper curve) and mouse (lower curve). [Figure 3] Inhibition of PD1 and PD-L1 binding using a variant (B11) protein that binds to PD-L1. [Figure 4] Inhibition of PD1 and PD-L1 binding using a variant (B11) protein that binds to PD-L1, either alone (triangle) or as a dimer (inverted triangle). Pembrolizumab (square) and atezolizumab (round) were used as controls. [Figure 5] Mean tumor weight (mg) measured in different experimental groups at the end of the study. NF1 = anti-HSP110 variant. NF2: anti-PD-L1 variant. NF1-NF2: dimer of both variants. [Figure 6] Data analysis of chicken CD3 expression in tumors. [Figure 7] Data analysis of chicken MMD expression in tumors. [Figure 8] The number of dead and surviving embryos at the end of the experiment. [Figure 9] Mean tumor weight (mg) measured in different experimental groups 10 days after CAM treatment. Mean ± SEM of tumor weight measured for each experimental group. [Figure 10] Mean tumor weight (mg) measured in different experimental groups 10 days after CAM treatment. Mean ± SEM of tumor weight measured for each experimental group. [Figure 11] Mean tumor weight (mg) measured in different experimental groups at the end of the study. Mean tumor weight (mg) ± SEM. [Figure 12] Relative levels of chicken CD3 expression in tumors by group (±SEM). [Figure 13] Data analysis of chicken MMD expression in tumors. Relative levels of chicken MMD expression in tumors by group (±SEM). [Figure 14]From right to left, the representations of the PDL1 structure with its two immunoglobulin-like domains (Ig-like type V (aa19~aa127) and Ig-like type C2 (aa133~aa225)) in complexes with PD1 (PDB 4ZQK, A), atezolizumab (PDB 5XXY, B), avelumab (PDB 5GRJ, C), and durvalumab (PDB 5XJ4, D). [Examples]

[0230] Example 1. Characterization of a variant of Sac7d that binds to PD-L1. The efficacy of PDL1-neutralizing nanophytin was evaluated using Promega's PD1 / PDL1 blocking bioassay. This assay consists of two genetically modified cell lines provided in a thawed form: Jurkat T cells (PD-1 effector cells) expressing human PD-1 and a luciferase reporter driven by an NFAT response element, and CHO-K1 cells (PD-L1 aAPC / CHO-K1 cells) expressing human PD-L1 and a modified cell surface protein designed to activate the congeneral TCR in an antigen-independent manner. When these two cell lines are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Neutralization of the PD1 / PDL1 interaction releases the inhibitory signal, leading to TCR activation and NFAT-RE-mediated luminescence.

[0231] In short, 0.5 mL of the thawed PD-L1 aAPC / CHO-K1 cell suspension was transferred to 14.5 mL of cell recovery medium (90% Ham's F12 / 10% FBS) and mixed thoroughly by gently inverting it once or twice. 100 μL of the cell suspension was dispensed into the inner 60 wells of two 96-well white flat-bottom assay plates. 100 μL of cell recovery medium was added to each of the outer wells of the assay plate. The assay plates were then covered with lids and incubated in a 37°C, 5% CO2 incubator for 16–20 hours. After overnight incubation, the medium was removed by flicking the plates. 40 μL of the test item concentration range and 40 μL of the thawed PD-1 effector cell suspension were all prepared in assay buffer (99% RPMI1640 / 1% FBS) and immediately added to the inner wells. Subsequently, 80 μL of assay buffer was added to the outer wells, the plate was covered, and incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the plate was allowed to equilibrate to ambient temperature for 5–10 minutes. 80 μL of Bio-Glo® reagent was added to the inner 60 wells of the assay plate, and the plate was incubated at ambient temperature for 5–30 minutes. Finally, luminescence was measured, and data analysis was performed using GraphPad Prism software V6.

[0232] The Sac7d variant (B11, SEQ ID NO: 21) was found to inhibit PD1 / PD-L1 binding (Figure 3). A correlation between affinity and neutralizing efficacy was also observed (not shown). The dimer of B11 was also shown to be as effective as the control (Figure 4). B11 is also referred to as NF2 in the examples.

[0233] Furthermore, this variant was found to interact with both human and mouse PD-L1 proteins (Figure 2). This property is particularly interesting and useful for preclinical studies, as such interspecies cross-reactivity is rarely observed with PD-L1 binders. The affinity of B11 for PD-L1 protein (surface plasmon resonance) is K D = approx. 1.5×10 -8 M (Human) and KD = 3.2 × 10 -8 It is M (mouse).

[0234] Protein maturation allowed us to determine that the key amino acids necessary for binding to human PD-L1 are I8, I26, L31, M42, and L44. R24, Y29, and K33 are further required for binding to mouse PD-L1.

[0235] Example 2. Characterization of Sac7d variants bound to HSP110 The Sac7d variant (A-C2, SEQ ID NO: 27) was found to bind to HSP110 and inhibit HSP110-Stat3 binding. A-C2 was favorable for the expansion and proliferation of antitumor M1 macrophages. A-C2 is also referred to as NF1 in the examples.

[0236] Through protein maturation, it became possible to determine that the key amino acids necessary for binding to human PD-L1 are W8, R9, W21, D22, W24, Y31, H33, M40, and K44.

[0237] Example 3. In vivo experiment - Anti-HSP110 variant alone or in combination with an anti-PD-L1 variant material and method All in vivo experiments were performed using CAM technology, which is equivalent to transplanting cancer cells into the chorioalangiomolecule (CAM) of chicken embryos (see Kroiss et al, Oncogene. 2015 May 28;34(22):2846-55; Green et al, PLoS One. 2009 Aug 21;4(8):e6713).

[0238] The MDA-MB-231 human breast cancer cell line was transplanted into CAM cells of 9-day-old chicken embryos. Treatment was administered between day 10 and day 17 (1-8 days before transplantation), and then analyses were performed for toxicity and tumor growth inhibition.

[0239] In short, white Leghorn fertilized eggs were incubated at 37.5°C and 50% relative humidity for 9 days. At that point (E9), a small hole was made in the air sac through the eggshell to release the chorioalinous membrane (CAM), and 1 cm of the eggshell was placed on top of the CAM. 2 The window was opened. At least 20 eggs were used in each group (more than 20 per group depending on the embryo survival rate at 9 days of development). The inoculation material was 1 × 10 6 Individual MDA-MB-231 cells were added to the CAM of each egg.

[0240] Keytruda® (pembrolizumab) was used as the reference compound. Six experimental groups are described in Table 2.

[0241] (Table 2) Test Group TIFF0007879809000029.tif49128

[0242] Tumors became detectable on day E10. Treatments for different groups are detailed in Table 3. After each treatment, the eggs were individually examined daily for death or visible abnormalities (visual inspection).

[0243] (Table 3) Explanation of treatment groups TIFF0007879809000030.tif136146

[0244] Quantitative evaluation of tumor growth On day 18 of embryonic development, the upper part of the CAM (containing the tumor) was removed from all viable embryos with tumors, washed with PBS buffer, and then directly transferred to PFA (fixed for 48 hours). Subsequently, the tumors were carefully separated from the normal CAM tissue and weighed. A one-way ANOVA analysis with post-hoc testing was performed on the data.

[0245] Quantitative evaluation of immune cell infiltration On the 18th day, six tumor samples were collected per group to evaluate the infiltration of immune cells. Each tumor sample was excised, washed with PBS buffer, and then transferred directly into 4% PFA (fixed for 48 hours). Subsequently, genomic RNA was extracted from the fixed tumors (using a commercial kit) and analyzed by RT-qPCR using specific primers for chicken CD3 and MMD sequences.

[0246] In all qPCR runs, the expression of GAPDH was also analyzed as a reference gene expression and used to normalize the immune biomarker expression between samples. One-way ANOVA analysis with post hoc testing was performed on the data.

[0247] Quantitative evaluation of embryotoxicity Embryo viability was examined daily. The number of embryos that died at E18 was also counted, and finally, in combination with the observation of visible macroscopic abnormalities, the embryotoxicity by the treatment was evaluated.

[0248] Significance of statistical analysis For all analyses, the statistical significance between groups was made visible on the graph by the presence of asterisks with the following meanings: · Without asterisk: No statistical significance (p-value > 0.05); · * : 0.05 ≥ p-value > 0.01; · ** : 0.01 ≥ p-value > 0.001; · *** : 0.001 ≥ p-value.

[0249] result Quantitative evaluation of tumor growth Figure 5 shows the average tumor weight (mg) measured in different experimental groups at the end of the test.

[0250] Quantitative evaluation of immune cell infiltration Chicken CD3 expression in tumors Figure 6 shows the data analysis of chicken CD3 expression in tumors.

[0251] Chicken MMD expression in tumors Figure 7 shows the data analysis of chicken MMD expression in tumors.

[0252] Quantitative evaluation of embryotoxicity Figure 8 shows the number of dead and surviving embryos at the end of the experiment.

[0253] conclusion The objective of this project was to evaluate the toxicity and efficacy of anti-HSP110 (NF1, A-C2) alone or as a dimer with anti-PD-L1 (NF2, B11) against tumors originating from the MDA-MB-231 human breast cancer cell line in a CAM model. Keytruda® was used as a reference compound.

[0254] Regarding tumor growth, both the NF1 compound and the NF1-NF2 compound induced significant inhibition of tumor growth at high doses (20 mg / kg) compared to the negative control. The effect of the NF1-NF2 compound was dose-dependent, and its performance at 20 mg / kg was clearly better than at 2 mg / kg.

[0255] Regarding immune cell infiltration in tumors, an increasing trend in CD3-positive cell infiltration was observed in the presence of Keytruda, an increasing trend in MMD-positive cells was observed in the presence of the test compound, and NF1 alone showed a dose-response effect. None of these increases were statistically significant.

[0256] Finally, in both treatment with NF1 compounds and treatment with NF1-NF2 compounds, no apparent embryotoxicity was induced at any of the test doses compared to the negative control.

[0257] Example 4. Efficacy testing of NF-B11 compound against breast cancer tumors originating from the MDA-MB-231 cell line. The CAM model described in Example 3 was used.

[0258] treatment On day 10 (E10), tumors became detectable. Subsequently, they were treated every two days for 10 days (E11, E13, E15, E17) by injecting 100 μL of medium (PBS), a reference compound (tecentriq / atezolizumab), and NF-B11 at two different doses (see Table 4 for concentrations) onto the tumors.

[0259] (Table 4) Experiment TIFF0007879809000031.tif37143

[0260] result Tumor growth Table 5 shows the mean tumor weights for different experimental groups at E18.

[0261] (Table 5) Mean tumor weight (mg), SD, SEM, and p-value for each experimental group TIFF0007879809000032.tif37143

[0262] Figure 9 shows the mean tumor weight (mg) measured in different experimental groups 10 days after CAM treatment.

[0263] toxicity Table 6 shows the number of dead and surviving embryos 10 days after treatment in different experimental groups.

[0264] (Table 6) Number of dead and surviving embryos in each experimental group. TIFF0007879809000033.tif35146

[0265] Regarding the results, Tecentriq did not show a statistically significant effect compared to the negative control, but NF-B11 treatment showed a 47% reduction in tumor weight when administered at 50 times the IC50. At 10 times the IC50, the tumor reduction was around 24%, but this was not statistically significant compared to the negative control.

[0266] Example 5. Efficacy test of NF-B11 compound against breast cancer tumors originating from the MDA-MB-231 cell line. treatment On day 10 (E10), tumors became detectable. These were then treated every two days (E11, E13, E15, E17) for 10 days by dropping 100 μL of medium (PBS), a reference compound (Tecentriq), and NF-B11 or NF-B11-B11 compound (see Table 7 for concentrations).

[0267] (Table 7) Test Group TIFF0007879809000034.tif38145

[0268] Tumor growth analysis On day 18 (E18), the upper part of the CAM was removed, washed with PBS, and then directly transferred to PFA (fixed for 48 hours). The tumor was then carefully separated from the normal CAM tissue. The tumor was then weighed. One-way ANOVA analysis with post-hoc tests was performed on these data.

[0269] Statistical significance Regarding tumor weight and metastasis, statistically significant differences between groups are indicated on the graph by the presence of asterisks, which have the following significance: - No asterisk: No statistically significant difference (p-value > 0.05); -1 star ( * ): 0.05 ≥ p-value > 0.01; -2 stars ( ** ): 0.01 ≥ p-value > 0.001; -3 stars ( *** ): 0.001 ≥ p-value.

[0270] toxicity Acute toxicity is assessed by the number of dead embryos 10 days after treatment.

[0271] result Tumor growth Figure 10 shows the mean tumor weight (mg) measured in different experimental groups 10 days after CAM treatment.

[0272] toxicity Table 8 shows the number of dead and surviving embryos 10 days after treatment in different experimental groups.

[0273] (Table 8) Number of dead and surviving embryos in each experimental group TIFF0007879809000035.tif40146

[0274] This study investigated the efficacy of anti-PD-L1 (NF-B11) compounds against tumors originating from MDA-MB-231 cells.

[0275] In this study, Tecentriq and NF-B11-B11 at 10 times the IC50 showed a significant effect on tumor weight (16-17% reduction).

[0276] Regarding toxicity, the rate of embryo death was high in the NF-B11-B11 treatment group at 10 times the IC50, but low in the NF-B11-B11 treatment group at 100 times the IC50, indicating no specific toxicity from the treatment (at the test dose).

[0277] Example 6. Evaluation of in vivo toxicity and efficacy of two compounds in tumors derived from the MDA-MB-231 human breast cancer cell line in a CAM model.

[0278] (Table 9) Compounds and Concentrations TIFF0007879809000036.tif38128NF1: Anti-HSP110 Sac7d variant (A-C2) NF2: Anti-PD-L1 Sac7d variant (B11) NF3: A variant of Sac7d that binds to EGFR.

[0279] treatment Tumors became detectable on day E10. Treatments for different groups are detailed in Table 10. After each treatment, the eggs were individually examined daily for death or visible abnormalities (visual inspection).

[0280] (Table 10) Explanation of treatment groups TIFF0007879809000037.tif96150

[0281] result Quantitative evaluation of tumor growth Figure 11 shows the mean tumor weight (mg) measured in different experimental groups at the end of the study. Mean tumor weight (mg) ± SEM.

[0282] Quantitative evaluation of immune cell infiltration Chicken CD3 expression in tumors Figure 12 shows the data analysis of chicken CD3 expression in tumors.

[0283] Chicken MMD expression in tumors Figure 13 shows the data analysis of chicken MMD expression in tumors.

[0284] Quantitative evaluation of embryotoxicity Table 11 shows the number of dead and surviving embryos at the end of the experiment.

[0285] (Table 11) Number of dead and surviving embryos by group TIFF0007879809000038.tif76138

[0286] Firstly, regarding tumor growth, both the NF2 compound and the NF1-NF3 compound did not affect tumor growth at low doses (2 mg / kg) compared to the negative control; at high doses (20 mg / kg), the inventors observed a tendency toward tumor growth regression with both compounds, but tumor growth inhibition was significant only with NF1-NF3.

[0287] Secondly, regarding tumor immune cell infiltration, the inventors observed an increasing trend in CD3-positive cell infiltration in the presence of Keytruda; an increasing trend in MMD-positive cells in the presence of the Sac7d variant; and a dose-effect relationship with NF2 alone. None of these increases were statistically significant.

[0288] Finally, neither treatment with the NF2 compound nor treatment with the NF1-NF3 compound induced significant embryotoxicity at any of the test doses compared to the negative control.

[0289] Example 7. Further elements Nanophytins were derived from ribosome display of human PDL1 using a 200 aa-length recombinant ECD from the R&D system (ref 156-B7), consisting of two immunoglobulin-like domains fused to an IgG1 Fc fragment. Numerous distinct nanophytins were isolated by specific signals in ELISA against human PDL1 immobilized by physical adsorption. These proteins could be classified into sequence clusters based on the homology of their variable domains. Next, candidates were screened for their ability to bind to their target in a biolayer interferometry setup (Octet RED96). In this setup, the target was immobilized on a protein A biosensor to ensure native and directional presentation of the target. In this setup, 60% of the candidates retained the ability to bind to human PDL1, and only 30% (16% of the total) cross-reacted with mouse PDL1. All cross-reactive nanophytins were confined to a single cluster, highlighting the differential drug potential of human and mouse PDL1.

[0290] The ability of nanophytin derived from this cluster to neutralize the interaction between PDL1 and PD1 was evaluated in a competitive ELISA and compared with candidates from other clusters. The competitive ELISA was performed using the following procedure: Human PD1 was immobilized on a NUNC maxisorp plate (100 μL, 1 μg / mL, 1 hour). The wells were blocked for 1 hour with TBS-BSA (20 mM Tris, 150 mM NaCl, 0.5% BSA, pH 7.4) (300 μL / well). Between each of the following steps, each well was washed three times with 300 μL of TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4). Nanophytin was mixed with biotinylated human PDL1 (100 nM) in TBS-T (final 100 μL) and applied to the wells. Subsequently, color development was performed by adding 100 μL of 1:10000 diluted streptavidin HRP conjugate to TBS-T for 1 hour, followed by the addition of 100 μL of 1 mg / ml o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich) solution in a color development buffer (0.05 M citrate, 0.05% hydrogen peroxide). The absorbance at 450 nm was measured using a Varioskan ELISA plate reader (Thermo Scientific).

[0291] Two nanophytin candidates derived from the aforementioned clusters were able to neutralize the interaction between human PDL1 and human PD1, and the neutralizing ability appeared to correlate well with their affinity, with the lowest affinity providing the highest neutralization efficiency. It was noteworthy that similar affinity did not directly translate to similar neutralizing ability between different clusters.

[0292] Two selected nanophytins (SEQ ID NO: 38 and SEQ ID NO: 59) were shown to target the Ig-like V domain of the PDL1 ECD on an epitope adjacent to or overlapping with the interaction region between human PDL1 and human PD1, and shared on mouse PDL1. These variants showed maximum affinity on human PDL1, with SEQ ID NO: 38 / SEQ ID NO: 41 showing the highest level of cross-reactivity on human and mouse PDL1, while SEQ ID NO: 68 / SEQ ID NO: 71 exhibited the best potency.

[0293] Modifications to D16, N36, and M56 (substitution with E, Q, and L, respectively) did not alter their affinity or neutralizing ability.

[0294] Modification of the mutant amino acids revealed that I8, I26, L31, M42, and L44 (and R24, Y29, and K33 in the case of mouse binding) are important for maintaining binding and neutralization to one type of nanophytin, while M8, L26, L31, L42, and F44 (and T24, A29, and R33 in the case of mouse binding) are important for maintaining binding and neutralization to the other type of nanophytin.

[0295] Example 8. Anti-HSP110 binder Furthermore, nanophytin was created as an Hsp110 binding agent. In short, a screening was performed using the Sac7d variant, and the identified and sequenced proteins were classified into four groups based on their sequence and signature. Within these groups, 20 different nanophytin binding site components were identified. The binding levels of these 20 nanophytin candidates were compared in ELISA at a constant concentration (1 μM).

[0296] The variant corresponding to SEQ ID NO: 80 (or SEQ ID NO: 27, A-C2) was determined to exhibit the best affinity and neutralizing ability. The amino acid substitutions described above indicated that W8, D22, W24 (which is conserved with respect to the wild-type protein), H33, and K44 are important for maintaining binding and affinity.

[0297] The affinity for this protein was less than 1 nM, indicating that it cannot enter eukaryotic cells.

[0298] The immunotherapy efficacy of the combination of A-C2 and anti-PD-L1 nanophytin was demonstrated in vivo in a chicken chorioalulia (CAM) model (see also Example 3 above).

[0299] Overall, these results demonstrate the therapeutic interest of A-C2 in cancer itself, albeit lacking internalization, through effects thought to involve macrophages, as well as its complementarity with proteins that inhibit the binding of PD-L1 and PD1 (particularly nanophytin anti-PD-L1).

Claims

1. A polypeptide comprising a variant of a member of the Sac7d family that binds to human and mouse PD-L1 and inhibits the binding of PD-L1 to PD1, wherein the variant is i) VKVKFVIGGEEKEVXTSKIKTVRRIGKYVLFKYDDXGKTGMGLVSEKDAPKELLDXLARAEREK (SEQ ID NO: 35), ii) SEQ ID NO: 35, amino acids 1-57, iii) TKVKFVIGGEEKEVXISKIKTVRRIGKYILFKYDDXGKTGMGLVSEKDAPKELLEKLK (SEQ ID NO: 44); iv) VKVKFVMGGEEKEVXTSKIRYVTRLGKAVLFRYDDXGKTGLGFVQEKDAPKELLDXLARAEREK (SEQ ID NO: 56); v) SEQ ID NO: 56 amino acids 1-57, and vi) TKVKFVMGGEEKEVXISKIRYVTRLGKAILFRYDDXGKTGLGFVQEKDAPKELLEKLK (SEQ ID NO: 65) A polypeptide selected from the group consisting of, where X at position 15 is selected from D or E, X at position 36 is selected from N or Q, and X at position 56 is selected from M or L.

2. The polypeptide according to claim 1, selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, and amino acids 1 to 57 of these sequences.

3. The polypeptide according to claim 1, selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 50, and amino acids 1 to 54 of these sequences.

4. The polypeptide according to claim 1, selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 62, and amino acids 1 to 57 of these sequences.

5. The polypeptide according to claim 1, selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 71, and amino acids 1 to 54 of these sequences.

6. The polypeptide according to any one of claims 1 to 5, wherein the variant of the member of the Sac7d family that binds to human and mouse PD-L1 is conjugated to an organic molecule.

7. The polypeptide according to any one of claims 1 to 5, wherein the variant of a member of the Sac7d family that binds to human and mouse PD-L1 is conjugated to (i) another variant of a protein of the Sac7d family and (ii) another polypeptide selected from an antibody.

8. The polypeptide according to claim 7, wherein the other polypeptide is another variant of the Sac7d family protein.

9. The polypeptide according to claim 8, wherein the other variant is a variant of the Sac7d family that binds to HSP110 or EGFR.

10. The polypeptide according to claim 9, wherein the other variant comprises sequence SEQ ID NO: 83 or SEQ ID NO: 80, or amino acids 1 to 57 of these sequences.

11. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 5 and 7 to 10.

12. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 10 or a nucleic acid according to claim 11, and a pharmaceutically acceptable carrier.

13. A method for producing a polypeptide according to any one of claims 1 to 5 and 7 to 10, a. A step of culturing a cell culture, wherein the cells are transformed with the nucleic acid described in claim 11, and b. The step of recovering the polypeptide. A method comprising the steps of:

14. A pharmaceutical product comprising the polypeptide described in any one of claims 1 to 10.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 10 for use in the treatment of cancer.

16. The pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high-frequency microsatellite instability cancer, bladder cancer, breast cancer, small cell lung cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

17. A pharmaceutical composition according to claim 15 or 16, to be used in combination with chemotherapy or CAR-T cell therapy.

18. A composition comprising a polypeptide according to any one of claims 1 to 10 and a chemotherapeutic agent or CAR-T cells for use simultaneously, separately, or sequentially (gradually) in the treatment of cancer.

19. The composition according to claim 18, wherein the cancer is selected from the group consisting of non-small cell lung cancer, urothelial carcinoma, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, primary mediastinal B-cell lymphoma, classical Hodgkin lymphoma, melanoma, Merkel cell carcinoma, cervical cancer, high-frequency microsatellite instability cancer, bladder cancer, breast cancer, small cell lung cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

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