Multispecific molecules

Abstract

The present invention relates to proteins with multispecific binding ability obtained by fusing OB-fold variants (particularly those derived from the Sac7d family) to the light or heavy chain of an antibody.

Description

[Technical Field]

[0001] This invention belongs to the field of molecular biology and, in particular, relates to the development of novel molecules and conjugates having multiple binding specificities. [Background technology]

[0002] Introduction Bispecific monoclonal antibodies (Mab) are artificial proteins that can simultaneously bind to two different antigens. They can be manufactured in multiple structural forms. They are generally used for cancer immunotherapy and drug delivery.

[0003] The following different types of bispecific antibodies exist: - Bispecific antibodies that maintain the complete IgG structure. These benefit from Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement binding, and FcRn-mediated recycling (responsible for a long half-life). They contain two Fab arms and one Fc region, with the two Fab sites binding to different antigens. Generally, each heavy and light chain pair is derived from a unique mAb. These bispecific antibodies are often manufactured using the quadroma or hybrid hybridoma method. - Fc-deletion antibodies. These include chemically linked Fabs consisting only of the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFv), or fusion proteins that mimic the variable domains of two antibodies.

[0004] Fully IgG bispecific antibodies can be further distinguished depending on their engineering process, which results in either symmetric or asymmetric molecules, each presenting different challenges.

[0005] Asymmetric bispecific antibodies are the result of enzymatically assisted in vitro chain swapping or fusion of different hybridomas (known as quadromas). However, these techniques produce the desired bispecific molecules in a mixture with high concentrations of undesirable byproducts that must be separated. As an example, in quadroma techniques, random pairing of the heavy and light chains of two antibodies expressed in a single cell theoretically yields 16 different combinations (10 different molecules), of which only one is bispecific, while the remaining pair formations result in non-functional or monospecific molecules. Such a process yields a statistic (and recovery rate) of 12.5% ​​of bispecific molecules in solution.

[0006] Next, the main challenge lies in the ability to drive the precise pairing of heavy and light chains. This is evaluated by techniques such as Knob-into-hole for heavy chains and Crossmab for light chains. Implementing these techniques makes it possible to obtain a significant reduction in the number of byproducts during the manufacturing process, but this achievement is only possible through the insertion of numerous point mutations that support the precise association of heavy and light chains, and is not without causal relationship with the ability of the molecule to be further developed. Specifically, such antibodies may be immunogenic, with the Fc region causing harmful downstream immune responses.

[0007] Symmetrical bispecific antibodies, on the other hand, rely on the fusion of additional ScFv domains (tandem Fab-IgG, DVD-IG, CODV-Ig, etc.) to provide further specificity to the primary IgG. A significant challenge to consider here is the reliance on the linker that ligates the ScFv to IgG, and the intrinsic stability of the ligated ScFv, which can affect manufacturability and production. This is especially true for bispecific antibodies and becomes even more challenging when considering triplicate or quadruplicate antibodies.

[0008] Bispecific molecules, and in particular antibodies, can be used whenever it is necessary to target multiple targets on the same pathway, parallel pathways, or different pathways. Specifically, such bispecific antibodies have been developed in cancer immunotherapy and bind to both tumor cells and cytotoxic cells. Bispecific antibodies also possess high cytotoxicity and bind to antigens that are relatively weakly expressed. Furthermore, targeting two or more molecules can be useful in avoiding the modulation of parallel pathways and preventing resistance to treatment.

[0009] WO 2012 / 009705 lists afitin (based on Sac7d from hyperthermia archaea) as an alternative scaffold for a complex containing one or more modular recognition domains. However, this document itself does not provide any information about the properties of so-called afitin, specifically its specific structure, nor does it describe any of these properties. Furthermore, this document does not actually demonstrate that any of the complexes fold properly and maintain their activity. In summary, the teachings in this document are incomplete and uncertain.

[0010] Yu et al (MAbs. 2014;6(6):1598-607 (Non-Patent Literature 1)) D2 discloses adalimumab (a humanized antibody that binds to TNFa) fused to the IL6-binding affibody ZIL6. The affibody molecule is an antibody mimetic consisting of a small protein based on a 3-helix bundle domain engineered to bind to the target protein with high affinity, and is distinct from the OB-fold domain disclosed herein. This document does not describe any information relating to yield.

[0011] Brack et al (Mol Cancer Ther. 2014 Aug;13(8):2030-9 (Non-Patent Literature 2)) disclose a bispecific Her2 targeting fusion protein comprising the anti-Her2 antibody pertuzumab and a FynSH3-derived binding protein, which is a 7 kDa globular protein derived from the SH3 domain of human Fyn kinase.

[0012] Jarviluoma et al (PLoS One. 2012;7(7):e40331 (Non-Patent Literature 3)) disclose neffin, a single-domain antibody fragment (Lama Ig heavy chain variable domain, VHH) fused to a 57-amino acid SH3 domain engineered to bind to multiple proteins of interest (see abstract). The single-chain neffin protein disclosed in this document should not be confused with modified antibodies containing two heavy and light chains.

[0013] Spangler et al (J Mol Biol. 2012 Sep 28;422(4):532-44 (Non-Patent Literature 4)) disclose a cetuximab-based anti-EGFR antibody fused to an engineered EGFR-binding variant of the type 10 domain of human fibronectin. These complexes are different from the complexes disclosed herein.

[0014] WO 2008 / 100470 (Patent Document 1) discloses an Ig fusion protein but does not describe the OB folded protein and variants disclosed herein, nor does it describe their use in the production of modified antibodies.

[0015] Thus, multispecific molecules that can be easily produced in excellent yield and are expected to be as stable as antibodies are still under development.

[0016] The applicant proposes generating such molecules by creating novel artificial proteins containing an antibody (a conventional antibody or a bispecific antibody) in which an OB-fold domain is located at at least one N-terminus or C-terminus of the antibody's heavy or light chain. The OB-fold domain is preferably fused or linked to the antibody chain by the following genetic manipulation: the sequence encoding the domain is genetically fused to the 5' and / or 3' ends of the antibody's heavy and / or light chain sequences, and this modified sequence is introduced into a suitable cell line or bacterial line to generate the artificial protein. Note that several amino acids (linkers) may be added between the antibody and the OB-fold domain.

[0017] The examples demonstrate that polypeptides of the present disclosure can be obtained in higher or at least similar or equivalent yields compared to antibodies without fused OB domains. The examples demonstrate that such effects can be obtained with various constructs. This indicates that the antibodies and OB folded domains (particularly variants of the Sac7d family of proteins) retain their functionality and thus retain their structure. It is thought and assumed that the OB folded domains, when selected particularly as proteins of the Sac7d family, can stabilize the antibody chain during the production process, thereby enabling improved yields. Thus, the effects observed with the various complexes in the examples can be generalized to all other antibodies and OB folded variants having the same structure. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] WO 2008 / 100470 [Non-patent literature]

[0019] [Non-Patent Document 1] MAbs. 2014;6(6):1598-607

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0020] The present invention thus relates to a polypeptide comprising a modified antibody. The antibody is preferably modified by the fusion of at least one variant of an OB-fold domain to at least one of the heavy or light chains of an immunoglobulin monomer at the N-terminus or C-terminus of the heavy or light chain.

[0021] The fusion is preferably made by genetic engineering, although chemical ligation of the OB-fold variant and the heavy or light chain of the antibody is also envisaged.

[0022] Preferably, the variant contains 5 to 20 mutated residues at the binding site of the OB-fold domain to its natural ligand.

[0023] The OB-fold domain and the antibody may bind to different targets or different epitopes of the same target, whereby the proteins disclosed herein will be able to bind to these multiple targets or multiple epitopes of a given target.

[0024] As shown in Figure 1 (which illustrates only symmetric molecules), two or more variants of an OB-fold domain antibody may bind to the light or heavy chain of the antibody. It is preferable that the protein of the present invention is symmetric (i.e., composed of the same heterodimer), i.e., that the same OB-fold variant is fused to two light or heavy chains of the protein. However, embodiments also include those in which only one OB-fold variant is bound to one of the heavy or light chains, and those in which one OB-fold variant is bound to one light or heavy chain of the protein, and another (different) OB-fold variant is bound to the other light or heavy chain. Thus, multiple combinations are possible, and Figure 1 is not exhaustive.

[0025] Specifically, as shown, the OB-fold variant is fused to two heavy or light chains of the antibody. The OB-fold variant may be the same or different on each of the heavy or light chains. In another embodiment, the OB-fold variant is fused to at least one heavy chain and at least one light chain of the antibody. The OB-fold variant may be the same or different on the heavy and light chains.

[0026] In another embodiment, two OB-fold (same or different) variants are fused to the same light or heavy chain (at the N-terminus and C-terminus of the chain).

[0027] A protein may contain one OB-fold variant. It may contain two OB-fold variants (preferably these are identical and symmetrical with respect to the protein). It may contain three OB-fold variants. It may contain four OB-fold variants (preferably two pairs of OB-fold variants and the protein is symmetrical). It may contain five OB-fold variants. It may contain six OB-fold variants (preferably three pairs of OB-fold variants and the protein is symmetrical). It may contain seven OB-fold variants. It may contain eight OB-fold variants (preferably four pairs of OB-fold variants; in this case, the protein is symmetrical). It is worth noting that a symmetrical protein is a protein made from the same heavy and light chains.

[0028] The present invention a. The sequence encoding the antibody heavy chain, which is fused at its 3' end with the sequence encoding a variant of the OB folded protein. b. The sequence encoding the antibody heavy chain, which is fused at its 5' end with the sequence encoding the variant of the OB folded protein. c. The sequence encoding the antibody light chain, which is fused at its 3' end with the sequence encoding a variant of the OB folded protein. d. The sequence encoding the antibody light chain, which is fused at its 5' end with the sequence encoding a variant of the OB folded protein. This also relates to gene constructs containing DNA sequences selected from a group consisting of the following:

[0029] These gene sequences, when introduced into a suitable host cell along with their complementary antibody gene sequences (a or b, the light chain encoding sequence; c or d, the heavy chain encoding sequence), enable the production of the proteins disclosed herein. The present invention also relates to vectors comprising such gene constructs, as well as host cells (particularly eukaryotic cells) whose genomes contain such gene constructs of the present invention (preferably together with complementary sequences).

[0030] The present invention also, a. A step of culturing a cell culture, wherein the cells are transformed with the described gene construct and its complementary antibody sequence, and b. A step to recover the polypeptide produced as a result. The present invention also relates to a method for producing polypeptides, which includes a step comprising the steps described above.

[0031] Such cultivation is carried out under conditions that allow for the expression of gene constructs and the construction of proteins expressed from these gene constructs. This method is particularly suitable when polypeptides are secreted into the culture medium.

[0032] The present invention also, (a) A step of culturing cells that have been transduced by the gene construct described and its complementary antibody sequence; (b) the process of collecting cells; and (c) A step of destroying cells to obtain a crude extract containing the polypeptide described. The present invention also relates to a method for producing polypeptides, which includes a step comprising the steps described above.

[0033] The present invention also includes a method for producing a gene construct for producing the described polypeptide, comprising the steps of genetically fusing a sequence encoding a variant of the OB fold domain to at least the 5' or 3' end of the heavy or light chain of an antibody, and recovering the resulting gene construct.

[0034] The present invention also relates to a complex comprising polypeptides disclosed herein, which are bound to at least one target by an OB-fold variant or antibody-binding site. Such a target is the antigen of the antibody or the target to which the OB-fold variant binds.

[0035] The present invention is particularly interesting because it makes it possible to obtain bispecific or multispecific molecules by a single and very simple method (a simple gene modification consisting of the fusion of an antibody sequence (light chain or heavy chain coding sequence) and an OB fold coding sequence).

[0036] In a preferred embodiment, this method ensures that 100% of the proteins recovered after production in cells transformed with a suitable gene vector are of the same type, thereby solving the problem of purifying bispecific antibodies recalled above, where the bispecificity statistic (and recovery rate) is generally about 12.5% ​​in solution. Furthermore, the method disclosed herein makes it possible to obtain multispecificity molecules with a degree of diversity comparable to that of antibodies (chimeric, human, mouse-derived, rat-derived, or derived from any idiotype). It should also be noted that, since the overall structure of the antibody is not modified, the molecule retains all the characteristics and properties of the antibody (including any effects by non-Fab fragments, such as those by the Fc fragment). It is also quite remarkable that the molecule disclosed herein can bind to both the antibody and the OB folded domain binding sites, despite the modification of the antibody. Therefore, such bispecificity binding ability suggests that the proper folding of the polypeptide fragment molecule (antibody portion and OB folded domain portion) is maintained. Finally, it was also remarkable that the yield of the molecule was maintained (compared to the antibody production yield), and even improved. [Invention 1001] A polypeptide comprising a modified antibody, wherein at least one variant of a Sac7d family protein is fused to at least one of the heavy chain or light chain of the antibody, and the variant contains 4 to 20 or 5 to 20 mutant residues at the binding site of the Sac7d family protein. [Invention 1002] The polypeptide of the present invention 1001, wherein the mutant amino acids of the variant are selected from the group consisting of 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 with reference to SEQ ID NO:1. [Invention 1003] The polypeptide of the present invention 1001 or 1002, wherein the variant contains 7 to 14 mutant amino acids selected from the group of amino acids corresponding to K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46 of Sac7d. [Invention 1004] A polypeptide according to any one of the invention 1001 to 1003, wherein at least one variant of the Sac7d family protein is fused to two heavy chains or two light chains of the antibody. [Invention 1005] A polypeptide according to any one of the present invention 1001 to 1004, wherein at least one variant of the Sac7d family protein is fused to one heavy chain of the antibody, and at least one variant of the Sac7d family protein is fused to one light chain of the antibody. [Invention 1006] A polypeptide according to any one of the present invention 1001 to 1004, wherein at least one heavy chain or at least one light chain of the antibody is fused with two variants of the Sac7d family protein. [Invention 1007] A polypeptide according to any one of the invention 1001 to 1004, wherein at least two different variants of the Sac7d family protein are fused to the heavy chain and / or light chain of the antibody. [Invention 1008] A polypeptide according to any one of the invention 1001 to 1007, wherein eight variants of the Sac7d family protein are fused to the heavy and light chains of the antibody. [Invention 1009] A polypeptide according to any of the invention 1001 to 1008, wherein the antibody is a therapeutic antibody. [Invention 1010] A polypeptide according to any of the invention 1001 to 1009 that binds to a protein selected from the group consisting of EGFR, VEGFR2, TfR, Her2, mesothelin, EpCam, CD38, CD3, CD7, PD1, PD-L1, CTLA4, OX40, VEGF, TNFα, IL17, IL4, IL13, IL23, IL12, MAdCam, and a4b7. [Invention 1011] A polypeptide according to any of the present invention 1001 to 1010, wherein a variant of the Sac7d family protein binds to IL17. [Invention 1012] The polypeptide of the present invention 1011, wherein a variant of the Sac7d family protein comprises sequence SEQ ID NO:35 or SEQ ID NO:36. [Invention 1013] A polypeptide of the present invention 1011 or 1012, comprising SEQ ID NO:37 or SEQ ID NO:38. [Invention 1014] A polypeptide according to any of the invention 1011 to 1013, wherein the antibody binds to a protein selected from the group consisting of TNFα, IL23, IL12, IL4, IL13, IL31 or its receptor, and tumor-specific antigens, particularly Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4. [Invention 1015] A polypeptide according to any of the present invention 1001 to 1010, wherein a variant of the Sac7d family protein binds to a subunit of TNFα, and preferably does not bind to the subunit of the protein if the subunit protein is contained in a fully formed polymer protein in its native state. [Invention 1016] A polypeptide according to the present invention 1015, comprising sequence SEQ ID NO:39 or SEQ ID NO:40. [Invention 1017] A polypeptide of the present invention 1015 or 1016, comprising sequence SEQ ID NO:41. [Invention 1018] A polypeptide according to any of the present invention 1015 to 1017, wherein the antibody binds to a protein selected from the group consisting of IL17, CD20, IL24, IL12, IL4, IL13, IL31 or its receptor, and tumor-specific antigens, particularly Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4. [Invention 1019] a. A sequence encoding the antibody heavy chain, which is fused at its 3' end with a sequence encoding a variant of a Sac7d family protein. b. A sequence encoding the antibody heavy chain, which is fused at its 5' end with a sequence encoding a variant of the Sac7d family of proteins. c. A sequence encoding the antibody light chain, which is fused at its 3' end with a sequence encoding a variant of a protein in the Sac7d family. d. The sequence encoding the antibody light chain, which is fused at its 5' end with the sequence encoding a variant of a protein in the Sac7d family. A gene construct containing a DNA sequence selected from the group consisting of the following. [Invention 1020] A vector comprising the gene construct of Invention 1019. [Invention 1021] A host cell containing the gene construct of Invention 1019 in its genome. [Invention 1022] A method for producing any polypeptide according to the present invention 1001 to 1018, a. A step of culturing a cell culture in which cells have been transformed with the gene construct of the present invention 1019 and a gene construct encoding a complementary antibody chain thereof, and b. Steps for recovering polypeptides The method comprising the steps of [Invention 1023] A method for maintaining or improving the yield of modified antibodies, a. A step of culturing a cell culture in which cells have been transformed with the gene construct of the present invention 1019 and a gene construct encoding a complementary antibody chain thereof, and b. A step of recovering the modified antibody generated after the expression of the gene construct in step a. The process includes the following steps: The method wherein the modified antibody recovered in step b comprises at least one variant of a protein of the Sac7d family, the variant comprises 5 to 20 mutant residues at the protein's binding site, and the yield of the recovered modified antibody is equal to or higher than the yield of the unmodified antibody produced under the same conditions. [Invention 1024] A polypeptide comprising a variant of a Sac7d family protein, wherein the variant contains 4 to 22 mutant amino acids at the binding site of the Sac7d family protein and binds to IL17. [Invention 1025] The polypeptide of the present invention 1024, wherein the mutated amino acid corresponds to an amino acid selected from the group consisting of Sac7d 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. [Invention 1026] The polypeptide of the present invention 1024 or 1025, wherein the Sac7d variant contains 4 to 17 mutant amino acids selected from the group corresponding to K7, Y8, K9, E11, K21, K22, W24, V26, M29, S31, T33, D35, T40, G41, R42, A44, and S46 of Sac7d. [Invention 1027] A polypeptide according to any of invention 1024 to 1026, comprising sequence SEQ ID NO:35 or SEQ ID NO:36. [Invention 1028] A polypeptide according to any of invention 1024 to 1027, comprising sequence SEQ ID NO:37 or SEQ ID NO:38. [Invention 1029] A polypeptide according to any of the Invention 1024-1027, comprising sequence SEQ ID NO:37 or SEQ ID NO:38, wherein 1 to 8 amino acids selected from the group consisting of V7, M8, F9, K11, Q26, L29, E35, D41, F44, and P46 are replaced with other amino acids. [Invention 1030] A polypeptide according to any of the invention 1024 to 1029, wherein a variant of the Sac7d family protein that binds to IL17 is linked to or fused with another protein or polypeptide. [Invention 1031] The polypeptide of the present invention 1030, wherein the other protein or polypeptide comprises another variant of the Sac7d family of proteins. [Invention 1032] The polypeptide of the present invention 1030, wherein the other protein or polypeptide is an antibody, preferably bound to TNFα or Her2 / neu. [Invention 1033] A polypeptide according to any of invention 1024 to 1032, conjugated to an organic molecule. [Invention 1034] A gene construct comprising a DNA sequence encoding any polypeptide according to invention 1024 to 1032. [Invention 1035] A vector comprising the gene construct of the present invention 1034. [Invention 1036] A host cell containing the gene construct of the present invention 1034 in its genome. [Invention 1037] A method for producing any polypeptide according to invention 1024 to 1032, a. A step of culturing a cell culture in which cells have been transformed by the gene construct of the present invention 1034, and b. Steps for recovering polypeptides The method comprising the steps of [Invention 1038] A polypeptide according to any of invention 1024 to 1032 as a pharmaceutical. [Modes for carrying out the invention]

[0037] Detailed description of the invention OB Fold Domain As shown above, the OB fold domain can be manipulated into variants that bind to specific targets by introducing mutations within its binding site.

[0038] In the context of this application, the term “OB-folded domain” (or “OB-folded protein”) means a naturally occurring OB-folded protein, but also means a domain that has an OB fold and is isolated from more complex proteins. These OB-folded domains are specifically described in detail in WO 2007 / 139397 and WO 2008 / 068637. The term also includes polypeptides that can be obtained by genetic engineering by fusing an OB-folded protein or a domain having an OB fold at the N-terminal or C-terminal position to a protein or domain of interest, such as a tag that allows for better purification.

[0039] In the context of the present invention, however, if such polypeptides contain other sequences not present within the OB-fold domain, it is preferable to use only domains that have the topology of an OB-fold and do not contain a full-length protein. In fact, it is preferable to use the smallest possible protein, and preferably, the variant of the OB-fold domain used in the context of the present invention contains a maximum of 300 amino acids, preferably a maximum of 200 amino acids, preferably a maximum of 175 amino acids, more preferably a maximum of 150 amino acids, and more preferably a maximum of 100 amino acids. In one particular embodiment, it contains a maximum of 80 or a maximum of 70 amino acids.

[0040] OB fold domain binding site OB-fold proteins are known in the art. They are specifically described in the literature cited above and also in Arcus (Curr Opin Struct Biol. 2002 Dec; 12(6):794-801). OB-folds take the form of cylinders having five beta (β) sheets. Most OB-fold proteins use the same binding interface to their native ligands, which may be oligosaccharides, oligonucleotides, proteins, metal ions, or catalytic substrates. This binding interface mainly consists of residues located within the β-sheets. Certain residues located within the loops may also be involved in the binding of OB-fold proteins to their native ligands. Accordingly, applications WO 2007 / 139397 and WO 2008 / 068637 and the Arcus literature (2002, hereafter) describe the OB-fold protein domains for binding to their native ligands.

[0041] Specifically, reference WO 2008 / 068637 accurately describes a method for identifying the binding domain of the OB folded protein.

[0042] By 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), it is possible to identify the location of the binding domain, specifically the amino acids that can be modified, by superimposing multiple sequences and 3D structures of proteins containing the OB fold domain. Referring to the sequence of Sac7d (SEQ ID NO:1), these are residues V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50, and P51.

[0043] The binding domains of other OB-fold proteins can be identified as described in WO 2008 / 068637. This application demonstrates that it is possible to perform 3D structural superposition of OB-fold proteins or domains (in this application, 10 domains including Sac7d are used) using the DALI website (http: / / www.ebi.ac.uk / dali / interactive.html) (Holm and Sander, 1998, Nucleic Acids Res 26, 316-319). Therefore, for any OB-fold protein (or any OB-fold domain), it is easy to identify the amino acid contained in the binding site that corresponds to the aforementioned Sac7d amino acid. Thus, by providing an amino acid that can be mutated in one of these proteins, it becomes possible to identify the corresponding amino acid in any other OB-fold domain.

[0044] It is also possible to delete certain amino acids from the OB fold scaffold. Similarly, referring to this Sac7d sequence, the residues that can be deleted are A59, R60, A61, E62, R63, E64, and / or K66.

[0045] The teachings of WO 2008 / 068637 indicate that, optionally, amino acids can be inserted into loops of OB-fold proteins, specifically proteins of the Sac7d family; specifically, loop 3 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example, the region of residues 25-30 of Sac7d, preferably between residues 27 and 28, can be inserted; loop 4 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example, the region of residues 35-40 of Sac7d, preferably between residues 37 and 38, can be inserted; and loop 1 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example, the region of residues 7-12 of Sac7d, preferably between residues 9 and 10, can be inserted.

[0046] Obtaining variants of the OB fold domain WO 2007 / 139397 describes the use of a library of OB-fold proteins in which the OB domain has been modified by introducing mutations into the OB domain for binding to the OB-fold protein and its native ligand. Specifically, as predicted herein, the modified OB-fold domains include a) at least one modified amino acid residue in the β-strand of the binding surface of the OB-fold domain compared to the naturally occurring OB-fold domain, or b) at least one modified amino acid residue in the β-strand of the binding surface of the OB-fold domain and at least one modified amino acid residue in the strand of the loop region of the OB-fold domain, or c) at least one modified amino acid residue in the strand of the loop region of the OB-fold domain. Generally, the modified OB-fold domains have altered binding characteristics compared to the naturally occurring OB-fold domains.

[0047] WO 2008 / 068637 describes the use of a library based on the Sac7d protein to obtain ligands with affinity to a target of interest. The method described in WO 2008 / 068637 involves constructing a combinatorial library containing multiple DNA molecules that are all identical in sequence except for the presence of certain random mutations that result in the generation of a wild-type protein variant, which exhibits a mutation at a specific amino acid in the binding site of this wild-type OB-fold protein. Specifically, in the context of WO 2008 / 068637, the wild-type OB folded protein is a Sac7d protein in which mutations have been introduced to produce variability in amino acids selected from K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, S46, or other amino acids such as V26, G27, K28, M29, S31, R42, A44, S46, E47, and K48. These amino acids are based on the Sac7d sequence represented by SEQ ID NO:1.

[0048] WO 2012 / 150314 demonstrates that a mutation originating from one protein in the Sac7d family can be carried to another protein in the same family. This portability consequently allows for the creation of 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 described in WO 2008 / 068637.

[0049] It has already been shown that it is possible to obtain such variants against a given target (especially when the target is a protein or peptide). Examples include variants that bind to immunoglobulins (Behar et al, Protein Engineering, Design & Selection vol. 26 no. 4 pp. 267-275, 2013) or other proteins (WO 2008 / 068637). Gera et al (J Mol Biol. 2011 Jun 17;409(4):601-16) have also shown the possibility of obtaining such proteins starting from Sso7d. Gocha et al (Scientific Reports 7, Article number: 12021(2017)) have also reported that variants can be obtained from Sso7d.

[0050] Example of an OB fold domain Non-limiting examples of OB-fold proteins that can be used according to the present invention include Sac7d, Sso7d, the N-terminal domain of SEB (Papageorgiou et al., 1998), the A chain 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), Cdc13 (Mitton-Fry et al., 2002), and the cold-shock DNA-binding domain of human Y-box protein YB-1 (Kloks et al., Any of the proteins listed in Table 3 of the literature, such as 2002, the E. coli inorganic pyrophosphatase EPPase (Samygina et al., 2001), or (Arcus, 2002), for example, 1krs (Lysyl-tRNA synthetase LysS, E. coli), 1c0aA (Asp-tRNA synthetase, E. coli), 1b8aA (Asp-tRNA synthetase, P. kodakaraensis), 1lylA (Lysyl-tRNA synthetase LysU, E. coli), 1quqA (replica protein A, 32kDa subunit, human), 1quqB (replica protein A, 14kDa subunit, human), 1jmcA (replica protein A, 70kDa subunit (RPA70 fragment, human)), 1otc (telomere-binding protein, O. nova), 3ullA (mitochondrial ssDNA binding protein, human), 1prtF (pertussis toxin S5 subunit, Bordetella pertussis), 1bcpD (pertussis toxin S5 subunit (ATP-bound), Bordetella pertussis), 3chbD (cholera toxin, Vibrio cholerae), 1tiiD (thermally unstable toxin, Escherichia coli), 2bosA (verotoxin 1 / Shiga toxin, B-pentamer, Escherichia coli), 1br9 (TIMP-2, human), 1an8 (superantigen SPE-C, Streptococcus pyogenes (S.pyogenes), 3seb (superantigen SPE, Staphylococcus aureus), 1aw7A (toxic shock syndrome toxin, Staphylococcus aureus), 1jmc (major cold shock protein, Escherichia coli), 1bkb (translation initiation factor 5a, P. aerophylum), 1sro (S1 RNA binding domain of PNPase, Escherichia coli), 1d7qA (translation initiation factor 1, elF1a, human), 1ah9 (translation initiation factor 1, IF1, Escherichia coli), 1b9mA (Mo-dependent transcription regulator ModE, Escherichia coli), 1ckmA (RNA guanylyl transferase, Chlorella virus, PBCV-1), 1a0i (ATP-dependent DNA ligase, bacteriophage) These include T7), 1snc (Staphylococcal nuclease, Staphylococcus aureus), 1hjp (DNA helicase RuvA subunit, N-terminal domain, Escherichia coli), 1pfsA (gene V protein, Pseudomonas bacteriophage pf3), 1gvp (gene V protein, filamentous bacteriophage (f1, M13)), 1gpc (gene 32 protein (gp32) core, bacteriophage T4), 1wgjA (inorganic pyrophosphatase, budding yeast (S. cerevisiae)), and 2prd (inorganic pyrophosphatase, T. thermophilus).

[0051] Specific and preferred examples of OB fold domains The Sac7d family is defined as a family of 7 kDa DNA-binding proteins associated with the Sac7d protein, isolated from extremophilic bacteria.

[0052] These proteins and this family are specifically described 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 (obtained from SEQ ID NO:1 to SEQ ID NO:9 and SEQ ID NO:12 to SEQ ID NO:14. In this consensus sequence, the dash symbol - indicates no amino acids, and not all proteins are the same size). The Sac7d family specifically includes the Sac7d or Sac7e protein from Sulfolobus acidocaldarius, the Sso7d protein from Sulfolobus solfataricus, DBP 7 (also known as Sto7 protein) from Sulfolobus tokodaii, the Ssh7b protein from Sulfolobus shibatae, the Ssh7a protein from Sulfolobus shibatae, the Mse7 from Metallosphaera sedula, the Mcu7 from Metallosphaera cuprina, the Aho7a, Aho7b, or Aho7c from Acidianus hospitalis, and Sulfolobus islandica (Sulfolobus This includes Sis7a or Sis7b from *Islandicus* and p7ss proteins from *Sulfolobus solfatalicus*. Given the extensive sequence similarities of proteins in the Sac7d family, it is directly possible and easy to identify the amino acids of a different protein that correspond to a given amino acid in Sac7d.

[0053] It should be noted that the number of mutant residues in the variant (compared to the wild-type protein) is preferably 5 to 25. The present invention can be carried out by a variant having, preferably, at least 5, more preferably at least 7 or 8, and even more preferably at least 10 substituted amino acids, but generally less than 25, more preferably less than 24, even more preferably less than 20, or less than 15 or 14 substituted amino acids, compared to the wild-type OB-folded domain. It is preferable that 7, 8, 9, 10, 11, 12, 13, or 14 amino acids are mutated at the binding site of the OB-folded domain compared to the wild-type OB-folded 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).

[0054] In certain embodiments, the number of mutant amino acids is between 7 and 14 (including upper and lower limits).

[0055] In one particular embodiment, these variants may also include the amino acid insertions shown above.

[0056] As shown, the Sac7d family of proteins includes 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, Aho7b, or Aho7c from Acidianus hospitalis, Sis7a or Sis7b from Sulfolobus islandicas, 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.

[0057] This variant of the Sac7d family of proteins may also be called nanofitin. Thus, the present invention preferentially applies to protein variants represented by any of SEQ ID NO:1 to SEQ ID NO:14, or protein variants having sequence SEQ ID NO:15, specifically variants of Sac7d.

[0058] In a preferred embodiment, the mutant amino acids are selected from the group consisting of 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 (see SEQ ID NO: 1 and the alignment in Figure 2). From the alignment in Figure 2, it is possible to identify amino acids in any other protein that correspond to the amino acids identified above in Sac7d. Note that the variant contains mutant amino acids selected from these amino acids (preferably 7 to 12 as described above) and may also contain other mutant amino acids (preferably 0 to 5) in other regions (i.e., selected from other residues of Sa7d). As shown above, A59, R60, A61, E62, R63, E64, and / or K66 can be deleted.

[0059] In a preferred embodiment, the mutant amino acid is selected from the group consisting of K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46.

[0060] The variants disclosed herein can be obtained by the methods described in WO 2008 / 068637 (in particular by various numbers of enrichments of combinatorial libraries using ribosome display).

[0061] Therefore, the variant of the OB fold domain is preferably a variant of a Sac7d family protein (Sac7d variant) containing 5 to 20 (preferably 7 to 14) mutant amino acids at the protein binding site. Preferably, the mutant amino acids of the Sac7d variant are selected from the group consisting of 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 (see SEQ ID NO: 1 and alignment in Figure 2). Preferably, the Sac7d variant contains 7 to 14 mutant amino acids selected from the group consisting of K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46.

[0062] The advantage of the method described in WO 2008 / 068637 is that it makes it possible to obtain variants of OB-fold proteins by screening combinatorial libraries containing or expressing multiple variants in which a certain number of amino acids are "randomized," i.e., replaced with random amino acids. Screening these libraries makes it possible to identify variants of these proteins that bind specifically to targets of interest other than the natural ligands of the wild-type protein from which the combinatorial library was constructed, generally with strong affinity (application WO 2008 / 068637 actually describes affinity on the order of 1 nanomolar).

[0063] As shown above, the applicant was able to demonstrate that it is possible to produce a specific binding protein by fusing an OB-fold domain that binds to a given target to the heavy or light chain of an antibody that binds to either the same target (thus increasing specificity and affinity) or a different target (thus obtaining a multispecific binding protein).

[0064] This fusion can be performed at the N-terminus and / or C-terminus 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 of proteins, it is possible to obtain molecules with an antibody structure (two heavy chains paired with two light chains, and such dimers paired together) that has an antibody region and a further binding region consisting of a modified OB-fold domain.

[0065] antibody Antibodies are large, Y-shaped proteins that recognize antigens via a Fab variable region. They are typically made up of four polypeptide chains: two identical heavy chains (approximately 400-500 amino acids) and two identical light chains (approximately 211-217 amino acids) linked by disulfide bonds. Each chain consists of a structural domain called an immunoglobulin domain.

[0066] There are multiple different types of antibody heavy chains that define five different fragments (Fc) that may attach to antigen-binding fragments and allow antibodies to be grouped into five isotypes (IgA, IgD, IgE, IgG, and IgM). Each heavy chain has a constant region and a variable region; the constant region is the same for all antibodies of the same isotype, but differs for antibodies of different isotypes.

[0067] In certain embodiments, the antibody portion of the protein disclosed herein is an IgG molecule.

[0068] In another embodiment, the antibody portion of the protein disclosed herein is an IgA molecule.

[0069] In another embodiment, the antibody portion of the protein disclosed herein is an IgM molecule.

[0070] In another embodiment, the antibody portion of the protein disclosed herein is an IgD molecule.

[0071] In another embodiment, the antibody portion of the protein disclosed herein is an IgE molecule.

[0072] The antibody may be a human antibody, a rodent antibody (such as a mouse antibody or rat antibody), a cat antibody, a dog antibody, a chicken antibody, a goat antibody, a camel antibody (camel antibody, llama antibody, alpaca antibody, or nanobody), a shark antibody, or an antibody from any other species. It may be a chimeric antibody or a humanized antibody. As recalled on Wikipedia, a humanized antibody is an antibody from a non-human species whose protein sequence has been modified to increase its similarity to antibody variants that occur naturally in humans. A chimeric antibody contains sequences from different species.

[0073] The antibodies that are part of the molecules disclosed herein are preferably antibodies that contain two identical heavy chains (about 400-500 amino acids, generally about 450 amino acids) and two identical light chains. Thus, the antibodies contain the same Fab variable region. Therefore, these antibodies are monospecific antibodies in which both parts of the antibody (combination of light and heavy chains) bind to the same epitope of the antigen.

[0074] However, antibodies may have different heavy and / or light chains. Specifically, in some embodiments, antibodies are bispecific antibodies. Thus, the term “antibody” encompasses both the “classical antibodies” disclosed above, which have the same heavy and light chains, and engineered antibodies that have two or more specificities.

[0075] In a particular embodiment, an antibody may have one heavy chain and one light chain derived from one antibody, as well as another heavy chain and one light chain derived from another antibody.

[0076] Specifically, the antibodies usable in the molecules disclosed herein are Triomab (Trion Pharma), KIH (knobs-into-holes)IgG (Xu et al, MAbs. 2015; 7(1): 231-242, Dillon et al, MABS 2017, 9(2), 213-230) (possibly having a common light chain as shown in Klein et al MAbs. 2012 Nov 1; 4(6): 653-663), cross-Mab (Roche Technology, Klein et al, MAbs. 2016 Aug-Sep; 8(6): 1010-1020, Cain, Chris. (2011). Crossing over to bispecificity. Science-Business eXchange.), and ortho-Fab IgG (Lewis et al, Nat Biotechnol. These antibodies may be DVD(bivariable domain)IgG (developed by AbbVie), 2 in 1-IgG (developed by Genetech), IgG-scFv (Orcutt et al, Protein Eng Des Sel. 2010 Apr;23(4):221-8), or DNL-Fab3. All of these antibodies are disclosed in Figure 2 of Kontermann and Brinkmann (Drug Discovery Today, 20 (7), 2015, 838-847) or in Brinkmann and Kontermann (MABS, 2017, 9 (2), 182-212). Fan et al (Journal of Hematology & Oncology (2015) 8:130) also describe bispecific antibodies and their applications.

[0077] The Triomab® family of trifunctional, bispecific antibodies that maintain an IgG-like shape are chimeric, consisting of two semi-antibodies, each having one light chain and one heavy chain, originating from the parental mouse IgG2a and rat IgG2b isotypes.

[0078] In certain embodiments, the antibody is a therapeutic antibody. Such therapeutic antibodies can be used in humans to cure a disease, slow the progression of a disease, or alleviate the symptoms of a disease.

[0079] Therapeutic antibodies are preferably selected from the following group: 3F8, 8H9, avagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab penteate pentetate), Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atorolimumab, Avelumab, Bapineuzumab, Basil iximab), Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab meltansineMertansine), Bleserumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab labtansine ravtansine), Caplacizumab, Capromab pendetide, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immune complex, Cedelizumab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan tetraxetan), codrituzumab, coltuximab ravtansine, conatumumab, concizumab, CR6261, crenezumab, clotedumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegolpegol), daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dinutuximab, diridavumab, domagrozumab, dorlimomab aritox), drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab (Ef alizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, emicizumab, enavatuzumab, enfortumab Vedotin (Enfortumab vedotin), Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan), epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, facinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab uzumab), ficlatuzumab, figitumumab, firivumab, flambotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, flulanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab Ozogamicin (Gemtuzumab ozogamicin), Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetanTiuxetan), Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Inolimomab, Inotuzumab ozogamicin ozogamicin), intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keriximab, labetuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab emtansine emtansine), lebrikizumab, lemalesomab, lensalizumab, lensilumab, lerdelimumab, lexatumumab, livivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorbotuzumab meltansine (Lorvotuzumab) mertansine, lucatumumab, rulizumab pegolpegol), Lumiliximab, Lumretuzumab, MABp1, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Miratuzumab, Minretumomab, Milbetuximab, Sorabutansine (Mirvetuximab) soravtansine), Mitumomab, Mogamulizumab, Monalizumab, Morolimmumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximab emtansine emtansine), narnatumab, natalizumab, navicixizumab, navivumab, nevacumab, necitumumab, nemolizumab, nererimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, merpentan (Nofetumomab)Merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab Monatox Monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab Vedotin (Pinatuzumab vedotin), Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab Vedotin (PolatuzumabVedotin, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol pegol), Robatumumab, Rolledumab, Romosozumab, Rontalizumab, Rovalpituzumab tesirine, Robelizumab, Luprizumab (Ruplizumab), Sacituzumab govitecan, Samalizumab, Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab Vedotin (Sofituzumab vedotin), Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tamtuvetmab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomabaritox), tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tisilimmab (=tremelimumab), tigatuzumab, tildrakizumab, timolumab, tisotumab vedotin Vedotin, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin), tuvirumab, ublituximab, urocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talirine, vandortuzumab vedotinVedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vorsetuzumab mahodotin Mafodotin, botumumab, xentuzumab, zalutumumab, zanolimmumab, zatuximab, ziralimumab, and zolimomab aritox.

[0080] Specifically, such therapeutic antibodies include adalimumab (marketed as Humira® for TNFα); infliximab (marketed as Remicade® for TNFα); cetuximab (marketed as Erbitux® for EGFR); secukinumab (marketed as Cosentyx® for IL17); pembrolizumab (marketed as Keytruda® for PD1); and bevacizumab (marketed as Avastin® for VEGF-A). Sold as; etrolizumab (against a4b7); vedolizumab (against a4b7); tremelimumab (against CTLA4); ipilimumab (against CTLA4, sold as Yervoy®); necitumumab (against EGFR, sold as Portrazza®); panitumumab (against EGFR, sold as Vectibix®); lebrikizumab (against IL-13); tralokinumab (against IL-13); ixekizumab (against IL-17, sold as Taltz®) (Sold as) Brodalumab (for IL17R, sold as Lumicef®); Dupilumab (for IL4R, sold as Dupixent®); Guselkumab (for IL23); Childrakizumab (for IL23); Risankizumab (for IL23); Briakinumab (for IL12 and IL23); Ustekinumab (for IL12 and IL23); Nivolumab (for PD1, sold as Opdivo®); Atezolizumab (for PD-L1, sold as Tec Sold as entriq (registered trademark); avelumab (for PD-L1); ranibizumab (for VEGF-A, sold as Lucentis (registered trademark)); brolucizumab (for VEGF-A); trastuzumab (for Her2, Herceptin (registered trademark)); amatsuximab (for mesothelin); tavolixizumab (for OX40); pogalizumab (for OX40); urerumab (for 4-1BB); utomirumab (for 4-1BB); BMS 986016 (for LAG3); lirirumab (for KIR); MEDI 570 (for ICOS);The group is selected from LY3321367 (for TIM3); rulizumab (for CD28); TAB08 (for CD28); and onartuzumab (for cMet, MetMab).

[0081] target The target of the multispecific molecule of the present invention may be any molecule or antigen of interest. Specifically, the multispecific molecule targets cancer or hyperproliferative diseases, such as lymphoma (e.g., non-Hodgkin lymphoma), renal cell carcinoma, prostate cancer, ovarian cancer, breast cancer, colorectal cancer, neuroendocrine cancer, endometrial cancer, pancreatic cancer, leukemia, lung cancer, glioblastoma multiforme, gastric cancer, liver cancer, sarcoma, bladder cancer, testicular cancer, esophageal cancer, head and neck cancer, and leptomeningeal carcinomatosis.

[0082] In a preferred embodiment, the OB fold domain binds to the targets disclosed below.

[0083] It can be selected from the following group: cell surface receptors Insulin receptor, low-density lipoprotein receptor-related protein 1, transferrin receptor, epidermal growth factor receptor, epidermal growth factor receptor variant III, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, Her2, Her3, Her4, PMSA, IGF-1R, GITR, RAGE, CD28. cell surface proteins : Mesothelin, EpCam, CD19, CD20, CD38, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, MadCam, a4b7, CD7, CD4, CD138. Angiogenesis or 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 protein:TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Bace1, α-synuclein, tau, amyloid.

[0084] In a preferred embodiment, the antibody portion of the molecule binds to a target selected from the group consisting of: cell surface receptors Insulin receptor, low-density lipoprotein receptor-related protein 1, transferrin receptor, epidermal growth factor receptor, epidermal growth factor receptor variant III, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, Her2, Her3, Her4, PMSA, IGF-1R, GITR, RAGE, CD28. cell surface proteins : Mesothelin, EpCam, CD19, CD20, CD38, CD3, TIM-3, CEA, cMet, ICAM1, ICAM3, MadCam, a4b7, CD7, CD4, CD138. Angiogenic factors or 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 protein :TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Bace1, α-synuclein, tau, amyloid.

[0085] In a preferred embodiment, the molecule consists of the following pair of targets (one of these described targets may be a target of the OB fold domain, and the other may be a target of the antibody moiety): - EGFR / EGFRvIII - EGFR / Her2 - VEGFR2 / PD1 - EGFR / PD1 - VEGF / PD-L1 - PD1 / OX40 - PD1 / CTLA4 - EGFR / CD3 - TNFa / IL17 - IL13 / IL4

[0086] Complexes in which an antibody binds to TNF-alpha and the OB-fold domain binds to IL-17, and the reverse complex, are of particular interest and preferred.

[0087] Further examples of target pairs are disclosed below. Note that the antibody portion of the molecule may bind to any one of the listed targets, while the OB-fold variant may bind to other targets. Therefore, any of the targets listed above is effective for either the antibody portion or the OB-fold variant portion of the molecule.

[0088] Artificial Molecules The vectors that enable the generation of the molecules disclosed herein are prepared by conventional molecular genetic methods.

[0089] In summary, the gene sequence encoding the Sca7d variant is ligated to the 5' or 3' end of the gene sequence encoding the heavy or light chain of the antibody by any method known in the art. To obtain a fusion protein, it is possible to introduce a linker between the two gene sequences, provided that a frameshift or stop codon is not introduced.

[0090] Therefore, the expressed fusion protein should be one of the following (from the N-terminus to the C-terminus): (a) Antibody heavy chain-(linker, if present)-OB fold domain variant, (b) OB folded domain variant - (linker if present) - heavy chain of antibody, (c) Antibody light chain-(linker, if present)-OB folded domain variant (d) OB fold domain variant - (linker, if present) - light chain of antibody

[0091] It is clear that further genetic modifications, such as the introduction of additional "tag" molecules at the C-terminus or N-terminus, can be predicted, which would improve purification, force the orientation of the OB fold, enable protease-mediated release of the OB fold, alter the pharmacokinetics of the compound, target specific types of tissue, chelate lantanides or radionuclides, or enable binding to payloads that may be mytansin or auristatin derivatives.

[0092] It should be noted that the codons in the gene sequence can be optimized for further production depending on the cells used for further production (see, for example, the OptimumGene® codon optimization technology from GenSript (Piscataway, NJ USA)).

[0093] Other nucleic acid molecules, such as nucleic acid molecules encoding the following, can be obtained, as shown below: (a) OB folded domain variant - (linker if present) - antibody heavy chain - (linker if present) - OB folded domain variant, (b) OB-fold domain variant - (linker if present) - antibody light chain - (linker if present) - OB-fold domain variant

[0094] These recombinant DNA constructs, containing one or more of the nucleotide sequences described above, are used in conjunction with vectors such as plasmids, phagemids, phages, or viral vectors.

[0095] These recombinant acid molecules can be produced by the technique 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.

[0096] The recombinant constructs of the present invention include an expression vector capable of expressing RNA, thereby resulting in the production of a protein from the gene sequence described above. The vector may further include a control sequence comprising a suitable promoter functionally linked to an open reading frame (ORF) of the gene sequence disclosed herein. The vector may further include a selectable marker sequence, such as an antibiotic resistance gene. Specific initiation and bacterial secretion signals may also be required for efficient translation of the coding sequence when bacteria are used as the expression host.

[0097] Molecular generation Cells are transfected or transformed with a vector containing sequences encoding the heavy and light chains of the antibody. At least one of the sequences contains a variant of the OB-fold protein discussed above.

[0098] Cells are cultured under conditions in which proteins are expressed and preferably secreted. These cell culture conditions are those commonly used for recombinant antibody production and are known in the art. Such known conditions can be optimized by those skilled in the art as needed. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461) outline such methods and provide a sufficient description therein.

[0099] Bacterial, phage (Shukra et al, Eur J Microbiol Immunol(Bp). 2014; 4(2): 91-98), or eukaryotic synthesis systems can be used.

[0100] To obtain appropriate post-translational modifications such as glycosylation, it is preferable to use eukaryotic cells.

[0101] Specifically, CHO (Chinese hamster ovary) cells, PER.C6 cells (human cell line, Pau et al, Vaccine. 2001 21;19(17-19):2716-21), HEK 293b cells (human embryonic kidney cell 293), NS0 cells (cell line derived from non-secreting mouse myeloma), or EB66 cells (duck cell line, Valneva, Lyons, France) can be used.

[0102] This disclosure also provides a host cell containing at least one of the DNA constructs described above. The host cell may be any cell on which the expression vector is available. As shown 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.

[0103] The introduction of recombinant constructs into host cells is carried out by any method known in the art (e.g., 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 (e.g., bacterial or yeast artificial chromosome). If introduced into the cellular genome, such introduction may be random or targeted using methods known in the art (e.g., homologous recombination).

[0104] Bacterial host and expression Expression vectors useful for bacterial use are constructed by inserting a recombinant DNA sequence, along with appropriate translation start and termination signals, into a functional leading phage having a functional promoter. The vector includes one or more phenotypic selectable markers and origins of replication that ensure vector retention and, if desired, lead to amplification in the host.

[0105] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0106] Eukaryotic host and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. Specifically, the cells described above can be used.

[0107] Transformed or transfected cells are cultured by methods known in the art, and polypeptides are recovered from the intracellular or extracellular fraction (depending on whether they are secreted or not).

[0108] Molecular isolation The resulting recombinant multispecific proteins can be separated and purified from the intracellular or extracellular fraction by any of the various known separation methods that utilize the physical or chemical properties of the proteins.

[0109] Specifically, methods such as precipitation, ultrafiltration, various types of liquid chromatography, such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and combinations thereof can be used.

[0110] In general, any known method for purifying recombinant antibodies and any method used therefor is applicable to the purification of the molecules disclosed herein.

[0111] If the tag is introduced within the recombinant sequence (e.g., a polyhistidine tag), the molecule can be purified using this tag. However, it is preferable to purify the molecule using affinity.

[0112] If the antibody portion of the molecule is of the IgG type, affinity chromatography using protein A can be used (see Fahrner et al Biotechnol Appl Biochem. 1999 Oct;30(Pt 2):121-8 in particular).

[0113] Alternatively, the method disclosed in Jiang et al. (Protein Expression and Purification, Volume 76, Issue 1, March 2011, Pages 7-14), which discloses a purification process for recombinant monoclonal antibodies expressed in glycosylated Pichia pastoris, can be used.

[0114] The method described by Maria et al (J Chromatogr A. 2015 May 8;1393:57-64) for the purification process of recombinant monoclonal antibodies by mixed-mode chromatography, or the method described by Liu et al (MAbs. 2010 Sep-Oct; 2(5): 480-499) for the recovery and purification process for monoclonal antibody production, can also be used.

[0115] In particular, the molecules generated herein can be isolated using any affinity method (affinity column, FACS, beads) by utilizing the fact that they bind to specific targets (antigens of antibodies and targets of OB fold variants).

[0116] One particular advantage of the method of the present invention is that all of the resulting molecules produced by the cells are multispecific molecules.

[0117] Generation of bispecific molecules Specifically, the following will be introduced into the cells: - Gene sequence encoding the light chain of an antibody - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 5' end of the heavy chain.

[0118] The resulting molecule, after the association of the heavy and light chains, becomes a bispecific molecule that binds to the antigen of the antibody and to the target of the variant of the OB-fold domain at the N-terminus of the heavy chain.

[0119] As shown in Figure 1.A, note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB folded domain variant binding sites at the N-terminus of the heavy chain).

[0120] In another embodiment, the following shall be introduced into the cell: - Gene sequence encoding the light chain of an antibody - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 3' end of the heavy chain.

[0121] The resulting molecule is a bispecific molecule that, after the association of the heavy and light chains, binds to the antigen of the antibody and to the target of the variant of the OB-fold domain at the C-terminus of the heavy chain.

[0122] As shown in Figure 1.B, note that the resulting molecule is symmetrical and has four binding domains (two antibody Fab binding sites and two OB folded domain variant binding sites at the C-terminus of the heavy chain).

[0123] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain in which a variant of the OB folded domain has been introduced at the 5' end of the light chain. - The gene sequence that codes for the heavy chain of an antibody.

[0124] The resulting molecule is a bispecific molecule that, after the association of the heavy and light chains, binds to the antigen of the antibody and to the target of the variant of the OB-fold domain at the N-terminus of the light chain.

[0125] As shown in Figure 1.C, note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB folded domain variant binding sites at the N-terminus of the light chain).

[0126] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which an OB-folded domain variant is introduced at the 3' end of the light chain. - The gene sequence that codes for the heavy chain of an antibody.

[0127] The resulting molecule is a bispecific molecule that, after the association of the heavy and light chains, binds to the antigen of the antibody and to the target of the variant of the OB-fold domain at the C-terminus of the light chain.

[0128] As shown in Figure 1.D, note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB folded domain variant binding sites at the C-terminus of the light chain).

[0129] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which an OB-folded domain variant is introduced at the 3' end of the light chain. - Gene sequence encoding the heavy chain of an antibody - The gene sequence that codes for the light chain of an antibody.

[0130] The resulting products after the association of the heavy and light chains are as follows: - A symmetrical, bispecific molecule (expected percentage 25%) that binds to the antigen and variant of the OB-fold domain at the C-terminus of the light chain of an antibody. - An asymmetric bispecific molecule (expected percentage 50%) that binds to the target of an OB-fold domain variant located at the C-terminus of only one of the antibody's antigens or light chains. - Symmetrical antibodies (expected percentage 25%).

[0131] Note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB folded domain variant binding sites at the C-terminus of the light chain).

[0132] Other combinations can be performed when transforming cells as follows: - A gene sequence encoding an antibody light chain, in which a variant of the OB folded domain is introduced at the 5' end of the light chain. - Gene sequence encoding the heavy chain of an antibody - Gene sequence encoding the light chain of an antibody, or - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 3' end of the light chain. - Gene sequence encoding the heavy chain of an antibody - Gene sequence encoding the light chain of an antibody, or - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 5' end of the light chain. - Gene sequence encoding the heavy chain of an antibody - The gene sequence that codes for the light chain of an antibody.

[0133] Generation of multispecific molecules In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which a variant of the OB folded domain is introduced at the 5' end of the light chain. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 5' end of the heavy chain.

[0134] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to variant targets of the OB-fold domain located at the N-terminus of the light chain and the N-terminus of the heavy chain.

[0135] Note that, as illustrated in Figure 1.G, the resulting molecule has six binding sites (two antibody Fab binding sites, and binding sites for the N-terminus of the light chain and for each of the four OB-fold domain variants located at the N-terminus of the light chain).

[0136] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which a variant of the OB-fold domain is introduced at the 3' end of the light chain. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 5' end of the heavy chain.

[0137] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to variants of the OB-fold domain located at the C-terminus of the light chain and the N-terminus of the heavy chain.

[0138] Note that, as illustrated in Figure 1.E, the resulting molecule has six binding sites (two antibody Fab binding sites, and four binding sites for variants of the OB folded domain located at the C-terminus and N-terminus of the light chain).

[0139] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which a variant of the OB folded domain is introduced at the 5' end of the light chain. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 3' end of the heavy chain.

[0140] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to variants of the OB-fold domain located at the N-terminus of the light chain and the C-terminus of the heavy chain.

[0141] As shown in Figure 1.I, the resulting molecule has six binding sites (two antibody Fab binding sites, and four binding sites for variants of the OB folded domain located at the N-terminus and C-terminus of the light chain).

[0142] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding an antibody light chain, in which a variant of the OB-fold domain is introduced at the 3' end of the light chain. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB folded domain is introduced at the 3' end of the heavy chain.

[0143] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to the variant targets of the OB-fold domain located at the C-terminus of the light chain and the C-terminus of the heavy chain.

[0144] Note that, as illustrated in Figure 1.K, the resulting molecule has six binding sites (two antibody Fab binding sites, as well as binding sites for the C-terminus of the light chain and for each of the four OB-fold domain variants located at the C-terminus of the light chain).

[0145] In another embodiment, the following shall be introduced into the cell: - A gene sequence encoding the light chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the light chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the light chain encoding sequence. - The gene sequence that codes for the heavy chain of an antibody.

[0146] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to variants of the OB-fold domains located at the N-terminus and C-terminus of the light chain.

[0147] As shown in Figure 1.J, note that the resulting molecule has six binding sites (two antibody Fab binding sites, and four binding sites for variants of the OB folded domain located at the N-terminus and C-terminus of the light chain).

[0148] In another embodiment, the following shall be introduced into the cell: - The gene sequence encoding the heavy chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the heavy chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the heavy chain encoding sequence. - The gene sequence that codes for the light chain of an antibody.

[0149] The resulting molecule, after the association of the heavy and light chains, becomes a multispecific molecule that binds to the antibody antigen and to variants of the OB-fold domains located at the N-terminus and C-terminus of the heavy chain.

[0150] Note that, as illustrated in Figure 1.F, the resulting molecule has six binding sites (two antibody Fab binding sites, and four binding sites for variants of the OB folded domain located at the N-terminus and C-terminus of the heavy chain).

[0151] In the above embodiment, the variants of the OB-fold domains introduced at the ends of the heavy and light chains are either the same or different.

[0152] If the variants are different, they could be: - Based on different OB fold domains, yet binding to the same target. - Based on different OB fold domains and binding to different targets. - Based on the same OB fold domain, but binding to different targets. - Based on the same OB fold domain and binding to the same target (but with different mutations).

[0153] Cells may be transformed or transfected with other coding sequences to obtain asymmetric molecules.

[0154] Other embodiments for generating multispecific molecules Three gene sequences In a further embodiment, cells are transformed or transfected with the following three gene sequences: (i) Gene sequence encoding the light chain of an antibody (ii) A gene sequence encoding the antibody heavy chain, fused to a gene sequence encoding a variant (A) of the OB fold domain (at the 3' or 5' end). (iii) A gene sequence encoding the antibody heavy chain, fused (at the 3' or 5' end) to a gene sequence encoding another variant (B) of the OB fold domain.

[0155] In another embodiment, the three gene sequences are as follows: (i) Gene sequence encoding the heavy chain of the antibody (ii) A gene sequence encoding the antibody light chain, fused to a gene sequence encoding a variant (A) of the OB fold domain (at the 3' or 5' end). (iii) A gene sequence encoding the antibody light chain, fused (at the 3' or 5' end) to a gene sequence encoding another variant (B) of the OB fold domain.

[0156] Therefore, logically, cells produce three different types of multispecific proteins in the following proportions: - Bispecific protein (which binds to the antigen and variant (A) targets of the antibody): 25% - Bispecific protein (which binds to the antigen and variant (B) targets of the antibody): 25% - Triple-specific proteins (which bind to the antigen and variant (A) and (B) targets of the antibody): 25%.

[0157] With respect to the above, variants (A) and (B) of the OB fold domain may be the same or different.

[0158] If the variants are different, they could be: - Based on different OB fold domains and binding to different targets. - Based on different OB fold domains and binding to different targets. - Based on the same OB fold domain, but binding to different targets. - Based on the same OB fold domain and binding to the same target (but with different mutations).

[0159] Multiple fused gene sequences It is also possible to fuse a gene sequence encoding an OB-fold domain variant to both the 5' and 3' ends of the gene sequence encoding the light or heavy chain of an antibody.

[0160] The gene sequences fused here can encode the same or different variants, as shown above.

[0161] The specific details are as follows: To produce a protein with six binding sites (if symmetrical, these proteins will be quadruple-specific if the OB fold variant is different), - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the heavy chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the heavy chain encoding sequence. - A variant of the OB-fold domain is introduced at the 5' end of the light chain encoding gene sequence, This results in the protein shown in Figure 1.N. - A gene sequence encoding the light chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the light chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the light chain encoding sequence. - A variant of the OB-fold domain is introduced at the 5' end of the heavy chain encoding sequence of an antibody, This results in the protein shown in Figure 1.0. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the heavy chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the heavy chain encoding sequence. - A gene sequence encoding the light chain of an antibody, in which a variant of the OB-fold domain is introduced at the 3' end of the sequence encoding the light chain. This results in the protein shown in Figure 1.M. - A gene sequence encoding the light chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the light chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the light chain encoding sequence. - A variant of the OB-fold domain is introduced at the 3' end of the heavy chain encoding sequence of an antibody, This results in the protein shown in Figure 1.L.

[0162] Cells transformed or transfected with these gene sequences result in a multispecific protein that presents eight binding sites (two antibody Fab binding sites and six binding sites for variants of the OB folded domain).

[0163] By transforming or transfecting cells as described below, a protein with 10 binding sites can also be obtained. - A gene sequence encoding the heavy chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the heavy chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the heavy chain encoding sequence. - A gene sequence encoding the light chain of an antibody, in which a variant of the OB-fold domain is introduced at the 5' end of the light chain encoding sequence, and a variant of the OB-fold domain (same or different) is introduced at the 3' end of the light chain encoding sequence. This results in the protein shown in Figure 1.H.

[0164] As described above, in all embodiments, the variants of the OB fold domain may be the same or different.

[0165] If the variants are different, they could be: - Based on different OB fold domains, yet binding to the same target. - Based on different OB fold domains and binding to different targets. - Based on the same OB fold domain, but binding to different targets. - Based on the same OB fold domain and binding to the same target (but with different mutations).

[0166] It is further predicted that cells will be transformed or transfected with the multiple gene sequences described above, thereby resulting in a mixture of various multispecific proteins (some of which are asymmetric).

[0167] Applications of molecules The targets and applications are found in Table 1 of Fan et al. Journal of Hematology & Oncology (2015) 8:130, which is incorporated herein by reference. Any combination of the targets listed in this table for bispecific antibodies can be used in the multispecific molecules disclosed herein. Targets and applications are also described in Yang et al. (Int J Mol Sci. 2017 Jan; 18(1):48), which is also incorporated by reference. Specifically, the combinations of targets and potential applications are listed below.

[0168] Cytotoxic T lymphocytes play a crucial role in the immune response against cancer, and since tumor cells may evade this immune response, one strategy is to recruit T cells near tumor cells using bispecific molecules. Activation and proliferation of T cells leads to tumor cell lysis.

[0169] One target of this multispecific molecule is tumor-associated antigens, and its second target is CD3 on T cells. It may also bind to type I (CD64), type IIα (CD32a), and type III (CD16) Fcγ receptors (FcγR) on accessory cells such as macrophages, dendritic cells, and NK cells, potentially improving the immune response.

[0170] Cancer immunotherapy TIFF0007896996000001.tif92148TIFF0007896996000002.tif181148TIFF0007896996000003.tif129148

[0171] Modification of host responses to drug resistance / metabolic pathways Bispecific antibodies are the best choice in that they can simultaneously inhibit two correlated signaling molecules, and specifically, inhibitory checkpoint molecules that are the main inhibitors impeding immunotherapy. TIFF0007896996000004.tif33147TIFF0007896996000005.tif198147

[0172] HER2 is an effective target for a number of cancers. HER3 signaling is an important mechanism of drug resistance to HER2 inhibitors. Dual targeting of HER2 / HER3 may lead to a more effective response.

[0173] Deregulated EGFR-dependent and HER3-dependent signaling is involved in the lesion formation of human cancers such as head and neck cancer and colorectal cancer.

[0174] Targeting HER2 and HER3 restored the sensitivity to GDC-0941 and enabled the re-arrest of prostate cancer growth by GDC-0941 (Poovassery et al, Int. J. Cancer. 2015;137:267-277).

[0175] anti-angiogenesis TIFF0007896996000006.tif146147

[0176] Multiple angiogenic factors including vascular endothelial growth factor receptor 2 (VEGFR2), VEGFR3, vascular endothelial growth factor A (VEGFA), angiopoietin, and platelet-derived growth factor (PDGF) are involved in tumor angiogenesis. Many cancer therapies disrupt angiogenesis by depleting these proteins. Dual targeting of angiogenic factors yields excellent results (Biel and Siemann Cancer Lett. 2016 Oct 1; 380(2):525-33).

[0177] Adoptive T cell transfer for cancer immunotherapy The targets are PD-1, tumor antigens, and molecules expressed on the surface of T lymphocytes.

[0178] The presence of bispecific molecules and T lymphocytes against cancer cells in vitro allows for effective priming of T lymphocytes subsequently injected into the patient.

[0179] Bispecific antibodies such as PD-1 / CD3, tumor antigen / CD3, and HER2 / CD3 can be used. The teachings of Urbanska et al (J. Transl. Med. 2014;12:347, using bispecific antibodies (CD20 / CD3 or HER2 / CD3) and engineered T cells) can also be incorporated.

[0180] Binding to cytokines TIFF0007896996000007.tif62147

[0181] Several cytokines have been identified as important mediators in inflammatory and autoimmune diseases. Therefore, blocking these cytokines has therapeutic potential. For example, inhibition of TNF-α has shown significant therapeutic effects on psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, juvenile arthritis, and numerous other diseases. Other effective cytokines include IL-6, IL-17, IL-1, IL-12, TGF-β, IL-4, and IL-13.

[0182] Payload delivery TIFF0007896996000008.tif89147

[0183] Interesting applications include the delivery of payloads such as drugs, radiolabeled substances, and nanoparticles. The payload is administered immediately after the unbound bispecific molecule is removed from the bloodstream. The bispecific molecule can be used to concentrate the payload at the tumor site. This strategy significantly extends serum retention time and improves the tumor / blood ratio. CEA and 99mOne example is a bispecific TF2 construct that specifically binds to the T-labeled hapten histamine succinylglycine (HSG) and is used in tumor imaging and radioimmunotherapy. In preclinical studies, TF2 was injected first, followed by... 99m T-labeled HSG was administered after removal of bsAb from the blood. A high tumor / blood ratio was observed. 99m This was observed due to T's high tumor uptake. TF2 is currently in Phase I trials in patients with colorectal cancer. Other applications of TF2 include radioimmunotherapy in patients with colorectal neoplasms. 177Lu HSG / 111In Targeting of HSG and CEA, and immunopositron emission tomography 68Ga This includes targeting of HSG and CEA (from Fan et al. Journal of Hematology & Oncology (2015) 8:130).

[0184] Digoxigenin (Dig) as a hapten can be used as a payload scaffold to mount on several cytotoxic components such as fluorophores, chelating agents, chemotherapeutic agents, nucleic acids, lipids, nanoparticles, or peptides and proteins, ultimately forming compounds such as Dig-Cy5, Dig-doxorubicin, and Dig-GFP. Hapten-based bispecific antibodies have also been shown to be effective siRNA delivery systems; specifically, siRNA digoxigenized at its 3' end and formulated into nanoparticles conjugates to bispecific antibodies that bind to tumor antigens such as HER2, IGF1-R, CD22, and LeY, specifically delivering the siRNA to cells expressing the corresponding antigen, resulting in internal translocation into endosomes and separation of Dig-siRNA from the bispecific antibody (Schneider et al, Mol. Ther. Nucleic Acids. 2012;1:e45).

[0185] Bacterial minicells are anucleate nanoparticles generated by inactivation of genes that control normal bacterial cell division. Chemotherapeutic agents can be packaged into the minicells and then linked to bispecific molecules that also bind to antigens on the membranes of cells (such as cancer cells), resulting in endocytosis, intracellular degradation, and drug release (Solomon et al, PLoS ONE. 2015;10:e0144559).

[0186] Crossing the blood-brain barrier TIFF0007896996000009.tif68148

[0187] Couch et al and Yu et al. designed bsAbs that bind to the transferrin receptor (TfR) and β-site APP cleaving enzyme 1 (BACE1) and cross the blood-brain barrier.

[0188] Diagnostic assay TIFF0007896996000010.tif61148

[0189] Treatment of infectious diseases TIFF0007896996000011.tif176148

[0190] Other uses TIFF0007896996000012.tif75148

[0191] [[ID=​​​​​​Treatment methods are also part of the present invention. The present invention also encompasses methods for treating patients in need, comprising the step of administering a therapeutically effective amount of the molecules disclosed herein. A “therapeutably effective” amount as herein is defined as an amount sufficient to obtain a clinical effect (reduction of an adverse condition). This can be determined by a Phase II clinical trial. It may be administered alone or in combination with another active agent, as a single dose or according to a multi-dose regimen. It is preferably non-toxic or has toxicity that is acceptable in light of the benefit to the patient’s health. The subject may be human or a non-human animal (e.g., rabbit, rat, mouse, monkey, or other lower primate).

[0193] The molecules disclosed herein will be formulated using one or more physiologically acceptable carriers or excipients known in the art and administered by any suitable means and through any suitable route. Therefore, parenteral (e.g., intramuscular, intravenous, intra-arterial, coma, or subcutaneous), intrapulmonary, and intranasal administration, and preferably intrafocal administration in topical immunosuppressive therapy, are anticipated. Infusion (particularly intravenous infusion) is preferred. The route of administration may vary depending on the disease being treated.

[0194] Molecules targeting TNF-α are generally used to treat inflammatory diseases (arthritis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, rhinitis, psoriasis, Crohn's disease) as well as certain cancers (colorectal cancer, breast cancer, bladder cancer, glioblastoma, other solid tumors) or other immunological disorders.

[0195] Molecules targeting IL-17 are also generally used to treat the same types of inflammatory diseases (arthritis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, rhinitis, psoriasis, Crohn's disease, ankylosing spondylitis) as well as some cancers (colorectal cancer, breast cancer, bladder cancer, glioblastoma, other solid tumors) or other immunological diseases (including multiple sclerosis).

[0196] Therefore, a complex containing an antibody that binds to TNFα and an OB fold variant (or a protein derived from the Dac7d family) that binds to IL17 (or vice versa) can be used to treat such diseases.

[0197] Specific composition Specifically, the present invention relates to a polypeptide disclosed above in which a variant of a protein of the Sac7d family binds to a subunit of TNFα. Preferably, if the subunit protein is contained within a fully formed polymer protein in its native state, it does not bind to the subunit of that protein.

[0198] In fact, the inventors have identified variants of the Sac7d family of proteins that bind to the soluble subunit of TNFα and either prevent the formation of a biologically active multimeric protein or shift the equilibrium towards a monomeric (or multimeric-inactive) subunit.

[0199] Specifically, such variants are arrays Includes TIFF0007896996000013.tif44143.

[0200] Specifically, 1 to 13 amino acids, preferably 1 to 11, more preferably 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, M8, F9, K11, V21, Q26, L29, E35, D41, F44, and P46, are replaced by another amino acid in SEQ ID NO:41.

[0201] In this embodiment, it is preferable that the antibody binds to a protein selected from the group consisting of IL17, CD20, IL24, IL12, IL4, IL13, IL6, IL31, or their receptors, and tumor-specific antigens, particularly Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4.

[0202] Specifically, the antibodies bind to IL17 (and more specifically, infliximab, adalimumab, certolizumab pegol, orgolimumab, secukinumab, afasebikuma, bimekizumab, or vunakizumab).

[0203] Specifically, the antibodies bind to CD20 (and specifically, bronzbetomab, FBTA05, ibritumomab tiuxetan, mosunetuzumab, obinutuzumab, okalatuzumab, ocrelizumab, ofatumumab, rituximab, tositumomab, or bertuzumab).

[0204] Specifically, the antibody binds to IL24.

[0205] Specifically, the antibody binds to IL-12.

[0206] Specifically, the antibody binds to IL-4 (and specifically to dupilumab).

[0207] Specifically, the antibody binds to IL-13.

[0208] Specifically, the antibody binds to IL-6 (and more specifically, siltuximab, olokizumab, or bovalilizumab).

[0209] Specifically, the antibody binds to IL31 or its receptor.

[0210] Specifically, the antibody binds to the tumor-specific antigen disclosed above.

[0211] Specifically, the antibodies bind to Her2 (specifically, DS-8201, erzmaxomab, gancotamab, margetuximab, pertuzumab, timigutuzumab, trastuzumab, or trastuzumab emtansine).

[0212] Specifically, the antibody binds to PDL1 (programmed death ligand 1, CD274).

[0213] Specifically, the antibody binds to CTLA4 (and more specifically, tremelimumab).

[0214] In another embodiment, variants of the Sac7d family of proteins bind to IL17.

[0215] In this aspect, variants of the Sac7d family of proteins are defined by the following sequence It is preferable to include one of the TIFF0007896996000014.tif84143.

[0216] In this embodiment, the antibody is TNFα (specifically infliximab, adalimumab, certolizumab pegol, orgolimumab), IL23 (specifically brazicumab, guselkumab, mirikizumab, risankizumab, or tildrakizumab), IL12 (specifically briakinumab, ustekinumab), IL4 (specifically dupilumab), IL13 (specifically anlukinzumab), IL31, or their receptor, and tumor-specific antigen, specifically Her2 (specifically DS-8201, erzmakisomab, gangcotamab, margetuximab, pertuzumab, timigutuzumab, trastuzumab, or trastuzumab) It is preferable that the protein binds to a protein selected from the group consisting of emtansine, PDL1 (programmed death ligand 1, CD274), or CTLA4 (specifically tremelimumab).

[0217] Specifically, the antibodies bind to TNFα (and specifically, infliximab, adalimumab, certolizumab pegol, and orgolimumab).

[0218] Specifically, the antibody binds to IL23 (and specifically brazicumab, guselkumab, mirikizumab, risankizumab, or tildrakizumab).

[0219] Specifically, the antibody binds to IL-12 (and more specifically, briakinumab or ustekinumab).

[0220] Specifically, the antibody binds to IL-4 (and specifically to dupilumab).

[0221] Specifically, the antibody binds to IL-13 (and specifically to anlukinzumab).

[0222] Specifically, the antibody binds to IL31 or its receptor.

[0223] Specifically, the antibody binds to the tumor-specific antigen disclosed above.

[0224] Specifically, the antibody binds to Her2 (specifically DS-8201, erzmaxomab, gancotamab, margetuximab, pertuzumab, timigutuzumab, trastuzumab, or trastuzumab emtansine).

[0225] Specifically, the antibody binds to PDL1 (programmed death ligand 1, CD274).

[0226] Specifically, the antibody binds to CTLA4 (specifically, tremelimumab).

[0227] Specific variants of Sac7d that bind to IL17 The present invention also relates to a polypeptide comprising a variant of a Sac7d family protein, wherein the variant specifically comprises 4 to 22 mutant amino acids at the binding site of an IL17-binding Sac7d family protein as disclosed herein, and thus antibody production is improved or stabilized. The sequence of Sac7d is Please note that the filename is TIFF0007896996000015.tif11143.

[0228] It is possible to obtain the above polypeptide in which the mutant amino acids of the Sac7d variant are selected from the group consisting of Sac7d 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.

[0229] It is possible to obtain the above polypeptide in which the Sac7d variant contains 4 to 17 mutant amino acids selected from the group corresponding to K7, Y8, K9, E11, K21, K22, W24, V26, M29, S31, T33, D35, T40, G41, R42, A44, and S46 of Sac7d.

[0230] In this aspect, a variant of the Sac7d family protein is the following sequence: It is preferable to include one of the following: TIFF0007896996000016.tif84143

[0231] Specifically, such polypeptides include sequence SEQ ID NO:37 or SEQ ID NO:38, 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, M8, F9, K11, Q26, L29, E35, D41, F44, and P46 are replaced with other amino acids. In fact, the applicant has shown that such residues can be modified without altering the observed binding. This was done by replacing these residues with alanine and confirming the binding. Since there was no loss of binding, this suggests that these residues are not important for binding to IL17.

[0232] In certain embodiments, the particular Sac7d variant is linked to or fused to another protein or polypeptide. Specifically, the other protein or polypeptide comprises another variant of a protein in the Sac7d family. As indicated above, it is also expected that the other protein or polypeptide may be an antibody (preferably bound to TNFα or Her2 / neu).

[0233] In another embodiment, such variants of the Sac7d family are conjugated to organic molecules. This can be done by any method known in the art. Specifically, molecules can be chemically linked to proteins. Examples of molecules include antiproliferative agents (cytotoxic agents and cell division 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.

[0234] Specifically, examples include DNA-binding agents or alkylating agents, such as anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and their analogues, 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 diseases, cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitors, quercetin, and / or resveratrol can also be used as molecules conjugated to polypeptides containing variants.

[0235] The present invention also relates to gene constructs comprising DNA sequences encoding polypeptides (variants of the Sac7d family of proteins that bind to IL17) as disclosed herein.

[0236] The present invention also includes a vector comprising the gene construct disclosed above, a host cell containing such gene construct in its genome, and the following steps: a. A step of culturing a cell culture in which cells have been transformed by a disclosed gene construct, and b. Steps for recovering polypeptides The present invention also relates to a method for generating such variants of IL17-binding Sac7d family proteins, which includes a step comprising the steps described above. .

[0237] The identified variant sequences can be cloned into any suitable vector by any molecular genetic method known in the art.

[0238] These recombinant DNA constructs, containing nucleotide sequences encoding polypeptides with the variants described above, are used in conjunction with vectors such as plasmids, phagemids, phages, or viral vectors.

[0239] These recombinant acid molecules can be produced by the technique 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.

[0240] The recombinant constructs of the present invention include a vector capable of expressing RNA, thereby resulting in the production of a protein from the gene sequence described above. The expression vector may further include a control sequence comprising a suitable promoter functionally linked to an open reading frame (ORF) of the gene sequence disclosed herein. The vector may further include a selectable marker sequence, such as an antibiotic resistance gene. Specific initiation and bacterial secretion signals may also be required for efficient translation of the coding sequence when bacteria are used as the expression host.

[0241] Molecular generation Cells are transfected or transformed with a vector containing a sequence encoding a polypeptide comprising the variant disclosed above.

[0242] Cells are cultured under conditions in which proteins are expressed and preferably secreted. These cell culture conditions are those commonly used for recombinant antibody production and are known in the art. Such known conditions can be optimized by those skilled in the art as needed. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461) outline such methods and provide a sufficient description thereof.

[0243] Bacterial, phage (Shukra et al, Eur J Microbiol Immunol(Bp). 2014; 4(2): 91-98), or eukaryotic synthesis systems can be used.

[0244] To obtain appropriate post-translational modifications such as glycosylation, it is preferable to use eukaryotic cells.

[0245] Specifically, CHO (Chinese hamster ovary) cells, PER.C6 cells (human cell line, Pau et al, Vaccine. 2001 21;19(17-19):2716-21), HEK 293b cells (human embryonic kidney cell 293), NS0 cells (cell line derived from non-secreting mouse myeloma), or EB66 cells (duck cell line, Valneva, Lyons, France) can be used.

[0246] This disclosure also provides a host cell containing at least one DNA construct encoding a polypeptide comprising the variants disclosed herein. The host cell may be any cell on which the expression vector is available. As shown 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.

[0247] The introduction of recombinant constructs into host cells is carried out by any method known in the art (e.g., 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 (e.g., bacterial or yeast artificial chromosome). If introduced into the cellular genome, such introduction may be random or targeted using methods known in the art (e.g., homologous recombination).

[0248] Bacterial host and expression Expression vectors useful for bacterial use are constructed by inserting recombinant DNA sequences, along with appropriate translation start and termination signals, into a functional leading phage having a functional promoter. The vector includes one or more phenotypic selectable markers and origins of replication to ensure vector retention and, if desired, amplification in the host.

[0249] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella tiphimurum, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0250] Eukaryotic host and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. Specifically, the cells described above can be used.

[0251] Transformed or transfected cells are cultured by methods known in the art, and polypeptides are recovered from the intracellular or extracellular fraction (depending on whether they are secreted or not).

[0252] Molecular isolation The resulting recombinant multispecific proteins can be separated and purified from the intracellular or extracellular fraction by any of the various known separation methods that utilize the physical or chemical properties of the proteins.

[0253] Specifically, methods such as precipitation, ultrafiltration, various types of liquid chromatography, such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and combinations thereof can be used.

[0254] In general, any known method for purifying recombinant polypeptides and any method used therefor is applicable to the purification of the molecules disclosed herein.

[0255] If the tag is introduced within the recombinant sequence (e.g., a polyhistidine tag), the molecule can be purified using this tag. However, it is preferable to purify the molecule using affinity.

[0256] Specifically, the fact that the molecules generated herein bind to specific targets allows for the isolation of such molecules using any affinity method (affinity column, FACS, beads).

[0257] One particular advantage of the molecules disclosed herein is that they do not need to be glycosylated to become active, and therefore can be produced in any type of cell, and do not need to be produced in eukaryotic cells. They are particularly well produced in bacterial cells.

[0258] The present invention also relates to such variants of the Sac7d family of proteins that bind to IL17 as a pharmaceutical. [Brief explanation of the drawing]

[0259] [Figure 1-1]Schematic diagrams of 15 types of proteins according to the present invention. These proteins are symmetrical. A. OB-fold variant at the N-terminus of the heavy chain. A. OB-fold variant at the C-terminus of the heavy chain. C. OB-fold variant at the N-terminus of the light chain. D. OB-fold variant at the C-terminus of the light chain. E. OB-fold variant at the N-terminus of the heavy chain and the C-terminus of the light chain. F. OB-fold variant at the N-terminus and C-terminus of the heavy chain. G. OB-fold variant at the N-terminus of the heavy chain and the N-terminus of the light chain. H. OB-fold variant at the N-terminus and C-terminus of the heavy chain and the light chain. I. OB-fold variant at the N-terminus of the heavy chain and the N-terminus of the light chain. J. OB-fold variant at the N-terminus and C-terminus of the light chain. K. OB-fold variant at the C-terminus of the heavy chain and the C-terminus of the light chain. L. OB-fold variant at the C-terminus of the heavy chain and the N-terminus and C-terminus of the light chain. M. OB fold variants at the N-terminus and C-terminus of the heavy chain and the C-terminus of the light chain. N. OB fold variants at the N-terminus and C-terminus of the heavy chain and the N-terminus of the light chain. O. OB fold variants at the N-terminus of the heavy chain and the N-terminus and C-terminus of the light chain. [Figure 1-2] The continuation of Figure 1-1 is shown below. [Figure 2] Alignment of Sac7d family proteins. [Figure 3] Loading of antibody-Sac7d variant constructs into a protein A biosensor using octet RD96. A5-HC: Sac7D variant at the N-terminus of the heavy chain. HC-A5: Sac7D variant at the C-terminus of the heavy chain. A5-LC: Sac7D variant at the N-terminus of the light chain. LC-A5: Sac7D variant at the C-terminus of the light chain. Naked antibody: Control antibody. [Figure 4] Diagram illustrating the functionality of antibodies when the Sac7d variant (H4) is fused to either the N-terminus or C-terminus of either the heavy chain (HC) or the light chain (LC). HC-H4: Sac7D variant at the C-terminus of the heavy chain. H4-LC: Sac7D variant at the N-terminus of the light chain. LC-H4: Sac7D variant at the C-terminus of the light chain. [Figure 5]Diagram illustrating the functionality of the Sac7d(H4) variant when fused to the Sac7d variant (H4) at either the N-terminus or C-terminus of either the heavy chain (HC) or light chain (LC). HC-H4: Sac7D variant at the C-terminus of the heavy chain. H4-LC: Sac7D variant at the N-terminus of the light chain. LC-H4: Sac7D variant at the C-terminus of the light chain. [Figure 6] Figure showing the double-binding capacity of various bispecific constructs by biolayer interferometry on octet RED96. HC-H4: Sac7D variant at the C-terminus of the heavy chain. H4-LC: Sac7D variant at the N-terminus of the light chain. LC-H4: Sac7D variant at the C-terminus of the light chain. [Figure 7] Figure showing the final productivity (total yield) of various constructs. H4-HC: Sac7D variant at the N-terminus of the heavy chain. HC-H4: Sac7D variant at the C-terminus of the heavy chain. H4-LC: Sac7D variant at the N-terminus of the light chain. LC-H4: Sac7D variant at the C-terminus of the light chain. HC-LC control: Control antibody. [Figure 8] Relative protein production of various antibody chimeras (with Sac7d variants against IL17) compared to naked antibodies. (A) Adalimumab and (B) Infliximab. [Examples]

[0260] Bispecific antibodies created by gene fusion of the OB fold can yield four different bispecific constructs if the OB fold is inserted into either the N-terminus or C-terminus of either the heavy or light chain. The following experiments illustrate the fusion of the Sac7d variant to IgG1κ antibody. These experiments are intended to provide proof of concept and demonstrate that it is possible to maintain the binding specificity of both the antibody fragment and the OB fold variant, as well as the level of fusion protein production (such production being equivalent to or even improved compared to the yield of antibodies without the OB fold variant fusion).

[0261] Stationary fragments of both the heavy and light chains were obtained using the commercially available encoding vectors pFUSE-CHIg-hIG1(InvivoGen) and pFUSE2ss-CLIg-hk(InvivoGen), respectively.

[0262] Each variable domain was obtained by gene synthesis (using Eurofins).

[0263] The DNA coding sequence of the Sac7d variant was directly amplified by PCR from the subcloning product in a pQE30 (Qiagen)-derived expression vector.

[0264] Next, the sequences of the constant fragment, variable domain, and Sac7d variant were assembled using Gibson Assembly to create six different vectors: three derived from the pFUSE-CHIg-hIG1 vector, and the other three forming the pFUSE2ss-CLIg-hk vector. In all cases, the vectors differed in the localization of the Sac7d variant sequence either upstream (fused at the N-terminus) or downstream (fused at the C-terminus) of the antibody chain sequence, or in its absence (no fusion).

[0265] In summary, the six types of vectors code the following: - Only the light chain of the antibody - Light chain of antibody fused to the Sac7d variant at the N-terminus - Light chain of antibody fused to the Sac7d variant at the C-terminus - Only the heavy chain of the antibody - Heavy chain of antibody fused to the Sac7d variant at the N-terminus - Heavy chain of antibody fused to the Sac7d variant at the C-terminus

[0266] The following examples illustrate the construction of polypeptides intended in this specification.

[0267] Other such polypeptides have been obtained with other Sac7d variants and antibodies, and their analysis yielded similar results to those reported below (ease of preparation, binding to both the Sac7d variant target and the antibody target).

[0268] Sac7d variants were constructed using the method disclosed in WO 2008 / 068637, employing a ribosome display to isolate variants against a given target from a library containing up to 14 mutant amino acids.

[0269] In summary, more than eight Sac7d variants were used in various experiments. Each variant bound to a different target or to a different epitope of the same target. These variants had 14 mutant amino acids (residues 7, 8, 9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, and 46), 11 mutant amino acids (residues 7, 22, 24, 26, 29, 31, 33, 38, 42, 44, and 46), 10 mutant amino acids (residues 21, 22, 24, 26, 29, 31, 33, 42, 44, and 46), 10 mutant amino acids (residues 21, 22, 24, 26, 29, 31, 33, 40, 44, and 46), and 9 mutant amino acids (residues 21, 22, 24, 26, 31, 33, 42, 44, and 46) compared to Sac7d.

[0270] The three antibodies each bind to different targets or to different epitopes of the same target (circulating protein).

[0271] Twenty-eight different combinations of fusion polypeptides were prepared using methods similar to those described below, and the results were all the same.

[0272] Specifically, the Sac7d variants used were those targeting IL17 (particularly SEQ ID NO:37 and SEQ ID NO:38) and were fused to anti-TNFα. Other Sac7d variants were used (specifically, SEQ ID NO:41 which binds to TNFα or SEQ ID NO:42 which binds to lysozyme, referred to as H4). Other Sac7d variants targeting other targets were also used. As shown above, the yield of recovered polypeptide antibody-variants of Sac7d consistently matched or exceeded the yield of antibodies without Sac7d variants, and biaffinity was always confirmed when examined.

[0273] As shown above, it is hypothesized that the addition of the Sac7d variant may stabilize the overall structure of the complex (and thus lead to improved production). Since both the antibody and the Sac7d variant maintain their structures (as demonstrated by the double-binding assay showing that they retain their function), it is thought that the results provided for various species in the examples can be generalized to the entire genus.

[0274] Example 1. Fusion to the heavy chain (N-terminus or C-terminus) This document provides an explanation of the construction of a full-length heavy chain.

[0275] Fusion of the Sac7d variant sequence at the N-terminus of the full-length heavy chain is involved in the DNA reworking of the pFUSE-CHIg-hIG1 vector by Gibson Assembly, which includes preparation by PCR amplification of two fragments and a linearized vector.

[0276] pFUSE-CHIg-hIG1 is converted to a forward oligonucleotide by PCR program 1. TIFF0007896996000017.tif4128 and reverse oligonucleotides Linearization was performed by PCR amplification using TIFF0007896996000018.tif4128.

[0277] The DNA sequence encoding the variable domain is obtained using PCR program 2, and then converted to a forward oligonucleotide. TIFF0007896996000019.tif11128 and reverse oligonucleotides The data was amplified by PCR using TIFF0007896996000020.tif4128.

[0278] The DNA sequence of the Sac7d variant was obtained using PCR program 2, and then converted to a forward oligonucleotide. TIFF0007896996000021.tif4136 and reverse oligonucleotides The data was amplified by PCR using TIFF0007896996000022.tif11128.

[0279] Fusion of the Sac7d variant sequence at the C-terminus of the full-length heavy chain is accompanied by preparation by PCR amplification of three fragments and a linearized vector. pFUSE-CHIg-hIG1 is forward oligonucleotide by PCR program 1. TIFF0007896996000023.tif4128 and reverse oligonucleotides The result is linearized by PCR amplification using TIFF0007896996000024.tif4136.

[0280] The DNA sequence of the constant domain was obtained by PCR program 2, and the forward oligonucleotide oligoCH-1 (SEQ ID NO:16) and reverse oligonucleotide were obtained. The data was amplified by PCR using TIFF0007896996000025.tif18140.

[0281] The DNA sequence encoding the variable domain is obtained using PCR program 2, and then converted to a forward oligonucleotide. The signal was amplified by PCR using TIFF0007896996000026.tif4128 and reverse oligonucleotide OligoCH-3 (SEQ ID NO:19).

[0282] The DNA sequence of the Sac7d variant was obtained using PCR program 2, and then converted to a forward oligonucleotide. TIFF0007896996000027.tif11128 and reverse oligonucleotides The data was amplified by PCR using TIFF0007896996000028.tif4133.

[0283] Construction of the full-length heavy chain lacking fusion with the Sac7d variant coding sequence involved PCR amplification of a single fragment and a linearization vector. pFUSE-CHIg-hIG1 was linearized by PCR amplification using PCR program 3 with forward oligonucleotide oligoCH-1 (SEQ ID NO: 16) and reverse oligonucleotide IL2ss_Rev (SEQ ID NO: 23). The DNA sequence encoding the variable domain was amplified by PCR using PCR program 2 with forward oligonucleotide OligoCH-6 (SEQ ID NO: 25) and reverse oligonucleotide OligoCH-3 (SEQ ID NO: 19).

[0284] Next, the PCR products were validated on 1.5% agarose gels (variable fragment amplified sequence and Sac7d variant amplified sequence) and 0.8% agarose gels (linear plasmid) containing Gel Green nucleic acid stain (1×). Bands of interest were excised under UV light and purified using Wizard SV gel and the PCR Cleanup System Kit (Promega). To increase the amount of inserted material generated, a second PCR was performed on 5 ng of purified variable fragment PCR product (4 copies) using the same PCR mix and program. After validation of band size on the 1.5% agarose gel, the same reaction was pooled and purified using Wizard SV gel and the PCR Cleanup System Kit (Promega).

[0285] PCR program 1: 30 seconds at 98°C; 25 cycles of denaturation (10 seconds at 98°C), annealing (30 seconds at 60°C), and extension (2 minutes at 72°C); then 5 minutes at 72°C; and then cool to 14°C. PCR program 2: 30 seconds at 98°C; 25 cycles of denaturation (10 seconds at 98°C), annealing (30 seconds at 60°C), and extension (30 seconds at 72°C); then 5 minutes at 72°C; and then cool to 14°C. PCR program 3: 30 seconds at 98°C; 25 cycles of denaturation (10 seconds at 98°C), annealing (30 seconds at 69°C), and extension (2 minutes at 72°C); then 5 minutes at 72°C; and then cool to 14°C.

[0286] These PCRs formed double-stranded DNA with overlapping ends, enabling directional annealing by Gibson Assembly. A 20 μL Gibson Assembly mix was prepared containing 125 ng of DNA fragments with an insertion fragment / linearized plasmid molar ratio of 5, T5 exonuclease (0.08 U, New England Biolabs), DNA Phusion polymerase (0.5 U, New England Biolabs), and Taq DNA ligase (80 U, New England Biolabs) in ISO 1× buffer. An incubation of 1 hour at 50°C was used to perform Gibson Assembly (Gibson et al., 2009).

[0287] The resulting ligated vector was used to transform E. coli strain BL21(DE3)pLysS. Cell cultures of BL21(DE3)pLysS competent cells (Coger) in the exponential growth phase (OD 600nmCell 0.5) was transformed by heat shock with 10 μL of the above Gibson assembly reaction solution and control. The cells were plated on 2YT agar medium at pH 7.5 containing chloramphenicol (10 μg / mL, Sigma-Aldrich) and zeosin (25 μg / mL, InvivoGen). Minipreps were performed using the Pure Yield Plasmid Miniprep System (Promega), and samples of each clone were stored in 20% glycerol at -80°C. The sequences were then confirmed by Sanger sequencing.

[0288] Example 2. Fusion to the light chain (N-terminus or C-terminus) This document provides an explanation of how to construct a full-length light chain.

[0289] The strategy for preparing the full-length light chain is as follows: oligonucleotides named oligoCH-1~7 are each oligoCL-1~7: The heavy chain preparation was carried out according to the instructions, except that it was replaced with TIFF0007896996000029.tif44144 and pFUSE2ss-CLIg-hk was used instead of pFUSE-CHIg-hIG1.

[0290] In addition, transformation was performed in *E. coli* DH5α F'Iq strain with the following modification: cell cultures in the exponential growth phase of DH5α F'Iq competent cells (Life technologies) (OD 600nm Cells 0.5) were transformed with 10 μL of Gibson assembly reaction solution by heat shock. The cells were plated onto pH 8 2YT agar medium containing kanamycin (25 μg / mL, VWR) and blastosidine (100 μg / mL, InvivoGen).

[0291] Example 3. Fusion of one NF to an antibody (+ bispecificity) HEK293-E6 cells were cultured in 1125 ml of FreeStyle F17 medium supplemented with 4 mM Glutamax and 0.1% Pluronic F-68 at 37 °C and 110 rpm in a humidified atmosphere of 5% CO2. Transfection was carried out as follows: When the cell density reached 1.5 - 2×10 6 cells / ml, cells were transfected with different plasmid pairs at a total of 1 mg DNA / L forming complexes with polyethyleneimine (PEI, polysciences ref 23966) at a 1:2 DNA:PEI ratio and a 2:3 HC:LC ratio. Six hours after transfection, ultra-low IgG fetal bovine serum and valproic acid were added to each culture. Five days after transfection, the culture supernatant was collected by centrifugation at 2,000 g for 20 minutes at 4 °C. The culture supernatant was filtered through 0.2 μm and loaded onto a Hitrap Protein A HP 5 ml column (GE healthcare) pre-equilibrated with PBS + 500 mM NaCl pH 7.2 binding buffer. Elution was carried out with 0.1 M citrate buffer pH 3.0, the fractions were neutralized with TRIS buffer pH 9.0, the fractions containing the product were pooled together and injected onto a Superdex 200 2660 gel filtration column. The fractions containing the product with a retention volume of 150 - 160 ml were pooled together and concentrated to 1 - 2 mg / ml with a 50 kDa membrane cut-off using a vivapsin centrifugal device.

[0292] Downstream purification of full-length IgG antibodies is a well-established process and usually involves an affinity chromatography step on a Protein A column.

[0293] The ability to capture different bispecific antibodies made by fusion of Sac7d variants at either the N-terminus or C-terminus of either the heavy or light chain was demonstrated by biolayer interferometry on an octet RED96.

[0294] One of the four bispecific constructs was loaded into a protein A biosensor at 25 nM for 300 seconds. All steps were performed at 30°C in TBS (Tris 20 mM, NaCl 150 mM, pH 7.4) with 0.01% BSA and 0.002% tween 20, while shaking at 1000 rpm. In all cases, rapid capture was observed with virtually no dissociation, reflecting the binding stability (Figure 3).

[0295] Example 4. Fusion of one NF to an antibody: Binding of either the antibody or the Sac7d variant to a target. The ability of antibodies and Sac7d variants to bind to their respective targets when involved in bispecific constructs formed by gene fusion of a Sac7d variant at either the N-terminus or C-terminus of either the heavy or light chain of an antibody was demonstrated using biolayer interferometry on octet RED96 (Fortebio).

[0296] The bispecific constructs were loaded onto a protein A sensor (Fortebio). Association and dissociation were induced for 300 and 900 seconds, respectively, 180 seconds after baseline. All steps were performed at 30°C in TBS (Tris 20 mM, NaCl 150 mM, pH 7.4) with 0.01% BSA and 0.002% tween 20, while shaking at 1000 rpm. Concentration ranges of 150 nM, 125 nM, 100 nM, 75 nM, and 50 nM were used for the Sac7d variant target. Concentration ranges of 600 nM, 200 nM, 66.66 nM, 22.22 nM, 7.40 nM, 2.46 nM, and 0.82 nM were used for the antibody target. After each run, the sensor was regenerated by 3 cycles of glycine 10 mM pH2 (10 seconds) and TBS (10 seconds).

[0297] Both the antibody and the Sac7d variant remained fully functional when ligated together, regardless of their fusion site (Figures 4 and 5).

[0298] Example 5. Fusion of one NF to an antibody: bispecificity One of the advantages of bispecific molecules lies in their ability to bind to two different targets simultaneously.

[0299] The ability of the Sac7d variant and antibody to simultaneously bind to their respective targets was demonstrated by biolayer interferometry using octet RED96. The bispecific construct was loaded onto a protein A sensor (Fortebio). After 180 seconds from baseline, a 300-second association time with the antibody target (200 nM) was performed, followed by a 300-second association time with the Sac7d variant target (300 nM). All steps were performed at 30°C in TBS (Tris 20 mM, NaCl 150 mM, pH 7.4) with 0.01% BSA and 0.002% tween 20, while shaking at 1000 rpm.

[0300] Figure 6 shows that the Sac7d variant and the antibody bind simultaneously to their respective targets.

[0301] Example 6. Increased production compared to antibody without NF or similar production Final productivity was measured, which corresponds to the amount of product obtained after the entire refining process.

[0302] Figure 7 shows that the amount of recovered product is equivalent to or better than that obtained with antibody alone.

[0303] Example 7. Fusion of two or more NFs to an antibody (to obtain specificity of 3 or more) The construction of multispecific molecules will be carried out using the same strategy as described above for bispecific molecules.

[0304] Further PCR-amplified DNA coding fragments of one Sac7d variant per additional specificity are used and added to the final molecule in the Gibson Assembly mix.

[0305] The linker and overlapping compatible ends required for the Gibson Assembly are delivered by forward and reverse oligonucleotides and added to the Sac7d variant coding sequence during the PCR amplification step. The multispecific molecule is purified as in Example 3.

[0306] Example 8. Complex formation in CHO-K1(AFG) cells Chinese hamster ovary cells (CHO-K1, ATCC CCL-61) were cultured in F-12K medium containing 1% L-glutamine and supplemented with 10% (v / v) inactivated ultra-low IgG fetal bovine serum and 1% (v / v) penicillin-streptomycin. The cells were expanded in tissue culture dishes and maintained at 37°C in a constant humidified atmosphere of 95% air and 5% CO2.

[0307] The day before transfection, cells were trypsinized and counted using 0.2% erythrosine B 1:1 (v / v). To achieve 70-90% confluence at transfection, cells were placed in 10 layers of cell culture factories at a rate of 126.4 × 10⁶ cells. 6 The seeds were seeded individually.

[0308] For each cell factory, 2 mg of DNA (1.2 mg of LC and 0.8 mg of HC) was diluted in 200 mL of Opti-MEM® I low-serum medium. Then, 2 mL of 2 mg / mL polyethyleneimine (PEI, linear, MW 25,000) was added, and the mixture was incubated at room temperature for 25 minutes. Cells were transfected by adding the DNA / PEI complex to fresh full growth medium.

[0309] Twenty-four hours after transfection, the culture medium was replaced with F-12K complete growth medium supplemented with antibiotics (blastosidine (10 μg / mL) and zeosin (300 μg / mL)) and valproic acid (final concentration 0.5 mM). Seven days after transfection, the supernatant was collected, and the protein concentration was quantified by biolayer interference technique using a protein A biosensor, according to an established standard method using naked antibodies.

[0310] The yield was compared to that of two commercially available therapeutic antibodies (adalimumab and infliximab). Gene constructs were created to generate the heavy chains of these antibodies fused to a variant of the Sac7d protein containing 12 mutations in the protein binding domain. The variant was located at the N-terminus or C-terminus of the adalimumab heavy chain and at the N-terminus of the infliximab heavy chain.

[0311] Figure 8 shows that the yield of the recovered polypeptide was maintained or improved in chimeras containing variants fused to either the N-terminus or C-terminus of the heavy chain of either (A) adalimumab or (B) infliximab.

[0312] Example 9. Use of SHuffle (AFG) technology Antibody and other complex proteins that are sensitive to oxidative stress and require post-translational modification for their effector function have, for a long time, been produced only in mammalian cell lines, HEK293, CHO, and eukaryotic expression hosts such as yeast. More recently, engineered bacterial strains such as SHuffle have also been demonstrated to be applicable to the intracellular production of functional antibodies, also known as cyclonal antibodies.

[0313] The production of chimeras formed by an antibody (adalimumab) and a variant of the Sac7d protein (nanophytin) was investigated in a SHuffle-expressing host using a bicistronic vector, as described by Robinson et al. (Nature Comm., 2015; 6:8072).

[0314] We constructed naked antibodies and three different antibody-nanophytin chimeras, each with a nanophytin fusion at either the N-terminus of the light chain, the C-terminus of the light chain, or the C-terminus of the heavy chain.

[0315] In all antibody-nanophytin chimeras, a 15-mer linker-coding sequence was inserted between the nanophytin DNA sequence and the antibody DNA sequence.

[0316] Competent T7 Shuffle express pLysY was grown in 2YT medium supplemented with chloramphenicol (10 μg / mL) at 37°C. The culture was divided into 1 mL fractions at 0.4–0.5 OD 600 nm and centrifuged at 4500 g for 5 minutes. The pellet was resuspended in 200 μL of chilled TSS buffer, then 0.5 μL of the expression vector to be transformed was added, and the mixture was incubated on ice for 30 minutes. The suspension was subjected to heat shock at 42°C for 30 seconds, followed by 5 minutes on ice. The suspension was supplemented with 800 μL of 2YT medium and incubated at 37°C with continuous shaking at 200 rpm. After 1 hour, the suspension was centrifuged at 4500 g for 5 minutes. The pellet was resuspended in 20 μL of 2YT medium, and the bacteria were spread onto a plate packed with 2YT agar supplemented with chloramphenicol (10 μg / mL) and ampicillin (100 μg / mL). The plate was incubated overnight at 37°C.

[0317] Single colonies were seeded in 10 mL of 2YT medium supplemented with chloramphenicol (10 μg / mL), ampicillin (100 μg / mL), and 1% glucose. The preculture was incubated overnight at 37°C with continuous shaking at 200 rpm. Next, 10 mL of the preculture was seeded into 200 mL of 2YT medium supplemented with chloramphenicol (10 μg / mL), ampicillin (100 μg / mL), and 0.1% glucose. The culture was incubated at 30°C with continuous shaking at 200 rpm until the OD 600 nm reached 0.7–0.8. Protein synthesis was then induced by adding IPTG (final concentration 1 mM), and the culture was maintained at 30°C for a further 16 hours with shaking at 200 rpm. The culture was stopped by centrifugation at 4°C, 3214 g, for 30 minutes. The pellet was resuspended in lysis buffer (PBS 1×, 5 mM EDTA, Bugbuster® 1×, 5 μg / mL de DNase I) and incubated for 1 hour with shaking at 200 rpm. The cell debris was then pelletized by centrifugation at 4°C, 3220 g, for 45 minutes. The supernatant was collected.

[0318] The expressed antibody chimeras were isolated by affinity chromatography using protein A resin (Pierce protein A agarose, ThermoFisher Scientific). 200 μL of 50% resin slurry was applied to a disposable 10 mL polypropylene column. The resin was washed with 10 columns of water and equilibrated with 10 columns of PBS (phosphate-buffered saline, sigma-aldrich, P4417). The supernatant was applied to the column and then washed with PBS to which 10 columns of 5 mM EDTA had been added. 0.1 M glycine pH 2-3 was used for elution. A 100 μL fraction of the eluate was collected and immediately neutralized by adding 10 μL of Tris 1M (pH 8.8). The amount of recovered material was then evaluated by spectrophotometric measurement at 280 nm. Sample purity was evaluated using a 10% SDS-PAGE gel under reducing conditions.

[0319] In this generation system, the yield of chimeras is intuitively equivalent to the yield of naked antibodies (there is no significant difference between the yields obtained).

[0320] Furthermore, evaluation of the double-binding properties of nanophytin-antibody chimeras by biolayer interferometry showed that the antibody and Sac7d variant maintained their affinity binding. To do this, various nanophytin-antibody chimeras were loaded into a protein A biosensor at 1.5 nM and incubated sequentially with TNFα (20 nM) and IL17 (125 nM) for 420 seconds, followed by a dissociation phase (900 seconds).

Claims

1. SEQ ID NO: A polypeptide that binds to IL17, including polypeptide 37.

2. SEQ ID NO: A polypeptide containing polypeptide 38 that binds to IL17.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide of SEQ ID NO: 37 or SEQ ID NO: 38 is fused to another protein or polypeptide.

4. The polypeptide according to claim 3, wherein the other protein or polypeptide comprises a variant of a protein of the Sac7d family, the variant comprising 4 to 20 or 5 to 20 mutant residues at the binding site of the protein of the Sac7d family, and the mutant amino acids correspond to amino acids selected from the group consisting of 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 of Sac7d, with reference to SEQ ID NO:

1.

5. The polypeptide according to claim 4, wherein a variant of the Sac7d family protein binds to TNFα.

6. The polypeptide according to claim 3, wherein the other protein is an antibody.

7. The polypeptide according to claim 6, wherein the polypeptide of SEQ ID NO: 37 or SEQ ID NO: 38 is fused to two heavy chains or two light chains of the antibody.

8. The polypeptide according to claim 6, wherein the polypeptide of SEQ ID NO: 37 or SEQ ID NO: 38 is fused to one heavy chain and one light chain of the antibody.

9. The polypeptide of SEQ ID NO: 37 or SEQ ID NO: 38 and a variant of the Sac7d family protein are fused to the heavy and / or light chain of the antibody, and the variant contains 4 to 20 or 5 to 20 mutant residues at the binding site of the Sac7d family protein, and the mutant amino acids are (SEQ ID A polypeptide according to any one of claims 6 to 8, corresponding to an amino acid selected from the group consisting of 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 of Sac7d (see NO: 1).

10. The polypeptide according to any one of claims 6 to 9, wherein the antibody is a therapeutic antibody.

11. The polypeptide according to any one of claims 6 to 10, wherein the antibody binds to a protein selected from the group consisting of TNFα, IL23, IL12, IL4, IL13, IL31, IL31 receptor, and tumor-specific antigens.

12. The polypeptide according to any one of claims 6 to 10, wherein the antibody binds to a protein selected from the group consisting of Her2, PDL1 (programmed death ligand 1, CD274), and CTLA4.

13. A polypeptide according to claim 1 or 2, conjugated to an organic molecule.

14. A gene construct comprising a DNA sequence encoding a polypeptide according to any one of claims 1 to 5.

15. a. A sequence encoding the heavy chain of an antibody, which is fused at its 3' end with a sequence encoding the polypeptide described in any one of claims 1 to 5. b. A sequence encoding the heavy chain of an antibody, which is fused at its 5' end with a sequence encoding the polypeptide described in any one of claims 1 to 5. c. A sequence encoding the light chain of an antibody, which is fused at its 3' end with a sequence encoding the polypeptide described in any one of claims 1 to 5. d. A sequence encoding the light chain of an antibody, which is fused at its 5' end with a sequence encoding the polypeptide described in any one of claims 1 to 5. A gene construct containing a DNA sequence selected from the group consisting of the following.

16. A vector comprising the gene construct according to claim 14 or 15.

17. A host cell comprising the gene construct described in claim 14 or 15 in its genome.

18. A method for producing a polypeptide according to any one of claims 1 to 5, a. A step of culturing a cell culture in which cells have been transformed by the gene construct described in claim 14, and b. Steps for recovering polypeptides The method comprising the steps of:

19. A method for producing a polypeptide according to any one of claims 6 to 12, a. A step of culturing a cell culture in which cells have been transformed with the gene construct described in claim 15 and a gene construct encoding a complementary antibody chain thereof, and b. Steps for recovering polypeptides The method comprising the steps of:

Images