Multispecific molecules

Abstract

The present invention provides proteins with multispecific binding capacity by fusion of OB-fold variants (in particular from the Sac7d family) to the light or heavy chains of antibodies.SOLUTION: Provided are polypeptides comprising modified antibodies, wherein at least one variant of a Sac7d family of proteins is fused to at least one of the heavy or light chains of the antibodies, wherein the variant comprises 4-20 or 5-20 mutated residues in the binding sites of the Sac7d family of proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of molecular biology and, in particular, relates to the development of novel molecules and conjugates with multiple binding specificities. [Background technology]

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

[0003] There are different types of bispecific antibody formats: - Bispecific antibodies maintain an intact IgG structure. They benefit from Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement fixation, and FcRn-mediated recycling (responsible for their long half-life). They contain two Fab arms and an Fc region, with the two Fab portions binding different antigens. Typically, each heavy and light chain pair is derived from a unique mAb. These bispecific antibodies are often produced by quadroma or hybrid hybridoma techniques. - Fc-deleted antibodies, which include chemically linked Fabs consisting of only the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFv), or fusion proteins mimicking the variable domains of two antibodies.

[0004] Full IgG bispecific antibodies can be further distinguished depending on their engineering process, resulting in either symmetric or asymmetric molecules, which pose different challenges.

[0005] Asymmetric bispecific antibodies are the result of enzymatically assisted in vitro chain swapping or the fusion of different hybridomas (called quadromas). However, these techniques produce the expected bispecific molecules in a mixture with a high content of undesired by-products that must be separated. As an example, quadroma technology randomly pairs the heavy and light chains of two antibodies expressed in a single cell, theoretically resulting in 16 different combinations (10 different molecules), of which only one is bispecific and the remaining pairs are nonfunctional or monospecific molecules. Such a process produces a statistical yield (and recovery rate) of bispecific molecules in solution of 12.5%.

[0006] The key challenge then lies in the ability to drive the correct pairing of heavy and light chains. This has been assessed by techniques such as knob-into-hole for heavy chains and crossmab for light chains. The implementation of these techniques allows for a significant reduction in the number of by-products during the production process, but this achievement is only possible through the insertion of multiple point mutations that support the correct association of heavy and light chains, which does not have any consequences for the ability of the molecule to be further developed. Specifically, such antibodies may be immunogenic, with the Fc region eliciting harmful downstream immune responses.

[0007] Symmetric bispecific antibodies, on the other hand, rely on the fusion of additional ScFv domains (tandem Fab-IgG, DVD-IG, CODV-Ig...) to provide additional specificity to the initial IgG. Significant challenges to consider here rely on the linker connecting the ScFv to the IgG and the inherent stability of the linked ScFv, which may affect manufacturability and production. This is particularly true for bispecific antibodies and becomes even more challenging when considering tria- or tetraspecific 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, binding to both tumor cells and cytotoxic cells. Bispecific antibodies also have high cytotoxicity and bind to relatively weakly expressed antigens. Furthermore, targeting two or more molecules can be useful for avoiding the regulation of parallel pathways and avoiding resistance to treatment.

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

[0010] Yu et al. (MAbs. 2014;6(6):1598-607) D2 discloses adalimumab (a humanized antibody that binds to TNFα) fused to the IL6-binding affibody ZIL6. Affibody molecules are antibody mimetics consisting of small proteins based on a three-helix bundle domain engineered to bind to target proteins with high affinity, and are distinct from the OB-fold domain disclosed herein. This document does not describe any information related to yield.

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

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

[0013] Spangler et al. (J Mol Biol. 2012 Sep 28;422(4):532-44) discloses cetuximab-based anti-EGFR antibodies fused to an engineered EGFR-binding variant of the 10th type III domain of human fibronectin. These conjugates differ from the conjugates disclosed herein.

[0014] WO 2008 / 100470 (Patent Document 1) discloses Ig fusion proteins and does not describe the OB-fold proteins and variants disclosed herein, nor their use in generating engineered antibodies.

[0015] Thus, multispecific molecules that are easily produced with good yields and that are also expected to be as stable as antibodies are still under development.

[0016] Applicant proposes to generate such molecules by creating novel engineered proteins comprising antibodies (conventional or bispecific) in which an OB-fold domain is located at the N- or C-terminus of at least one of the antibody's heavy or light chains. The OB-fold domain is preferably fused or linked to the antibody chains by the following genetic manipulation: the protein engineered protein is generated by genetically fusing the domain-encoding sequence to the 5' and / or 3' ends of the antibody heavy and / or light chain sequences and introducing this modified sequence into an appropriate cell line or bacterial strain. It should be noted that a few amino acids (linker) may be added between the antibody and the OB-fold domain.

[0017] The examples show that polypeptides of the present disclosure can be obtained in higher or at least similar or equivalent yields compared to antibodies not fused with an OB domain. The examples show that such effects can be obtained with various constructs. Since the antibodies and OB-fold domains (especially variants of proteins of the Sac7d family) retain their functionality, this indicates that they retain their structure. It is believed and hypothesized that the OB-fold domain, particularly when selected as a protein of the Sac7d family, may stabilize the antibody chains during the production process, thereby enabling improved yields. Therefore, the effects observed with the various conjugates in the examples can be generalized to all other antibodies and OB-fold variants with 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] Mol Cancer Ther. 2014 Aug;13(8):2030-9 [Non-patent document 3] PLoS One. 2012;7(7):e40331 [Non-patent document 4] J Mol Biol. 2012 Sep 28;422(4):532-44 Summary of the Invention

[0020] The present invention thus relates to polypeptides comprising modified antibodies, which have been 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, preferably at the N-terminus or C-terminus of the heavy or light chain.

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

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

[0023] The OB-fold domains and antibodies may be capable of binding to different targets or different epitopes of the same target, thereby allowing the proteins disclosed herein to bind to multiple of these targets or multiple epitopes of a given target.

[0024] As shown in Figure 1 (which depicts only symmetric molecules), two or more variants of an OB-fold domain antibody may be attached to the light or heavy chain of the antibody. It is preferred that the proteins of the invention are symmetric (i.e., comprise the same heterodimer), i.e., that the same OB-fold variant is fused to both light or heavy chains of the protein. However, embodiments in which only one OB-fold variant is attached to only one of the heavy or light chains are also included, as are embodiments in which one OB-fold variant is attached to the light or heavy chain of the protein and another (different) OB-fold variant is attached to the other light or heavy chain. Thus, multiple combinations are possible, and Figure 1 is not intended to be exhaustive.

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

[0027] A protein may contain one OB-fold variant. It may contain two OB-fold variants (preferably if these are the same and present symmetrically about the protein). It may contain three OB-fold variants. It may contain four OB-fold variants (preferably if there are two pairs of OB-fold variants and the protein is symmetric). It may contain five OB-fold variants. It may contain six OB-fold variants (preferably if there are three pairs of OB-fold variants and the protein is symmetric). It may contain seven OB-fold variants. It may contain eight OB-fold variants (preferably if there are four pairs of OB-fold variants; in this case the protein is symmetric). It is recalled that a symmetric protein is one made up of the same heavy and light chains.

[0028] The present invention provides a. A sequence encoding the heavy chain of an antibody fused at its 3' end to a sequence encoding a variant of an OB-fold protein. b. A sequence encoding the heavy chain of an antibody fused at its 5' end to a sequence encoding a variant of an OB-fold protein. c. A sequence encoding the light chain of an antibody fused at its 3' end to a sequence encoding a variant of an OB-fold protein. d. A sequence encoding an antibody light chain fused at its 5' end to a sequence encoding a variant of an OB-fold protein. The present invention also relates to a genetic construct comprising a DNA sequence selected from the group consisting of:

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

[0030] The present invention also provides a. culturing a cell culture, wherein the cells have been transformed with the described gene construct and its complementary antibody sequence; and b. Recovering the resulting polypeptide. The present invention also relates to a method for producing a polypeptide of the present invention, comprising the steps of:

[0031] Such culturing is carried out under conditions that allow expression of the gene construct and the protein expressed from the gene construct, this method being particularly suitable when the polypeptide is secreted into the culture medium.

[0032] The present invention also provides (a) culturing cells that have been transduced with the described gene construct and its complementary antibody sequence; (b) harvesting the cells; and (c) disrupting the cells to obtain a crude extract containing the polypeptide described. The present invention also relates to a method for producing a polypeptide of the present invention, comprising the steps of:

[0033] The invention also includes methods for generating genetic constructs for producing the described polypeptides, comprising genetically fusing a sequence encoding a variant of an OB-fold domain to at least the 5' or 3' end of an antibody heavy or light chain, and recovering the resulting genetic construct.

[0034] The present invention also relates to complexes consisting of the polypeptides disclosed herein bound to at least one target by an OB-fold variant or antibody binding site, such target being therefore the antigen of an antibody or 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 genetic modification consisting of the fusion of an antibody sequence (light or heavy chain coding sequence) with an OB-fold coding sequence.

[0036] In a preferred embodiment, the present method ensures that 100% of the proteins recovered after production in cells transformed with the appropriate gene vector are of the same type, thereby solving the problem of bispecific antibody purification recalled above, where the statistics (and recovery rate) of bispecific molecules is generally approximately 12.5% ​​in solution. Furthermore, the methods disclosed herein make it possible to obtain multispecific molecules with the same degree of diversity as antibodies (chimeric, human, murine, rat, or any idiotypic origin). It should also be noted that the overall structure of the antibody is not altered, so the molecules retain all of the characteristics and properties of antibodies (including any effects due to non-Fab fragments, such as those due to the Fc fragment). It is also quite surprising that the molecules disclosed herein are able to bind at both the antibody and OB-fold domain binding sites, despite the modifications made to the antibody. Therefore, such bispecific binding ability suggests that the proper folding of the polypeptide fragment molecules (antibody and OB-fold domain portions) is maintained. Finally, it was surprising to observe that the production yield of the molecules is maintained (compared to the antibody production yield) and even improved. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention OB-fold domain As shown above, the OB-fold domain can be engineered into variants that bind to specific targets by introducing mutations within its binding site.

[0038] In the context of the present application, the term "OB-fold domain" (or "OB-fold protein") refers to naturally occurring OB-fold proteins, but also to domains having an OB-fold and isolated from more complex proteins. These OB-fold domains are specifically described in more 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-fold protein or a domain having an OB-fold at the N- or C-terminus to a protein or domain of interest, for example, a tag that allows for better purification.

[0039] In the context of the present invention, however, it is preferred to use only domains with the OB-fold topology and not the full-length protein, if such polypeptides contain other sequences not present in the OB-fold domain. Indeed, it is preferred to use proteins as small as possible, and preferably, the variant of the OB-fold domain used in the context of the present invention contains at most 300 amino acids, preferably at most 200 amino acids, preferably at most 175 amino acids, more preferably at most 150 amino acids, more preferably at most 100 amino acids. In one particular embodiment, it contains at most 80 or at most 70 amino acids.

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

[0041] In particular, document WO 2008 / 068637 describes precisely how to identify the binding domains of OB-fold proteins.

[0042] 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 with OB-fold domains. With reference to the sequence of Sac7d (SEQ ID NO:1), these are residues V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50, and P51.

[0043] Binding domains of other OB-fold proteins can be identified as described in WO 2008 / 068637. This application shows that it is possible to perform 3D structural superpositions 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). Thus, for any OB-fold protein (or any OB-fold domain), it is easy to identify the amino acids that are involved in the binding site and that correspond to the Sac7d amino acids mentioned above. Therefore, by providing amino acids that can be mutated in one of these proteins, it becomes possible to identify the corresponding amino acids in any other OB-fold domain.

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

[0045] The teachings of WO 2008 / 068637 also teach that amino acids can be inserted optionally into loops of OB-fold proteins, in particular proteins of the Sac7d family; in particular, insertions of 1 to 15 amino acid residues can be made in loop 3 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example in the region of residues 25 to 30 of Sac7d, preferably between residues 27 and 28; insertions of 1 to 15 amino acid residues can be made in loop 4 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example in the region of residues 35 to 40 of Sac7d, preferably between residues 37 and 38; and insertions of 1 to 20 residues can be made in loop 1 (as defined in Figures 1b and 2 of WO 2008 / 068637), for example in the region of residues 7 to 12 of Sac7d, preferably between residues 9 and 10.

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

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

[0048] WO 2012 / 150314 shows that mutations from one protein of the Sac7d family can be carried to another protein of the same family. This portability results in starting from a mutant of one protein of the Sac7d family to create a mutant of another protein of the Sac7d family. The initial mutant may be obtained, in particular, by carrying out the process of WO 2008 / 068637.

[0049] It has already been shown that it is possible to obtain such mutants for 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) also demonstrated the possibility of obtaining such proteins starting from Sso7d. Gocha et al. (Scientific Reports 7, Article number: 12021(2017)) also reported that mutants can be obtained from Sso7d.

[0050] Examples of OB-fold domains Non-limiting examples of OB-fold proteins that can be used in accordance with the present invention include the N-terminal domains of Sac7d, Sso7d, SEB (Papageorgiou et al., 1998), the A chain of Shiga-like toxin IIe (PDB 2bosa), human Neutrophil Activatin Peptide-2 (NAP-2, PDB 1tvxA), the molybdenum-binding protein (modg) of Azotobacter vinelandii (PDB 1h9j), the N-terminal domain of SPE-C (Roussel et al., 1997), the B5 subunit of E. coli Shiga-like toxin (Kitov et al., 2000), Cdc13 (Mitton-Fry et al., 2002), and the cold shock DNA-binding domain of the human Y-box protein YB-1 (Kloks et al., 2002), the E. coli inorganic pyrophosphatase EPPase (Samygina et al., 2001), or any of the proteins listed in Table 3 of (Arcus, 2002), such as 1krs (Lysyl-tRNA synthetase LysS, E. coli), 1c0aA (Asp-tRNA synthetase, E. coli), 1b8aA (Asp-tRNA synthetase, P. kodakaraensis), 1lylA (Lysyl-tRNA synthetase LysU, E. coli), 1quqA (Replication protein A, 32 kDa subunit, human), 1quqB (Replication protein A, 14 kDa subunit, human), 1jmcA (Replication protein A, 70 kDa subunit (RPA70 fragment, human), 1otc (Telomere end binding protein, O. nova), 3ullA (mitochondrial ssDNA-binding protein, human), 1prtF (pertussis toxin S5 subunit, B. pertussis), 1bcpD (pertussis toxin S5 subunit (ATP-binding), B. pertussis), 3chbD (cholera toxin, V. cholerae), 1tiiD (heat-labile toxin, E. coli), 2bosA (Verotoxin 1 / Shiga toxin B-pentamer, E. coli), 1br9 (TIMP-2, human), 1an8 (superantigen SPE-C, Streptococcus pyogenes (S.pyogenes), 3seb (superantigen SPE, S. aureus), 1aw7A (toxic shock syndrome toxin, S. aureus), 1jmc (major cold shock protein, E. coli), 1bkb (translation initiation factor 5a, P. aerophylum), 1sro (S1 RNA-binding domain of PNPase, E. coli), 1d7qA (translation initiation factor 1, eIF1a, human), 1ah9 (translation initiation factor 1, IF1, E. coli), 1b9mA (Mo-dependent transcriptional regulator ModE, E. coli), 1ckmA (RNA guanylyltransferase, Chlorella virus, PBCV-1), 1a0i (ATP-dependent DNA ligase, bacteriophage T7), 1snc (staphylococcal nuclease, Staphylococcus aureus), 1hjp (DNA helicase RuvA subunit, N-terminal domain, 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, S. cerevisiae), and 2prd (inorganic pyrophosphatase, T. thermophilus).

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

[0052] These proteins and this family are specifically described in WO 2008 / 068637. Thus, within the context of the present invention, a protein belongs to the Sac7d family if it has one of the sequences SEQ ID NO:1 to SEQ ID NO:14 or if it has 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 which the dash - indicates no amino acid and the proteins do not all have the same size). This Sac7d family specifically includes the Sac7d or Sac7e protein from Sulfolobus acidocaldarius, the Sso7d protein from Sulfolobus solfataricus, the DBP 7 also called Sto7 protein from Sulfolobus tokodaii, the Ssh7b protein from Sulfolobus shibatae, the Ssh7a protein from Sulfolobus shibatae, the Mse7 from Metallosphaera sedula, the Mcu7 from Metallosphaera cuprina, the Aho7a or Aho7b or Aho7c from Acidianus hospitalis, and the Sac7d or Sac7e protein from Sulfolobus islandicus. These include Sis7a or Sis7b from Sac7d and the p7ss protein from Sulfolobus solfataricus. Given the extensive sequence similarity of the Sac7d family of proteins, it is straightforward and easy to identify the amino acid in other proteins that corresponds to a given amino acid in Sac7d.

[0053] It should be noted that the number of mutated residues in the variant (compared to the wild-type protein) is preferably between 5 and 25. The invention can be practiced with variants that preferably have at least 5, more preferably at least 7 or 8, and even more preferably at least 10 substituted amino acids compared to the wild-type OB-fold protein (or domain), but generally have fewer than 25, more preferably fewer than 24, and even more preferably fewer than 20, or fewer than 15 or 14 substituted amino acids. Preferred are cases where 7, 8, 9, 10, 11, 12, 13, or 14 amino acids are mutated in the binding site of the OB-fold domain compared to the wild-type OB-fold domain. These mutations are introduced at amino acids corresponding to V2, K3, K5, K7, Y8, K9, G10, E11, K13, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, Y34, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50, and P51 of Sac7d (SEQ ID NO:1).

[0054] In particular embodiments, the number of mutated amino acids is between 7 and 14 (inclusive).

[0055] In one particular embodiment, these variants may also contain the amino acid insertions set forth above.

[0056] As shown, the proteins of the Sac7d family are Sac7d or Sac7e from Sulfolobus acidocaldarius, Sso7d from Sulfolobus solfataricus, DBP 7 also called Sto7 from Sulfolobus tokodaii, Ssh7b from Sulfolobus shibatae, Ssh7a from Sulfolobus shibatae, Mse7 from Metallosphaera sedula, Mcu7 from Metallosphaera cuprina, Aho7a or Aho7b or Aho7c from Acidianus hospitalis, Sis7a or Sis7b from Sulfolobus islandicus, and p7ss from Sulfolobus solfataricus. The various sequences of 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] Variants of this Sac7d family of proteins may be called nanofitins. The invention is thus preferably carried out with respect to variants of the proteins represented by any of SEQ ID NO:1 to SEQ ID NO:14 or with the sequence SEQ ID NO:15, in particular variants of Sac7d.

[0058] In a preferred embodiment, the mutated 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 above-identified amino acids of Sac7d. It should be noted that the variant will contain mutated amino acids selected from among these amino acids (preferably 7 to 12 as described above) and may also contain other mutated amino acids (preferably 0 to 5) in other regions (i.e., selected from other residues of Sac7d). As noted above, A59, R60, A61, E62, R63, E64, and / or K66 can be deleted.

[0059] In preferred embodiments, the mutated amino acids are 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 rounds of enrichment of combinatorial libraries using ribosome display).

[0061] Thus, the OB-fold domain variant is preferably a variant of a protein of the Sac7d family (Sac7d variant) containing 5 to 20 (preferably 7 to 14) mutated amino acids in the binding site of the protein, preferably 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). Preferably, the Sac7d variant contains 7 to 14 mutated amino acids selected from the group consisting of K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44, and S46.

[0062] An 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 have been "randomized," i.e., replaced with random amino acids. Screening of these libraries makes it possible to identify variants of these proteins that specifically bind, typically with strong affinity (application WO 2008 / 068637 in fact describes affinities of the order of 1 nanomolar), to a target of interest other than the natural ligand of the wild-type protein from which the combinatorial library was generated.

[0063] As set out above, Applicants have been able to show that it is possible to create specific binding proteins by fusing an OB-fold domain that binds to a given target to the heavy or light chain of an antibody that binds either the same target (thereby increasing specificity and affinity) or to another target (thereby obtaining a multispecific binding protein).

[0064] This fusion can be at the N-terminus and / or C-terminus of the antibody chain (heavy and / or light chain). It should be noted that, in particular, when small OB-fold domains (approximately 70 amino acids) such as those from the Sac7d family of proteins are used, it becomes possible to obtain molecules with the structure of an antibody (two light chains paired with two heavy chains, and such dimers paired together) with an antibody region and an additional binding region consisting of a modified OB-fold domain.

[0065] antibody Antibodies are large, Y-shaped proteins that recognize antigens via the 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 is composed of structural domains called immunoglobulin domains.

[0066] There are several different types of antibody heavy chains that define five different fragments (Fc) to which an antigen-binding fragment may be attached, allowing 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 in all antibodies of the same isotype, but different in antibodies of different isotypes.

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

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

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

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

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

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

[0073] Preferably, the antibody that is part of the molecules disclosed herein is an antibody that contains two identical heavy chains (about 400-500 amino acids, generally about 450 amino acids) and two identical light chains. Therefore, the antibody contains the same Fab variable region. Therefore, this antibody is a monospecific antibody in which both parts of the antibody (combination of light and heavy chains) bind to the same epitope of an antigen.

[0074] However, antibodies may have different heavy and / or light chains. Specifically, in some embodiments, the antibody is a bispecific antibody. Thus, the term "antibody" encompasses both the "classical antibodies" disclosed above, which have the same heavy and light chains, as well as engineered antibodies with two or more specificities.

[0075] In certain embodiments, the antibodies have one heavy and light chain from one antibody and another heavy and light chain from another antibody.

[0076] Specifically, antibodies that can be used in the molecules disclosed herein include Triomab (Trion Pharma), knobs-into-holes (KIH) IgG (Xu et al., MAbs. 2015; 7(1): 231-242, Dillon et al., MABS 2017, 9(2), 213-230) (possibly sharing 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. 2014;32(2):191-8), DVD (dual variable 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 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 chimeras consisting of two half antibodies, each with one light 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 disease, slow the progression of disease, or alleviate symptoms of disease.

[0079] The therapeutic antibody is preferably selected from the group consisting of: 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate pentetate, Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab iximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab-mertansineMertansine, Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan Tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, CR6261, Crenezumab, Crotedumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumabpegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dinutuximab, diridavumab, domagrozumab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab Alizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab situxetanCituxetan, Epratuzumab, Erenumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fivatuzumab uzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetanTiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imicilomab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Inolimomab, Inotuzumab ozogamicin ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab emtansine Emtansine, Lebrikizumab, Remalesomab, Lendalizumab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetlaxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, lucatumumab, lulizumab pegolpegol, Lumiliximab, Lumretuzumab, MABp1, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximab emtansine Emtansine, Narnatumab, Natalizumab, Navicixizumab, Navivumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomabmerpentan, obiltoxaximab, obinutuzumab, ocralizumab, ocrelizumab, odulimomab, ofatumumab, olaritumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab , Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab Pinatuzumab vedotin, Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab vedotinvedotin), Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Risankizumab, Rituximab, Rivabazumab pegol pegol, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovalpituzumab tesirine, Rovelizumab, Ruplizumab (Ruplizumab), Sacituzumab govitecan, Samalizumab, Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tamtuvetmab, Tanezumab, Taplitumomab peptoxetane paptox, Tarextumab, Tefibazumab, TelimomabAritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Ticilimumab (= Tremelimumab), Tigatuzumab, Tildrakizumab, Timolumab, Tisotumab vedotin, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab Celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximab talirine, VandortuzumabVedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Borsetuzumab mafodotin, votumumab, xentuzumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

[0080] Specifically, such therapeutic antibodies include adalimumab (against TNFα, sold under the name Humira®); infliximab (against TNFα, sold under the name Remicade®); cetuximab (against EGFR, sold as Erbitux®); secukinumab (against IL17, sold as Cosentyx®); pembrolizumab (against PD1, sold as Keytruda®); bevacizumab (against VEGF-A, sold under the name Avastin®); etrolizumab (against a4b7); vedolizumab (against a4b7); tremelimumab (against CTLA4); ipilimumab (against CTLA4, marketed as Yervoy®); necitumumab (against EGFR, marketed as Portrazza®); panitumumab (against EGFR, marketed as Vectibix®); lebrikizumab (against IL13); tralokinumab (against IL13); ixekizumab (against IL17, marketed as Taltz®) brodalumab (directed against IL17R, marketed as Lumicef®); dupilumab (directed against IL4R, marketed as Dupixent®); guselkumab (directed against IL23); tildrakizumab (directed against IL23); risankizumab (directed against IL23); briakinumab (directed against IL12 and IL23); ustekinumab (directed against IL12 and IL23); nivolumab (directed against PD1, marketed as Opdivo®); atezolizumab (directed against PD-L1, marketed as Tec marketed as entriq®); avelumab (against PD-L1); ranibizumab (against VEGF-A, marketed as Lucentis®); brolucizumab (against VEGF-A); trastuzumab (against Her2, Herceptin®); amatuximab (against mesothelin); tavolixizumab (against OX40); pogalizumab (against OX40); urelumab (against 4-1BB); utomilumab (against 4-1BB); BMS 986016 (against LAG3); lirilumab (against KIR); MEDI 570 (against ICOS);Selected from the group consisting of LY3321367 (against TIM3); lulizumab (against CD28); TAB08 (against CD28); and onartuzumab (against cMet, MetMab);

[0081] target The target of the multispecific molecules of the present invention can be any molecule or antigen of interest. Specifically, the multispecific molecules target cancer or hyperproliferative diseases, such as lymphoma (e.g., non-Hodgkin's 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 preferred embodiments, the OB-fold domain binds to a target disclosed below.

[0083] It may be 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. 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 proteins:TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Base1, 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 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 proteins :TNFa, IL23, IL12, IL33, IL4, IL13, IL5, IL6, IL4, IFNg, IL17, RANKL, Base1, α-synuclein, tau, amyloid.

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

[0086] Of particular interest and preference are complexes in which the antibody binds to TNFalpha and the OB-fold domain binds to IL17, and vice versa.

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

[0088] Creation of artificial molecules Vectors that allow for the production of the molecules disclosed herein are produced by conventional molecular genetic methods.

[0089] In summary, the gene sequence encoding the Sca7d variant is linked 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, a linker can be introduced between the two gene sequences, as long as no frameshift or stop codon is introduced.

[0090] Thus, the expressed fusion protein should be (from N- to C-terminus) either: (a) antibody heavy chain-(linker, if present)-OB-fold domain variant; (b) the OB-fold domain variant-(and linker, if present)-antibody heavy chain; (c) Antibody light chain-(linker, if present)-OB-fold domain variant (d) OB-fold domain variant—(linker, if present)—antibody light chain

[0091] Clearly, further genetic modifications can be envisaged, such as the further introduction of "tag" molecules at the C- or N-terminus, which would improve purification, or force direction of the OB fold, or allow protease-mediated release of the OB fold, or modify the pharmacokinetics of the compound, or target specific types of tissue, or chelate lanthanides or radionuclides, or allow conjugation to a payload which may be a maytansine or auristatin derivative.

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

[0093] As shown below, other nucleic acid molecules can be obtained, such as those encoding: (a) OB-fold domain variant-(linker, if present)-antibody heavy chain-(linker, if present)-OB-fold 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 above-described nucleotide sequences are used in conjunction with a vector, such as a plasmid, phagemid, phage, or viral vector.

[0095] These recombinant nucleic acid molecules can be produced by the techniques described in Sambrook et al., 1989 (Sambrook J, Fritschi EF and Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York). Alternatively, the DNA sequences can be chemically synthesized, for example, using a synthesizer.

[0096] The recombinant constructs of the present invention include expression vectors capable of expressing RNA, thereby resulting in the production of proteins from the above-mentioned gene sequences. Thus, the vectors may further comprise control sequences, including a suitable promoter operably linked to the open reading frame (ORF) of the gene sequences disclosed herein. The vectors may further comprise selectable marker sequences, such as antibiotic resistance genes. Specific initiation and bacterial secretion signals may also be required for efficient translation of the coding sequence when bacteria are used as expression hosts.

[0097] Molecular generation Cells are transfected or transformed with vectors containing sequences encoding antibody heavy and light chains, at least one of which comprises a variant of an OB-fold protein as discussed above.

[0098] The cells are cultured under conditions that allow the protein to be expressed and preferably secreted. The cell culture conditions are those commonly used for recombinant antibody production and are known in the art. Such conditions, known in the art, can also be optimized by those skilled in the art as needed. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461) provide an overview of such methods and provide a thorough description thereof.

[0099] Bacterial, phage (Shukra et al, Eur J Microbiol Immunol(Bp). 2014; 4(2): 91-98), or eukaryotic production 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 293 cells), NS0 cells (a cell line derived from a non-secretory mouse myeloma), or EB66 cells (duck cell line, Valneva, Lyons, France) can be used.

[0102] The present disclosure also provides a host cell that contains at least one of the DNA constructs described above.Host cell can be any cell that expression vector can be used for.As shown above, it can be higher eukaryotic host cell such as mammalian cell, lower eukaryotic host cell such as yeast cell, or prokaryotic cell such as bacterial cell.

[0103] The introduction of recombinant constructs into host cells can be carried out by any method known in the art (for example, calcium phosphate transfection, lipofection, DEAE, dextran-mediated transfection, electroporation, or phage infection). The vector can be inserted into the genome of host cells, or can be maintained as an extragenomic vector (for example, bacterial artificial chromosome or yeast artificial chromosome, etc.). When introduced into the cell genome, this introduction can be random, or can be targeted using methods known in the art (such as homologous recombination).

[0104] Bacterial host and expression Expression vectors useful for bacterial use are constructed by inserting a recombinant DNA sequence into an operable leading phage carrying a functional promoter, along with appropriate translation initiation and termination signals. The vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide for amplification within 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 hosts and expression Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. Specifically, the cells described above can be used.

[0107] The transformed or transfected cells are cultured by methods known in the art and the polypeptide is recovered from the intracellular or extracellular fraction (depending on whether it is secreted or not).

[0108] Molecular isolation The produced recombinant multispecific protein can be separated and purified from the intracellular or extracellular fraction by any of a variety of known separation methods that exploit the physical or chemical properties of the protein.

[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] Generally, any method known and used to purify recombinant antibodies is adaptable to purify the molecules disclosed herein.

[0111] If a tag has been introduced into the recombinant sequence (e.g., a polyhistidine tag), the tag can be used to purify the molecule. However, it is preferred to purify the molecule using affinity.

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

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

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

[0115] In particular, the fact that the molecules generated herein bind to specific targets (antigens for antibodies and targets for OB-fold variants) can be used to isolate such molecules using any affinity-based method (affinity columns, FACS, beads).

[0116] One particular advantage of the methods 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 is introduced into the cells: - Gene sequence encoding the antibody light chain - A gene sequence encoding an antibody heavy chain in which a variant of the OB-fold domain has been introduced into the 5' end of the heavy chain.

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

[0119] Note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB-fold domain variants at the N-terminus of the heavy chain), as illustrated in Figure 1.A.

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

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

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

[0123] In another embodiment, the following is introduced into the cell: - A gene sequence encoding an antibody light chain, with a variant OB-fold domain introduced into the 5' end of the light chain. - The gene sequence that encodes the heavy chain of an antibody.

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

[0125] Note that the resulting molecule, as illustrated in Figure 1.C, is symmetric and has four binding sites (two antibody Fab binding sites and two OB-fold domain variants at the N-terminus of the light chain).

[0126] In another embodiment, the following is introduced into the cell: - A gene sequence encoding an antibody light chain, with a variant OB-fold domain introduced into the 3' end of the light chain. - The gene sequence that encodes the heavy chain of an antibody.

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

[0128] Note that the resulting molecule is symmetrical and has four binding sites (two antibody Fab binding sites and two OB-fold domain variants at the C-terminus of the light chain), as illustrated in Figure 1.D.

[0129] In another embodiment, the following is introduced into the cell: - A gene sequence encoding an antibody light chain, with a variant OB-fold domain introduced into the 3' end of the light chain. - Gene sequence encoding the antibody heavy chain - The gene sequence that encodes the light chain of an antibody.

[0130] The resulting product, after association of the heavy and light chains, is: - Symmetric bispecific molecules (expected percentage 25%) that bind to the antigen of the antibody and the target of a variant of the OB-fold domain at the C-terminus of the light chain. - Asymmetric bispecific molecules (expected percentage 50%) that bind to the antigen of the antibody and to the target of a variant of the OB-fold domain at the C-terminus of only one of the light chains. - Symmetric antibodies (expected percentage 25%).

[0131] Note that the resulting molecule is symmetric and has four binding sites: two antibody Fab binding sites and two OB-fold domain variants at the C-terminus of the light chain.

[0132] Other combinations can be performed when transforming cells with: - A gene sequence encoding an antibody light chain in which a variant OB-fold domain has been introduced into the 5' end of the light chain. - Gene sequence encoding the antibody heavy chain - the gene sequence encoding the light chain of the antibody, or - A gene sequence encoding an antibody heavy chain in which a variant OB-fold domain has been introduced at the 3' end of the light chain. - Gene sequence encoding the antibody heavy chain - the gene sequence encoding the light chain of the antibody, or - A gene sequence encoding an antibody heavy chain in which a variant OB-fold domain has been introduced at the 5' end of the light chain. - Gene sequence encoding the antibody heavy chain - The gene sequence that encodes the light chain of an antibody.

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

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

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

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

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

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

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

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

[0141] Note that the resulting molecule has six binding sites (two antibody Fab binding sites and binding sites for each of the four OB-fold domain variants at the N-terminus of the light chain and the C-terminus of the light chain), as illustrated in Figure 1.I.

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

[0143] The resulting molecule, after association of the heavy and light chains, is a multispecific molecule that binds to the antigen of the antibody and the target of the variants of the OB-fold domains at the C-terminus of the light chain and the C-terminus of the heavy chain.

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

[0145] In another embodiment, the following is introduced into the cell: - a gene sequence encoding the light chain of an antibody, wherein a variant of an OB-fold domain has been introduced at the 5' end of the light chain coding sequence and a variant of an OB-fold domain (either the same or a different one) has been introduced at the 3' end of the light chain coding sequence. - The gene sequence that encodes the heavy chain of an antibody.

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

[0147] Note that the resulting molecule has six binding sites (two antibody Fab binding sites and one each of the four OB-fold domain variants at the N- and C-termini of the light chain), as illustrated in Figure 1.J.

[0148] In another embodiment, the following is introduced into the cell: - a gene sequence encoding the heavy chain of an antibody, wherein a variant of an OB-fold domain has been introduced at the 5' end of the heavy chain coding sequence and a variant of an OB-fold domain (either the same or different) has been introduced at the 3' end of the heavy chain coding sequence. - The gene sequence that encodes the light chain of an antibody.

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

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

[0151] In the above embodiments, the variants of the OB-fold domain introduced at the termini of the heavy and light chains are the same or different.

[0152] If the variants are different, they can be: - Based on different OB-fold domains and 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 bind to the same target (with different mutations).

[0153] The cells may be transformed or transfected with other coding sequences to obtain the asymmetric molecule.

[0154] Other embodiments for generating multispecific molecules Three gene sequences In a further embodiment, the 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 an antibody heavy chain fused (at the 3' or 5' end) to a gene sequence encoding an OB-fold domain variant (A); (iii) A gene sequence encoding an antibody heavy chain fused (at the 3' or 5' end) to a gene sequence encoding another variant of the OB-fold domain (B).

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

[0156] Thus, the cells would theoretically produce three different types of multispecific proteins in the following proportions: - Bispecific proteins (binding to the antigen of the antibody and the target of variant (A)): 25% - Bispecific proteins (binding to the antibody's antigen and the variant's target): 25% - Trispecific proteins (binding to the antigen of the antibody and to the target of variants (A) and (B)): 25%.

[0157] In regard 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 can 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 bind to the same target (with different mutations).

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

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

[0161] Specific embodiments are as follows: To generate proteins with six binding sites (if symmetric, these proteins would be tetraspecific with different OB-fold variants), - a gene sequence encoding the heavy chain of an antibody, in which a variant of an OB-fold domain has been introduced at the 5' end of the sequence encoding the heavy chain and a variant of an OB-fold domain (either the same or a different one) has been introduced at the 3' end of the sequence encoding the heavy chain; - a gene sequence encoding an antibody light chain, wherein a variant of an OB-fold domain has been introduced into the 5' end of the sequence encoding the light chain; This results in the protein depicted in Figure 1.N. - a gene sequence encoding the light chain of an antibody, wherein a variant of an OB-fold domain has been introduced at the 5' end of the light chain coding sequence and a variant of an OB-fold domain (either the same or a different) has been introduced at the 3' end of the light chain coding sequence. - a gene sequence encoding an antibody heavy chain, in which a variant of an OB-fold domain has been introduced into the 5' end of the sequence encoding the heavy chain; This results in the protein depicted in Figure 1.O. - a gene sequence encoding the heavy chain of an antibody, in which a variant of an OB-fold domain has been introduced at the 5' end of the sequence encoding the heavy chain and a variant of an OB-fold domain (either the same or a different one) has been introduced at the 3' end of the sequence encoding the heavy chain; - a gene sequence encoding an antibody light chain, wherein a variant of an OB-fold domain has been introduced into the 3' end of the sequence encoding the light chain; This results in the protein depicted in Figure 1.M. - a gene sequence encoding the light chain of an antibody, wherein a variant of an OB-fold domain has been introduced at the 5' end of the light chain coding sequence and a variant of an OB-fold domain (either the same or a different) has been introduced at the 3' end of the light chain coding sequence. - a gene sequence encoding an antibody heavy chain, in which a variant of an OB-fold domain has been introduced into the 3' end of the sequence encoding the heavy chain; This results in the protein illustrated in Figure 1.L.

[0162] Transforming or transfecting cells with these gene sequences results in a multispecific protein displaying eight binding sites (two antibody Fab binding sites and six binding sites for variants of the OB-fold domain).

[0163] Proteins with 10 binding sites can also be obtained by transforming or transfecting cells with - a gene sequence encoding the heavy chain of an antibody, in which a variant of an OB-fold domain has been introduced at the 5' end of the sequence encoding the heavy chain and a variant of an OB-fold domain (either the same or a different one) has been introduced at the 3' end of the sequence encoding the heavy chain; - a gene sequence encoding the light chain of an antibody, in which a variant of an OB-fold domain has been introduced at the 5' end of the sequence encoding the light chain and a variant of an OB-fold domain (either the same or different) has been introduced at the 3' end of the sequence encoding the light chain; This results in the protein depicted 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 can be: - Based on different OB-fold domains and 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 bind to the same target (with different mutations).

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

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

[0168] Because cytotoxic T lymphocytes play a key role in the immune response against cancer and tumor cells can evade the immune response, one strategy is to use bispecific molecules to recruit T cells to the vicinity of tumor cells, where their activation and proliferation leads to tumor cell lysis.

[0169] One target of the multispecific molecule is a tumor-associated antigen, and the 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 TIFF2026021519000001.tif92148TIFF2026021519000002.tif181148TIFF2026021519000003.tif129148

[0171] Altered host response 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, which are the main inhibitors that hinder immunotherapy. TIFF2026021519000004.tif33147TIFF2026021519000005.tif198147

[0172] HER2 is a valid target for many cancers. HER3 signaling is an important mechanism of drug resistance to HER2 inhibitors. Dual targeting of HER2 / HER3 may result in more effective responses.

[0173] Deregulated EGFR- and HER3-dependent signaling contributes to the pathogenesis of human cancers such as head and neck and colorectal cancer.

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

[0175] anti-angiogenesis TIFF2026021519000006.tif146147

[0176] Multiple angiogenic factors, including endothelial growth factor receptor 2 (VEGFR2), VEGFR3, 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 has shown 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 the effective priming of T lymphocytes that are subsequently infused into the patient.

[0179] Bispecifics 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 adapted.

[0180] Cytokine binding TIFF2026021519000007.tif62147

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

[0182] Payload Delivery TIFF2026021519000008.tif89147

[0183] An interesting application is the delivery of payloads such as drugs, radiolabels, and nanoparticles. The payload is administered as soon as the unbound bispecific molecule is cleared 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 99mA bispecific TF2 construct, used in tumor imaging and radioimmunotherapy, specifically binds to the T-labeled hapten histamine succinylglycine (HSG). In preclinical studies, TF2 was first injected, followed by 99m T-labeled HSG was administered after removal of bsAb from the blood. A high tumor / blood ratio 99m High tumor uptake of T was observed. TF2 is in Phase I trials in patients with colorectal cancer. Other uses of TF2 include radioimmunotherapy in patients with colorectal neoplasms. 177Lu HSG / 111In Targeting HSG and CEA, and immunopositron emission tomography 68Ga These include targeting HSG and CEA (Fan et al. Journal of Hematology & Oncology (2015) 8:130).

[0184] Digoxigenin (Dig) as a hapten can be used as a payload scaffold to load several cytotoxic moieties, such as fluorophores, chelators, 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, digoxigenylated siRNA at its 3' end and formulated into nanoparticles was conjugated to bispecific antibodies that bind to tumor antigens such as HER2, IGF1-R, CD22, and LeY, resulting in specific delivery of the siRNA to cells expressing the corresponding antigen, leading to internalization into endosomes and detachment of Dig-siRNA from the bispecific antibody (Schneider et al., Mol. Ther. Nucleic Acids. 2012;1:e45).

[0185] Bacterial minicells are anuclear nanoparticles generated by inactivating genes that control normal bacterial cell division. Chemotherapeutic drugs can be packaged within minicells that are subsequently linked to bispecific molecules that also bind to antigens on the membrane 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 TIFF2026021519000009.tif68148

[0187] Couch et al. and Yu et al. designed a bsAb that binds to the transferrin receptor (TfR) and beta-APP cleaving enzyme 1 (BACE1) and crosses the blood-brain barrier.

[0188] Diagnostic Assays TIFF2026021519000010.tif61148

[0189] Treating infectious diseases TIFF2026021519000011.tif176148

[0190] Other uses TIFF2026021519000012.tif75148

[0191] Treatment method The present invention also relates to the multispecific molecules described above for their use as medicaments and / or for treating or preventing diseases in which inhibition of the target is required. As exemplified above, the choice of target will obviously depend on the disease it is desired to treat or prevent.

[0192] Therapeutic methods are also part of the present invention. The present invention also encompasses a method for treating a patient in need thereof, comprising administering a therapeutically effective amount of a molecule disclosed herein. A "therapeutically effective" amount according to the present invention is defined as an amount sufficient to obtain a clinical benefit (reduction of adverse conditions). It can be determined by a phase II clinical trial. It can be administered alone or in combination with another agent, as a single dose or according to a multiple dose regimen. It is preferably non-toxic or has a toxicity that is acceptable in view of the benefits to the patient's health. The subject may be a human or a non-human animal (e.g., rabbit, rat, mouse, monkey, or other lower primate).

[0193] The molecules disclosed herein will be formulated with one or more physiologically acceptable carriers or excipients known in the art, and will be administered by any suitable means and through any suitable route.Therefore, parenteral (for example, intramuscular, intravenous, intraarterial, intraluminal or subcutaneous), intrapulmonary and intranasal administration, and if desired for local immunosuppressive treatment, intralesional administration is expected.Infusion (especially intravenous infusion) is preferred.The route of administration can vary depending on the disease to be treated.

[0194] Molecules directed against 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 some cancers (colorectal cancer, breast cancer, bladder cancer, glioblastoma, other solid cancers) or other immunological diseases.

[0195] Molecules against IL17 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 cancers) or other immunological diseases (including multiple sclerosis).

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

[0197] Specific Compositions Specifically, the present invention relates to the polypeptides disclosed above, in which a variant of a protein of the Sac7d family binds to a subunit of TNFα, preferably without binding to the subunit of said protein when said subunit protein is comprised in its native, fully formed multimeric protein.

[0198] Indeed, the inventors have determined that it is possible to identify variants of the Sac7d family of proteins that bind to soluble subunits of TNFα and prevent the formation of biologically active multimeric proteins or shift the equilibrium towards monomeric (or multimeric inactive) subunits.

[0199] Specifically, such variants include those of the sequence Includes TIFF2026021519000013.tif44143.

[0200] Specifically, 1 to 13, 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, H22, M24, Q26, L29, E35, D41, F44, and P46, more preferably the group consisting of V7, M8, F9, K11, V21, Q26, L29, E35, D41, F44, and P46, are replaced with another amino acid in SEQ ID NO:41.

[0201] In this embodiment, it is preferred if 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, in particular Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4.

[0202] Specifically, the antibody binds to IL17 (and is specifically infliximab, adalimumab, certolizumab pegol, orgolimumab, secukinumab, afacevicumab, bimekizumab, or vunakizumab).

[0203] Specifically, the antibody binds to CD20 (and is specifically Brontuzumab, FBTA05, Ibritumomab Tiuxetan, Mosunetuzumab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Ofatumumab, Rituximab, Tositumomab, or Veltuzumab).

[0204] Specifically, the antibody binds to IL24.

[0205] Specifically, the antibody binds to IL12.

[0206] Specifically, the antibody binds to IL4 (and specifically is dupilumab).

[0207] Specifically, the antibody binds to IL13.

[0208] Specifically, the antibody binds to IL6 (and is specifically siltuximab, olokizumab, or bovalilizumab).

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

[0210] Specifically, the antibodies bind to the tumor-specific antigens disclosed above.

[0211] Specifically, the antibody binds to Her2 (specifically, DS-8201, Ertumaxomab, 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 is specifically tremelimumab).

[0214] In another embodiment, the variant of the Sac7d family of proteins binds to IL17.

[0215] In this embodiment, the variant of the Sac7d family of proteins has the sequence It is preferable if it contains one of the following: TIFF2026021519000014.tif84143.

[0216] In this embodiment, the antibody is selected from the group consisting of TNFα (specifically infliximab, adalimumab, certolizumab pegol, orgolimumab), IL23 (specifically brazikumab, guselkumab, mirikizumab, risankizumab, or tildrakizumab), IL12 (specifically briakinumab, ustekinumab), IL4 (specifically dupilumab), IL13 (specifically anrukinzumab), IL31 or its receptor, and tumor-specific antigens, specifically Her2 (specifically DS-8201, ertumaxomab, gancotamab, marjetuximab, pertuzumab, timigituzumab, trastuzumab, or trastuzumab). emtansine), PDL1 (programmed death-ligand 1, CD274), or CTLA4 (specifically tremelimumab).

[0217] Specifically, the antibody binds to TNFα (and specifically is infliximab, adalimumab, certolizumab pegol, orgolimumab).

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

[0219] Specifically, the antibody binds to IL12 (and is specifically briakinumab or ustekinumab).

[0220] Specifically, the antibody binds to IL4 (and specifically is dupilumab).

[0221] Specifically, the antibody binds to IL13 (and specifically is anrukinzumab).

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

[0223] Specifically, the antibodies bind to the tumor-specific antigens disclosed above.

[0224] Specifically, the antibody binds to Her2 (specifically DS-8201, ertumaxomab, gancotamab, marjetuximab, pertuzumab, timigituzumab, 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 polypeptides comprising variants of Sac7d family proteins, specifically those disclosed herein, which contain 4 to 22 mutated amino acids in the binding site of the Sac7d family proteins that bind to IL17, thereby improving or stabilizing antibody production. The sequence of Sac7d is Please note that the file is TIFF2026021519000015.tif11143.

[0228] The above polypeptide can be obtained, wherein the mutated 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, D35, D36, N37, G38, K39, T40, G41, R42, A44, S46, E47, K48, D49, A50, and P51 of Sac7d.

[0229] The above polypeptide can be obtained, wherein the Sac7d variant contains 4 to 17 mutated 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 embodiment, the variant of the Sac7d family protein has the following sequence: It is preferable if it contains one of the following: TIFF2026021519000016.tif84143.

[0231] Specifically, such polypeptides comprise the 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, or more preferably 1 amino acid selected from the group consisting of V7, M8, F9, K11, Q26, L29, E35, D41, F44, and P46 have been replaced with another amino acid. Indeed, Applicants have shown that such residues can be modified without altering the observed binding. This was done by replacing these residues with alanine and confirming binding. There was no loss of binding, suggesting that these residues are not important for binding to IL17.

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

[0233] In another embodiment, this variant of Sac7d family is conjugated with organic molecules.This can be carried out by any method known in the art.Specifically, the molecule can be chemically linked to protein.The molecule can include antiproliferative agents (cytotoxic and cytostatic agents), including cytotoxic compounds (for example, 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] Specific examples include DNA-binding or alkylating agents such as anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and their analogs, 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 are also included. Thalidomide and its derivatives, lenalidomide, and pomalidomide are also included. Cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitors, quercetin, and / or resveratrol can also be used as molecules conjugated to polypeptides containing variants to treat inflammatory diseases.

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

[0236] The present invention also relates to a vector comprising the genetic construct disclosed above, a host cell comprising such a genetic construct in its genome, and a method for producing a vector comprising the steps of: a. culturing a cell culture, wherein the cells have been transformed with the disclosed genetic construct; and b. Recovering the polypeptide The present invention also relates to a method for producing such a variant of a protein of the Sac7d family that binds to IL17, the method comprising the steps of: .

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

[0238] These recombinant DNA constructs, which contain a nucleotide sequence encoding a polypeptide, including the variants described above, are used in conjunction with a vector, such as a plasmid, phagemid, phage, or viral vector.

[0239] These recombinant nucleic acid molecules can be produced by the techniques described in Sambrook et al., 1989 (Sambrook J, Fritschi EF and Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York). Alternatively, the DNA sequences can be chemically synthesized, for example, using a synthesizer.

[0240] The recombinant construct of the present invention includes a vector capable of expressing RNA, thereby resulting in the production of a protein from the above-mentioned gene sequence. The expression vector may further comprise a control sequence, including a suitable promoter, operably linked to the open reading frame (ORF) of the gene sequence disclosed herein. The vector may further comprise 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 expression hosts.

[0241] Molecular generation Cells are transfected or transformed with vectors containing sequences encoding polypeptides, including the variants disclosed above.

[0242] The cells are cultured under conditions that allow the protein to be expressed and preferably secreted. The cell culture conditions are those commonly used for recombinant antibody production and are known in the art. Such conditions, known in the art, can also be optimized by those skilled in the art as needed. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451-3461) provide an overview of such methods and provide a thorough description thereof.

[0243] Bacterial, phage (Shukra et al, Eur J Microbiol Immunol(Bp). 2014; 4(2): 91-98), or eukaryotic production 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 293 cells), NS0 cells (a cell line derived from a non-secretory mouse myeloma), or EB66 cells (duck cell line, Valneva, Lyons, France) can be used.

[0246] The present disclosure also provides a host cell that contains at least one DNA construct that encodes polypeptides comprising variants disclosed herein.Host cell can be any cell that expression vector can be used for.As shown above, it can be higher eukaryotic host cell such as mammalian cell, lower eukaryotic host cell such as yeast cell, or prokaryotic cell such as bacterial cell.

[0247] The introduction of recombinant construct into host cell can be carried out by any method known in the art (for example, calcium phosphate transfection, lipofection, DEAE, dextran-mediated transfection, electroporation, or phage infection). The vector can be inserted into the genome of host cell, or can be maintained as an extragenomic vector (for example, bacterial artificial chromosome or yeast artificial chromosome, etc.). When introduced into the cell genome, this introduction can be random or can be targeted using methods known in the art (such as homologous recombination).

[0248] Bacterial host and expression Expression vectors useful for use in bacteria are constructed by inserting a recombinant DNA sequence into a functional leading phage carrying a functional promoter, along with appropriate translation initiation and termination signals. The vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide for amplification within the host.

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

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

[0251] The transformed or transfected cells are cultured by methods known in the art and the polypeptide is recovered from the intracellular or extracellular fraction (depending on whether it is secreted or not).

[0252] Molecular isolation The produced recombinant multispecific proteins can be separated and purified from the intracellular or extracellular fractions by any of a variety of known separation methods that exploit the physical or chemical properties of the protein.

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

[0255] If a tag has been introduced into the recombinant sequence (e.g., a polyhistidine tag), the tag can be used to purify the molecule. However, it is preferred to purify the molecule using affinity.

[0256] Specifically, the fact that the molecules produced herein bind to specific targets can be used to isolate such molecules using any affinity-based method (affinity columns, FACS, beads).

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

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

[0259] [Figure 1-1]Schematic representation of 15 proteins according to the invention. These proteins are symmetric. 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 and light chains. 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- and C-termini of the heavy chain and the C-termini of the light chain. N. OB-fold variants at the N- and C-termini of the heavy chain and the N-termini of the light chain. O. OB-fold variants at the N- and C-termini of the heavy chain and the N- and C-termini of the light chain. [Figure 1-2] A continuation of Figure 1-1 is shown. [Figure 2] Alignment of Sac7d family proteins. [Figure 3] Loading of antibody-Sac7d variant constructs onto Protein A biosensor in 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 of antibody functionality when fused to a Sac7D variant (H4) at either the N- 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 5]Diagram of the functionality of Sac7d (H4) variants when fused to Sac7d variants (H4) at either the N- 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] Diagram of the dual binding capacity of different bispecific constructs by biolayer interferometry in 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] Diagram of 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. [Example]

[0260] Bispecific antibodies created by genetic fusion of the OB-fold can result in four different bispecific constructs when the OB-fold is inserted at either the N- or C-terminus of either the heavy or light chain. The following experiments illustrate the fusion of a Sac7d variant to an IgG1κ antibody. These experiments serve as 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 production of the fusion protein, which is comparable to or even improved compared to the yield of an antibody not fused with the OB-fold variant.

[0261] Both heavy and light chain constant fragments were obtained by commercially available encoding vectors pFUSE-CHIg-hIG1 (InvivoGen) and pFUSE2ss-CLIg-hk (InvivoGen), respectively.

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

[0263] The DNA coding sequences of the Sac7d variants were amplified by PCR directly from the products of subcloning in an expression vector derived from pQE30 (Qiagen).

[0264] The constant fragment, variable domain, and Sac7d variant sequences, when available, were then assembled by Gibson Assembly to generate six different vectors, three of which were derived from the pFUSE-CHIg-hIG1 vector and three of which formed the pFUSE2ss-CLIg-hk vector. In all cases, the vectors differed in the location of the Sac7d variant sequence, either upstream (N-terminal fusion) or downstream (C-terminal fusion) of the antibody chain sequences, or its absence (no fusion).

[0265] In summary, the six vectors encode: - Only the light chain of the antibody - antibody light chain fused at its N-terminus to a Sac7d variant - antibody light chain fused at its C-terminus to a Sac7d variant - Antibody heavy chain only - antibody heavy chain fused at its N-terminus to a Sac7d variant - antibody heavy chain fused at its C-terminus to a Sac7d variant

[0266] The following examples illustrate the construction of polypeptides contemplated herein.

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

[0268] Sac7d variants were generated by the method disclosed in WO 2008 / 068637 using ribosome display to isolate variants against a given target from a library with up to 14 mutated amino acids.

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

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

[0271] Twenty-eight different combinations of fusion polypeptides were produced using methods similar to those described below, all with similar results.

[0272] Specifically, the Sac7d variants used were directed against IL17 (specifically SEQ ID NO:37 and SEQ ID NO:38) and fused to anti-TNFα. Other Sac7d variants were used (specifically SEQ ID NO:41, which binds to TNFα, or SEQ ID NO:42, designated H4, which binds to lysozyme). Other Sac7d variants against other targets were also used. As shown above, the yields of recovered polypeptide antibody-variants of Sac7d consistently matched or exceeded those of antibodies without the Sac7d variant, confirming dual affinity whenever examined.

[0273] As shown above, it is hypothesized that the addition of the Sac7d variant may stabilize the overall structure of the complex (thus resulting in improved production). Because both the antibody and the Sac7d variant maintain their structure (as demonstrated by dual binding assays showing that they retain their function), it is believed 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- or C-terminus) A description of the construction of a full-length heavy chain is provided.

[0275] Fusion of the Sac7d variant sequence at the N-terminus of the full-length heavy chain consisted in DNA re-engineering of the pFUSE-CHIg-hIG1 vector by Gibson Assembly, involving preparation by PCR amplification of the two fragments and the linearized vector.

[0276] pFUSE-CHIg-hIG1 was amplified by PCR using the forward oligonucleotide TIFF2026021519000017.tif4128 and reverse oligonucleotide It was linearized by PCR amplification using TIFF2026021519000018.tif4128.

[0277] The DNA sequences encoding the variable domains were amplified by PCR program 2 using the forward oligonucleotide TIFF2026021519000019.tif11128 and reverse oligonucleotide It was amplified by PCR using TIFF2026021519000020.tif4128.

[0278] The DNA sequences of the Sac7d variants were analyzed by PCR program 2 using the forward oligonucleotide TIFF2026021519000021.tif4136 and reverse oligonucleotide It was amplified by PCR using TIFF2026021519000022.tif11128.

[0279] Fusion of the Sac7d variant sequence at the C-terminus of the full-length heavy chain involved preparation by PCR amplification of the three fragments and the linearized vector. pFUSE-CHIg-hIG1 was amplified by PCR program 1 using the forward oligonucleotide TIFF2026021519000023.tif4128 and reverse oligonucleotide It is linearized by PCR amplification using TIFF2026021519000024.tif4136.

[0280] The DNA sequence of the constant domain was amplified by PCR program 2 using the forward oligonucleotide oligoCH-1 (SEQ ID NO: 16) and the reverse oligonucleotide It was amplified by PCR using TIFF2026021519000025.tif18140.

[0281] The DNA sequences encoding the variable domains were amplified by PCR program 2 using the forward oligonucleotide It was amplified by PCR with TIFF2026021519000026.tif4128 and the reverse oligonucleotide OligoCH-3 (SEQ ID NO: 19).

[0282] The DNA sequences of the Sac7d variants were analyzed by PCR program 2 using the forward oligonucleotide TIFF2026021519000027.tif11128 and reverse oligonucleotide It was amplified by PCR using TIFF2026021519000028.tif4133.

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

[0284] The PCR products were then verified on a 1.5% agarose gel (variable fragment amplified sequence and Sac7d variant amplified sequence) and a 0.8% agarose gel (linearized plasmid) containing Gel Green nucleic acid stain (1x). The bands of interest were excised under UV light and purified using the Wizard SV Gel and PCR Clean-up System Kit (Promega). To increase the amount of insert generated, a second PCR was performed on 5 ng of purified variable fragment PCR product (four replicates) using the same PCR mix and program. After verifying the band size on a 1.5% agarose gel, the same reactions were pooled and purified using the Wizard SV Gel and PCR Clean-up System Kit (Promega).

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

[0286] These PCR reactions resulted in the formation of double-stranded DNA with overlapping ends, which allowed for directional annealing by Gibson assembly. A 20 μL Gibson assembly mix was prepared containing 125 ng of DNA fragments at an insert / linearized plasmid molar ratio of 5 in ISO 1x buffer, 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). A 1-hour incubation at 50°C was used for Gibson assembly (Gibson et al., 2009).

[0287] The resulting ligated vector was transformed into E. coli strain BL21(DE3)pLysS. Exponentially growing cell cultures (OD ) of BL21(DE3)pLysS competent cells (Coger) were grown at 100°C. 600nm0.5) was transformed with 10 μL of the above Gibson assembly reaction and controls by heat shock. Cells were plated on 2YT agar medium (pH 7.5) containing chloramphenicol (10 μg / mL, Sigma-Aldrich) and Zeocin (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. Sequences were then confirmed by Sanger sequencing.

[0288] Example 2. Fusion to the light chain (N- or C-terminus) A description of the construction of a full-length light chain is provided.

[0289] The strategy for the preparation of the full-length light chain was to ligate oligonucleotides designated oligoCH-1 to 7 with each oligonucleotide designated oligoCL-1 to 7: The description of the heavy chain preparation was followed except that TIFF2026021519000029.tif44144 was exchanged and pFUSE2ss-CLIg-hk was used instead of pFUSE-CHIg-hIG1.

[0290] Additionally, transformation was performed in E. coli DH5α F'Iq strain with the following modifications: exponentially growing cell cultures (OD ) of DH5α F'Iq competent cells (Life technologies) were grown at 100°C. 600nm 0.5) was transformed with 10 μL of the Gibson assembly reaction by heat shock. Cells were plated on 2YT agar medium (pH 8) containing kanamycin (25 μg / mL, VWR) and blasticidin (100 μg / mL, InvivoGen).

[0291] Example 3. Fusion of one NF to an antibody (+ bispecific) HEK293-E6 cells were cultured in 1125 ml 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% CO. Transfections were performed as follows: when cell densities were 1.5–2 × 10 6 Cells were transfected with a total of 1 mg DNA / L of different plasmid pairs complexed with polyethyleneimine (PEI, Polysciences ref. 23966) at a DNA:PEI ratio of 1:2 and a HC:LC ratio of 2:3 when the total volume reached 1000 cells / mL. 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 xg for 20 minutes at 4°C. The culture supernatant was filtered through a 0.2 μm filter 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 performed with 0.1 M citrate buffer, pH 3.0. Fractions were neutralized with TRIS buffer, pH 9.0. Product-containing fractions were pooled and injected onto a Superdex 200 2660 gel filtration column. Product-containing fractions at a retention volume of 150-160 ml were pooled together and concentrated to 1-2 mg / ml using a vivapsin centrifugal device with a 50 kDa membrane cutoff.

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

[0293] The possibility of capturing different bispecific antibodies made by fusion of Sac7d variants at either the N- or C-terminus of either the heavy or light chain was demonstrated by biolayer interferometry with octet RED96.

[0294] One of the four bispecific constructs was loaded onto 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) supplemented with 0.01% BSA and 0.002% Tween 20, with shaking at 1000 rpm. In all cases, rapid capture was observed with minimal dissociation, reflecting the stability of the binding (Figure 3).

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

[0296] The bispecific constructs were loaded onto a Protein A sensor (Fortebio). After a 180-second baseline, association and dissociation were allowed to occur for 300 and 900 seconds, respectively. All steps were performed at 30°C in TBS (Tris 20 mM, NaCl 150 mM, pH 7.4) supplemented with 0.01% BSA and 0.002% Tween 20, with shaking at 1000 rpm. Concentration ranges of 150, 125, 100, 75, and 50 nM were used for the Sac7d variant targets. Concentration ranges of 600, 200, 66.66, 22.22, 7.40, 2.46, and 0.82 nM were used for the antibody targets. After each run, the sensor was regenerated with three cycles of glycine 10 mM pH 2 (10 seconds) and TBS (10 seconds).

[0297] Both the antibody and the Sac7d variant remained fully functional when linked together, regardless of the position of the fusion (Figs. 4 and 5).

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

[0299] The ability of the Sac7d variant and antibody to simultaneously bind to their respective targets was demonstrated by biolayer interferometry with octet RED96. The bispecific construct was loaded onto a Protein A sensor (Fortebio). After a 180-second 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 (20 mM Tris, 150 mM NaCl, pH 7.4) supplemented with 0.01% BSA and 0.002% Tween 20, with shaking at 1000 rpm.

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

[0301] Example 6. Increased or Similar Production of Antibody Without NF The final productivity was measured, which corresponds to the amount of product obtained after all purification steps.

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

[0303] Example 7. Fusion of two or more NFs to an antibody (gaining three or more specificities) The construction of multispecific molecules is carried out by the strategies described above for bispecific molecules.

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

[0305] The linkers and overlapping compatible ends required for Gibson Assembly are carried by the forward and reverse oligonucleotides and are added to the Sac7d variant coding sequence during the PCR amplification step. The multispecific molecules are 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 with 1% L-glutamine and supplemented with 10% (v / v) inactivated ultra-low IgG fetal bovine serum and 1% (v / v) penicillin-streptomycin. Cells were expanded in tissue culture dishes and maintained at 37°C in a humidified constant atmosphere of 95% air and 5% CO2.

[0307] The day before transfection, cells were trypsinized and counted using erythrosin B 0.2% 1:1 (v / v). Cells were plated at 126.4 x 10 per 10-layer cell culture factory to be 70-90% confluent at the time of transfection. 6 The cells were seeded at 1000 x g.

[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 reduced serum medium. 2 mL of 2 mg / mL polyethyleneimine (PEI, linear, MW 25,000) was then added, and the mixture was incubated at room temperature for 25 minutes. Cells were transfected by adding the DNA / PEI complex to fresh complete growth medium.

[0309] Twenty-four hours after transfection, the medium was replaced with F-12K complete growth medium supplemented with antibiotics (blasticidin (10 μg / mL) and zeocin (300 μg / mL)) and valproic acid (0.5 mM final concentration). Seven days after transfection, supernatants were collected, and protein concentrations were quantified by biolayer interference technology on a Protein A biosensor according to established standard methods using naked antibodies.

[0310] The production yields were compared with those of two commercially available therapeutic antibodies (adalimumab and infliximab). Genetic constructs were created to generate the heavy chains of these antibodies fused to variants of the Sac7d protein with 12 mutations in the binding domain of the protein. The variants were located at the N- or C-terminus of the heavy chain for adalimumab and at the N-terminus of the heavy chain for infliximab.

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

[0312] Example 9. Use of SHuffle (AFG) technology Complex proteins such as antibodies, which are sensitive to oxidative stress and require post-translational modifications for their effector functions, have long been produced exclusively in eukaryotic expression hosts such as mammalian cell lines, HEK293, CHO, and yeast. Recently, engineered bacterial strains such as SHuffle have also been demonstrated to be applicable for the intracellular production of functional antibodies, also termed cyclonal.

[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] Naked antibodies and three different antibody-nanophytin chimeras with nanophytin fusions at either the N-terminus of the light chain, the C-terminus of the light chain, or the C-terminus of the heavy chain were constructed.

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

[0316] Competent T7 SHuffle express pLysY was grown at 37°C in 2YT medium supplemented with chloramphenicol (10 μg / mL). The culture was divided into 1 mL fractions at an OD 600 nm of 0.4–0.5 and centrifuged at 4500 g for 5 min. The pellet was resuspended in 200 μL of chilled TSS buffer, and then 0.5 μL of the expression vector to be transformed was added and incubated on ice for 30 min. The suspension was heat-shocked at 42°C for 30 s, followed by 5 min 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 h, the suspension was centrifuged at 4500 g for 5 min. The pellet was resuspended in 20 μL of 2YT medium, and the bacteria were spread onto plates filled with 2YT agar supplemented with chloramphenicol (10 μg / mL) and ampicillin (100 μg / mL). The plates were incubated overnight at 37°C.

[0317] A single colony was inoculated into 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. 200 mL of 2YT medium supplemented with chloramphenicol (10 μg / mL), ampicillin (100 μg / mL), and 0.1% glucose was then inoculated with 10 mL of the preculture. The culture was incubated at 30°C with continuous shaking at 200 rpm until the OD 600 nm reached 0.7-0.8. Protein production was then initiated by the addition of IPTG (1 mM final concentration), and the culture was maintained at 30°C for an additional 16 hours with shaking at 200 rpm. The culture was terminated by centrifugation at 3214 g for 30 minutes at 4°C. The pellet was resuspended in lysis buffer (PBS 1x, 5 mM EDTA, Bugbuster® 1x, 5 μg / mL de DNase I) and incubated for 1 hour with shaking at 200 rpm. Cell debris was then pelleted by centrifugation at 3220 g for 45 minutes at 4°C. The lysis 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 column volumes of water and equilibrated with 10 column volumes of PBS (phosphate-buffered saline, Sigma-Aldrich, P4417). The lysate was applied to the column, which was then washed with 10 column volumes of PBS containing 5 mM EDTA. 0.1 M glycine pH 2–3 was used for elution. The eluate was collected in 100 μL fractions and immediately neutralized by adding 10 μL of Tris 1 M (pH 8.8). The amount of recovered material was then assessed by spectrophotometry at 280 nm. The purity of the samples was assessed on a 10% SDS-PAGE gel under reducing conditions.

[0319] In this production system, the production yield of the chimera is comparable to that of the naked antibody (no significant difference between the yields obtained).

[0320] Furthermore, evaluation of the dual binding properties of the nanophytin-antibody chimeras by biolayer interferometry showed that the antibody and Sac7d variants maintained their affinity binding. To do this, various nanophytin-antibody chimeras were loaded onto a Protein A biosensor at 1.5 nM and sequentially incubated with TNFα (20 nM) and IL-17 (125 nM) for 420 s, followed by a dissociation phase (900 s).

[0321] Sequence information SEQUENCE LISTING <110> AFFILOGIC <120> Multi-specific molecules <150> EP17306580.6 <151> 2017-11-14 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 66 <212> PRT <213> Sulfolobus acidocaldarius <400> 1 Met Val Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe 20 25 30 Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys Lys 65 <210> 2 <211> 64 <212> PRT <213> Sulfolobus solfataricus <400> 2 Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 3 <211> 65 <212> PRT <213> Sulfolobus acidocaldarius <400> 3 Met Ala Lys Val Arg Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe 20 25 30 Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Met Asp Met Leu Ala Arg Ala Glu Lys Lys 50 55 60 Lys 65 <210> 4 <211> 64 <212> PRT <213> Sulfolobus shibatae <400> 4 Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 5 <211> 64 <212> PRT <213> Sulfolobus shibatae <400> 5 Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp 1 5 10 15 Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 6 <211> 64 <212> PRT <213> Sulfolobus tokodaii <400> 6 Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Ser Gly Lys Lys 50 55 60 <210> 7 <211> 64 <212> PRT <213> Sulfolobus islandicus <400> 7 Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp 1 5 10 15 Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 8 <211> 64 <212> PRT <213> Sulfolobus islandicus <400> 8 Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp 1 5 10 15 Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 9 <211> 64 <212> PRT <213> Sulfolobus islandicus <400> 9 Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Gln Val Asp 1 5 10 15 Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys 50 55 60 <210> 10 <211> 62 <212> PRT <213> Metallosphaera sedula. <400> 10 Met Ala Thr Lys Ile Lys Phe Lys Tyr Lys Gly Gln Asp Leu Glu Val 1 5 10 15 Asp Ile Ser Lys Val Lys Lys Val Trp Lys Val Gly Lys Met Val Ser 20 25 30 Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Asn Met Ile Gly Lys Lys 50 55 60 <210> 11 <211> 62 <212> PRT <213> Metallosphaera cuprina <400> 11 Met Ala Thr Lys Ile Lys Phe Lys Tyr Lys Gly Gln Asp Leu Glu Val 1 5 10 15 Asp Ile Ser Lys Val Lys Val Trp Lys Val Gly Lys Met Val Ser 20 25 30 Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Ser Met Ile Gly Lys Lys 50 55 60 <210> 12 <211> 61 <212> PRT <213> Acidianus hospitals <400> 12 Met Thr Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe 20 25 30 Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys 35 40 45 Asp Wing Pro Lys Glue Lew Glue Lys Leo Glu Lys Lys 50 55 60 <210> 13 <211> 62 <212> PRT <213> Acidianus the Hospitaller <400> 13 Met Ala Thr Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val 1 5 10 15 Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser 20 25 30 Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Asp Lys Leu Glu Lys Lys 50 55 60 <210> 14 <211> 60 <212> PRT <213> Acidianus the Hospitaller <400> 14 Met Ala Thr Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val 1 5 10 15 Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser 20 25 30 Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu 35 40 45 Lys Asp Ala Pro Lys Glu Leu Leu Glu Lys Leu Lys 50 55 60 <210> 15 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence <220> <221> VARIANT <222> (2)..(2) <223> Xaa is V, A or T <220> <221> VARIANT <222> (3)..(3) <223> Xaa is T or K <220> <221> VARIANT <222> (4)..(4) <223> Xaa is - or K <220> <221> VARIANT <222> (6)..(6) <223> Xaa is R or K <220> <221> VARIANT <222> (15)..(15) <223> Xaa is E or Q <220> <221> VARIANT <222> (18)..(18) <223> Xaa is T or I <220> <221> VARIANT <222> (31)..(31) <223> Xaa is V or I <220> <221> VARIANT <222> (37)..(39) <223> Yes Yes Yes is EGG or DN- <220> <221> VARIANTS <222> (57)..(57) <223> Stone is M or L <220> <221> VARIANTS <222> (58)..(58) <223> Stone is Q, D or E <220> <221> VARIANTS <222> (59)..(59) <223> Stone is M or K <220> <221> VARIANTS <222> (61)..(67) <223> Stone is -------, EKS--GK, EK--QK, ARAEREK, ARA-EKK, E-----K, EX--G, ACAEREK or ACA-EKK <400> 15 Met No. Val No. Phe Lys Tyr Lys Gly Glu Glu Lys No. Val 1 5 10 15 Asp Valid Lys Ile Lys Val Trp Arg Val Gly Lys Met Valid 20 25 30 Phe Thr Tyr Asp Xaa Xaa Xaa Gly Lys Thr Gly Arg Gly Ala Val Ser 35 40 45 Glu Lys Asp Ala Pro Lys Glu Leu Br. 50 55 60 Yes Yes Yes Lys 65 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide oligoCH-1 <400> 16 gctagcacca agggccca 18 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide VHfuse_Rev <400> 17 tgaattcgtg acaagtgcaa gact 24 <210> 18 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide OligoCH-2 <400> 18 ggtagtgcag gctccggcag tggcggtagc gaggtgcagt tggtagagt 49 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> OligoCH-3 <400> 19 gcccttggtg ctagcact 18 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide VH_NF_For <400> 20 cttgtcacga attcagtcaa ggtgaaattc 30 <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Glink15.1_Rev <400> 21 ggagcctgca ctaccggaac cgcctgaacc tttctcgcgt tccgc 45 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide OligoCH-4 <400> 22 tgagtcctag ctggccaga 19 <210> 23 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide IL2ss_Rev <400> 23 tgaattcgtg acaagtgcaa gacttagtgc a 31 <210> 24 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide OligoCH-5 <400> 24 ggagcctgca ctaccggaac cgcctgaacc tttacccgga gacagggaga 50 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide OligoCH-6 <400> 25 cttgtcacga attcagaggt g 21 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Glink15.1_For <400> 26 ggtagtgcag gctccggcag tggcggtagc gtcaaggtga aattc 45 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide OligoCH-7 <400> 27 gccagctagg actcatttct cgcgttccgc 30 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-1 <400> 28 aaacgtacgg tggctgcacc a 21 <210> 29 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-2 <400> 29 ggtagtgcag gctccggcag tggcggtagc gacatccaga tgacacagtc 50 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-3 <400> 30 agccaccgta cgttttatct 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-4 <400> 31 tagagggagc tagctcgaca tg 22 <210> 32 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-5 <400> 32 ggagcctgca ctaccggaac cgcctgaacc acactctccc ctgttgaag 49 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-6 <400> 33 cttgtcacga attcagacat ccaga 25 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> oligoCL-7 <400> 34 agctagctcc ctctatttct cgcgttccgc 30 <210> 35 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to IL17 <220> <221> misc_feature <222> (7)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (29)..(29) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (44)..(44) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (46)..(46) <223> Xaa can be any naturally occurring amino acid <400> 35 Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Phe Asn Val Trp Arg Xaa Gly Lys Xaa Val Asn Phe 20 25 30 Met Tyr Asp Asp Asn Gly Lys Ile Gly Ile Gly Xaa Val Xaa Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys Lys 65 <210> 36 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to IL17 <220> <221> misc_feature <222> (7)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (44)..(44) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (46)..(46) <223> Xaa can be any naturally occurring amino acid <400> 36 Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Phe Asn Val Trp Arg Xaa Gly Lys Ser Val Asn Phe 20 25 30 Met Tyr Asp Asp Asn Gly Lys Ile Gly Ile Gly Xaa Val Xaa Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys Lys 65 <210> 37 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to IL17 <400> 37 Met Val Lys Val Lys Phe His Ala Lys Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Phe Asn Val Trp Arg Ser Gly Lys Ser Val Asn Phe 20 25 30 Met Tyr Asp Asp Asn Gly Lys Ile Gly Ile Gly His Val Asp Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys Lys 65 <210> 38 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to IL17 <400> 38 Met Val Lys Val Lys Phe Ser Arg Phe Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Phe Asn Val Trp Arg Ile Gly Lys Thr Val Asn Phe 20 25 30 Met Tyr Asp Asp Asn Gly Lys Ile Gly Ile Gly Ala Val Asp Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys Lys 65 <210> 39 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to TNF-alpha <220> <221> misc_feature <222> (7)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (21)..(22) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (24)..(24) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (26)..(26) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (29)..(29) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (44)..(44) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (46)..(46) <223> It can be any naturally occurring amino acid. <400> 39 Met Val Lys Val Lys Phe Free Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile No. Val No. Arg No. Gly Lys No. His Phe 20 25 30 Trp Tyr Glu Asp Asn Gly Lys Ile Asp Lys Gly Xaa Val Xaa Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys 65 <210> 40 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to TNF-alpha <220> <221> misc_feature <222> (7)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (21)..(21) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (29)..(29) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (44)..(44) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (46)..(46) <223> Xaa can be any naturally occurring amino acid <400> 40 Met Val Lys Val Lys Phe Xaa Xaa Xaa Gly Xaa Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Xaa His Val Met Arg Xaa Gly Lys Xaa Val His Phe 20 25 30 Trp Tyr Glu Asp Asn Gly Lys Ile Asp Lys Gly Xaa Val Xaa Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys 65 <210> 41 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to TNF-alpha <400> 41 Met Val Lys Val Lys Phe Val Met Phe Gly Lys Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Val His Val Met Arg Gln Gly Lys Leu Val His Phe 20 25 30 Trp Tyr Glu Asp Asn Gly Lys Ile Asp Lys Gly Phe Val Pro Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Lys 65 <210> 42 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Sequence binding to lysozyme <400> 42 Met Val Lys Val Lys Phe Phe Trp Asn Gly Glu Glu Lys Glu Val Asp 1 5 10 15 Thr Ser Lys Ile Val Trp Val Lys Arg Ala Gly Lys Ser Val Leu Phe 20 25 30 Ile Tyr Asp Asp Asn Gly Lys Asn Gly Tyr Gly Asp Val Thr Glu Lys 35 40 45 Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu 50 55 60 Light Light 65

Claims

1. A polypeptide comprising an engineered antibody, wherein at least one variant of a Sac7d family protein is fused to at least one of the heavy or light chains of the antibody, and the variant comprises 4 to 20 or 5 to 20 mutated residues in the binding site of the Sac7d family protein.

2. 2. The polypeptide of claim 1, wherein the mutated 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.

3. 3. The polypeptide of claim 1 or 2, wherein the variant contains 7 to 14 mutated 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.

4. 4. The polypeptide of claim 1, wherein at least one variant of a protein of the Sac7d family is fused to the two heavy chains or the two light chains of the antibody.

5. The polypeptide of any one of claims 1 to 4, wherein at least one variant of the Sac7d family of proteins is fused to one heavy chain of the antibody, and at least one variant of the Sac7d family of proteins is fused to one light chain of the antibody.

6. 5. The polypeptide of claim 1, wherein at least one heavy chain or at least one light chain of said antibody is fused to two variants of said Sac7d family protein.

7. The polypeptide of any one of claims 1 to 4, wherein at least two different variants of said Sac7d family protein are fused to the heavy and / or light chain of said antibody.

8. The polypeptide of any one of claims 1 to 7, wherein the eight variants of the Sac7d family of proteins are fused to the heavy and light chains of the antibody.

9. The polypeptide of any one of claims 1 to 8, wherein the antibody is a therapeutic antibody.

10. 10. The polypeptide of any one of claims 1 to 9, which 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.

11. The polypeptide of any one of claims 1 to 10, wherein the variant of a protein of the Sac7d family binds to IL17.

12. 12. The polypeptide of claim 11, wherein the variant of the Sac7d family protein comprises the sequence SEQ ID NO: 35 or SEQ ID NO:

36.

13. 13. The polypeptide of claim 11 or 12, comprising SEQ ID NO: 37 or SEQ ID NO:

38.

14. 14. The polypeptide of any one of claims 11 to 13, wherein the antibody binds to a protein selected from the group consisting of TNFα, IL23, IL12, IL4, IL13, IL31 or their receptors, and tumor-specific antigens, in particular Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4.

15. 11. The polypeptide of any one of claims 1 to 10, wherein the variant of a protein of the Sac7d family binds to a subunit of TNFα, and preferably does not bind to the subunit protein when the subunit protein is comprised in a fully formed multimeric protein in its native state.

16. 16. The polypeptide of claim 15, comprising the sequence SEQ ID NO: 39 or SEQ ID NO:

40.

17. 17. The polypeptide of claim 15 or 16, comprising the sequence SEQ ID NO:

41.

18. 18. The polypeptide of any one of claims 15 to 17, wherein the antibody binds to a protein selected from the group consisting of IL17, CD20, IL24, IL12, IL4, IL13, IL31 or their receptors, and tumor-specific antigens, in particular Her2, PDL1 (programmed death ligand 1, CD274), or CTLA4.

19. a. A sequence encoding the heavy chain of an antibody fused at its 3' end to a sequence encoding a variant of a protein of the Sac7d family. b. A sequence encoding the heavy chain of an antibody fused at its 5' end to a sequence encoding a variant of a protein of the Sac7d family. c) a sequence encoding the light chain of an antibody fused at its 3' end to a sequence encoding a variant of a protein of the Sac7d family; d. A sequence encoding the light chain of an antibody fused at its 5' end to a sequence encoding a variant of a protein of the Sac7d family. A genetic construct comprising a DNA sequence selected from the group consisting of:

20. A vector comprising the genetic construct of claim 19.

21. 20. A host cell comprising in its genome the genetic construct of claim 19.

22. A method for producing a polypeptide according to any one of claims 1 to 18, comprising: a. culturing a cell culture in which the cells have been transformed with the genetic construct of claim 19 and a genetic construct encoding the complementary antibody chain; b. Recovering the polypeptide The method comprising the steps of:

23. 1. A method for maintaining or improving the production yield of an engineered antibody, comprising: a. culturing a cell culture in which the cells have been transformed with the genetic construct of claim 19 and a genetic construct encoding the complementary antibody chain; b. Recovering the modified antibody produced after expression of the genetic construct of step a. The process comprises: The method, wherein the modified antibody recovered in step b comprises at least one variant of a protein of the Sac7d family, the variant comprising 5 to 20 mutated residues in the binding site of the protein, and the yield of the recovered modified antibody is equal to or higher than the yield of an unmodified antibody produced under the same conditions.

24. A polypeptide comprising a variant of a Sac7d family protein, wherein the variant comprises 4 to 22 mutated amino acids in the binding site of the Sac7d family protein and binds to IL17.

25. 25. The polypeptide of claim 24, wherein the mutated 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.

26. 26. The polypeptide of claim 24 or 25, 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.

27. 27. The polypeptide of any one of claims 24 to 26, comprising the sequence SEQ ID NO: 35 or SEQ ID NO:

36.

28. 28. The polypeptide of any one of claims 24 to 27, comprising the sequence SEQ ID NO: 37 or SEQ ID NO:

38.

29. 28. The polypeptide of any one of claims 24 to 27, comprising the sequence SEQ ID NO: 37 or SEQ ID NO: 38, in which 1 to 8 amino acids selected from the group consisting of V7, M8, F9, K11, Q26, L29, E35, D41, F44, and P46 have been replaced with another amino acid.

30. 30. The polypeptide of any one of claims 24 to 29, wherein said variant of a protein of the Sac7d family that binds to IL17 is linked or fused to another protein or polypeptide.

31. 31. The polypeptide of claim 30, wherein the other protein or polypeptide comprises another variant of the Sac7d family of proteins.

32. 31. The polypeptide of claim 30, wherein the other protein or polypeptide is an antibody, preferably binding to TNFα or Her2 / neu.

33. 33. The polypeptide of any one of claims 24 to 32, conjugated to an organic molecule.

34. A genetic construct comprising a DNA sequence encoding the polypeptide of any one of claims 24 to 32.

35. A vector comprising the genetic construct of claim 34.

36. A host cell comprising in its genome the genetic construct of claim 34.

37. A method for producing a polypeptide according to any one of claims 24 to 32, comprising: a. culturing a cell culture, wherein the cells have been transformed with the genetic construct of claim 34; and b. Recovering the polypeptide The method comprising the steps of:

38. A polypeptide according to any one of claims 24 to 32 as a medicament.

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