A fusion protein comprising an antigen binding domain and a cytokine trimer domain

Abstract

This invention relates to a novel form of fusion protein RACE (Receptor-Antibody Conjugated (Cell) Engager) prepared by linking a first monomer, a second monomer, and a third monomer via a mortise and tenon joint to a cytokine trimer domain, and replacing the constant region (Fc) of the antibody with the aforementioned cytokine trimer domain. It has been confirmed that the RACE of this invention not only exhibits superior binding ability to the target receptor compared to the parent antibody, but also demonstrates excellent simultaneous binding ability to both the antigen and the target receptor, thus making it useful for use in bispecific pharmaceutical compositions.

Description

Technical Field

[0001] This invention relates to a fusion protein comprising an antigen-binding domain and a cytokine trimer domain, a method for producing the same, and its uses. Prior Technology

[0002] The licensing of blincyto (Amgen), a CD19 / CD3 linker designed using anti-CD19 antibody and anti-CD3 antibody, has accelerated the development of bispecific immunotherapeutic agents that treat tumors by homing immune cells.

[0003] Anti-CD3 antibodies primarily target the CD3ε (CD3 epsilon, CD3e) molecule present on the surface of T cells, inducing T cell activation and then inducing apoptosis through bispecific antibodies targeting the expressed antigen. Antibodies targeting CD3 rapidly promote T cell activity to induce apoptosis of target cells within a short period, sometimes also inducing a surge in cytokine secretion. This is considered to be due to the inherent characteristics of the CD3 molecule, which resides on the T cell surface and induces the primary cellular activity.

[0004] The activity of immune cells, including T cells, is regulated by a balance between co-stimulatory and inhibitory factors. Co-stimulatory factors that aid immune cell activity are broadly classified into two families of proteins: the B7 receptor family, including CD28 and inducible costimulatory molecules (ICOS), and TNF receptor proteins, including 4-1BB and OX40. In particular, these co-stimulatory factor proteins are known to be inducible molecules expressed in activated T cells, which is believed to mitigate the toxicity associated with CD3 antibodies.

[0005] 4-1BB ligand (CD137L) inhibits the expression and signaling of 4-1BB receptors in antigen-presenting cells. 4-1BB ligands exist in two forms: a membrane protein contained within the cell membrane and a water-soluble form cleaved from the apical portion of the membrane-binding site. The water-soluble 4-1BB ligand does not participate in 4-1BB receptor signaling, while the membrane protein form strongly induces activation of T cells or natural killer (NK) cells expressing the 4-1BB receptor. It has been reported that, through this mechanism, promotion of 4-1BB signaling can inhibit tumor growth. Accordingly, various antibody molecules capable of functionally activating 4-1BB have been developed for tumor therapy.

[0006] Interestingly, activating antibodies that inhibit the binding of the 4-1BB ligand to the 4-1BB receptor and facilitate the aggregation of the 4-1BB receptor (e.g., utomilumab, Pfizer) exhibit partial activity when bound to the Fc receptor, although they alone do not possess sufficient activity. Conversely, activating antibodies that, by additionally binding to the 4-1BB receptor in the ligand-4-1BB ligand-4-1BB state, facilitate the aggregation of the 4-1BB receptor-ligand complex (e.g., urelumab, BMS-663513, BMS) exhibit sufficient activity even without the aid of the Fc receptor, but are observed to show strong toxicity in vivo due to the Fc receptor. In fact, urelumab's clinical trials were discontinued due to this strong toxicity (NCT00612664).

[0007] In addition, there have been attempts to design molecules with tumor transplantation efficacy by using the 4-1BB ligand itself, but no meaningful results have been achieved in the design of bivalent molecules that maintain the intrinsic structure of the ligand with minimal changes and can specifically act on tumors. Summary of the Invention

[0008] Technical issues

[0009] In the process of studying molecular design related to maintaining the intrinsic structure of ligands and being able to specifically act on tumors, the inventors developed a cytokine trimer domain, and in particular derived a method for preparing fusion proteins containing a cytokine trimer domain linked to an antigen-binding domain in a knock-in-hole structure. The binding ability of the fusion proteins prepared by this method to various targets was confirmed, thus completing the present invention.

[0010] Therefore, the object of the present invention is to provide a fusion protein.

[0011] Furthermore, another object of the present invention is to provide a polynucleotide encoding a fusion protein.

[0012] Furthermore, another objective of the present invention is to provide a vector containing a polynucleotide encoding a fusion protein.

[0013] Furthermore, another object of the present invention is to provide a method for producing fusion proteins.

[0014] Furthermore, another object of the present invention is to provide a method for producing a fusion protein of an antibody and a tumor necrosis factor superfamily trimer.

[0015] Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by the inactivation or damage of the host's immune cells.

[0016] Furthermore, another object of the present invention is to provide a method for treating diseases caused by the inactivation or damage of the host's immune cells.

[0017] Technical solution

[0018] To achieve the above objectives, the present invention provides a fusion protein comprising: an antigen-binding domain; and a cytokine trimer domain.

[0019] To achieve another objective mentioned above, the present invention provides a polynucleotide encoding the fusion protein of the present invention.

[0020] To achieve another objective mentioned above, the present invention provides a vector comprising a polynucleotide encoding the fusion protein of the present invention.

[0021] Meanwhile, the present invention provides a carrier composition for producing fusion proteins, comprising: a first carrier, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a first monomer and a second monomer linked by a linker; a second carrier, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a third monomer; and a third carrier comprising a polynucleotide encoding the light chain of Fab.

[0022] To achieve the other objective mentioned above, the present invention provides a method for producing a fusion protein, comprising the step of transfecting a vector containing a polynucleotide encoding the fusion protein of the present invention into a host cell.

[0023] Meanwhile, the present invention also provides a method for producing a fusion protein, comprising the step of transfecting a host cell with a vector composition for producing the fusion protein, the vector composition comprising: a first vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a first monomer and a second monomer linked by a linker; a second vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a third monomer; and a third vector, comprising a polynucleotide encoding the light chain of Fab.

[0024] To achieve another objective mentioned above, the present invention provides a method for producing a fusion protein of an antibody and a tumor necrosis factor superfamily trimer, wherein the tumor necrosis factor superfamily forms a trimer comprising a dimer and a third monomer of the tumor necrosis factor superfamily, the dimer comprising a first monomer and a second monomer of the tumor necrosis factor superfamily (TNFSF), and the production method comprising: step 1) selecting one of two surfaces present between the dimer and the third monomer; step 2) selecting an amino acid pair in the first monomer or the second monomer and the third monomer at a distance of less than 6 Å between the first monomer or the second monomer and the third monomer on the selected surface; step 3) inducing a mutation at the third monomer site in the selected amino acid pair to form a club; step 4) screening for amino acid residues of the first monomer or the second monomer that pair with the club of the third monomer and cause structural collision; step 5) inducing a mutation in the amino acid residues of the screened first monomer or the second monomer to form a mortar; and step 6) inducing a club-mortar interaction between the club of the third monomer and the mortar of the first monomer or the second monomer. The dimer and the third monomer mentioned above are linked with heavy chains of Fab.

[0025] To achieve yet another objective, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention for the prevention or treatment of diseases caused by the inactivation or damage of the function of host immune cells.

[0026] To achieve yet another objective, the present invention provides a method for treating diseases caused by the inactivation or damage of host immune cells, comprising the step of administering the fusion protein of the present invention to a subject.

[0027] The effects of the invention

[0028] This invention relates to a cytokine trimer domain comprising a first monomer and a second monomer (dimer) and a third monomer (monomer) connected by a linker. Preferably, it relates to a novel form of fusion protein RACE (Receptor-Antibody Conjugated (cell) Engager) that binds to the cytokine trimer domain by replacing the cytokine trimer domain and the constant region (Fc) of the antibody prepared by mortise and tenon binding the linker-connected first monomer and the second monomer and the third monomer (monomer) with the aforementioned trimer structure. The method for preparing RACE of this invention improves the purity and productivity of the trimer. It has been confirmed that the RACE prepared by this method not only exhibits superior binding ability to the target receptor compared to the parent antibody, but also exhibits excellent simultaneous binding ability to the antigen and the target receptor, and therefore can be usefully used as a bispecific pharmaceutical composition. Simple Explanation of the Diagram

[0029] Figure 1 is a diagram showing the unit proteins (part A) contained in the fusion protein of the present invention and the fusion protein (part B) containing the above-mentioned unit proteins. Figure 2 is a diagram illustrating the mortar and pestle structure of the cytokine trimer domain of the present invention. Figure 3 shows the interaction sites and distances between the dimers and monomers of the cytokine trimer domain of the present invention (part A) and the results of each interaction (part B). Figure 4 is a diagram comparing the case where the cytokine trimer domain of the present invention generates a pestle in only one direction or generates both a pestle and a mortar simultaneously. Figure 5 shows the results confirming the purity of three fusion proteins containing different antibody and cytokine trimer domains. Figure 6 shows the results of confirming the formation of the mortar and pestle structure (part A), production volume (part B), and purity (part C) in the fusion protein containing the antibody domain and the cytokine trimer domain of the present invention. Figure 7 shows the results of confirming the formation of a club-shaped structure between the dimer and the monomer by confirming the structure of the cytokine trimer domain of the present invention. Figure 8 is a diagram showing the structure of the cytokine trimer domain and the connector that links the dimers according to the present invention. Figure 9 shows the results of identifying disulfide bond proteins with mismatched protein hinges (Part A) and identifying the types and occurrence rates of mismatched hinges (Part B) in the cytokine trimer domain of the present invention without forming a mortar and pestle structure. Figure 10 shows the structure and size of the fusion protein containing the antibody domain and the cytokine trimer domain of the present invention. Figure 11 shows the results confirming the thermodynamic stability of the fusion protein containing antibody domains and cytokine trimers of the present invention. Figure 12 shows the results confirming the binding affinity of the fusion protein containing antibody domains and cytokine trimers of the present invention to the antigen. Figure 13 shows the results confirming the binding affinity of the fusion protein containing antibody domains and cytokine trimers of the present invention to the target receptor. Figure 14 shows the results confirming the simultaneous binding ability of the fusion protein of the present invention, which contains an antibody domain and a cytokine trimer, to both the antigen and the target receptor. Figure 15 shows the results of evaluating the 4-1BB signaling level of the fusion protein containing antibody domains and cytokine trimers of the present invention. Figure 16 shows the results of evaluating the on-target and off-target signal transduction of the fusion protein containing antibody domains and cytokine trimers of the present invention in solid cancer cell lines, where part A represents the results of RACE-02C and part B represents the results of RACE-02B(B). Figure 17 is a schematic diagram of a vector containing a polynucleotide encoding a fusion protein of the present invention, comprising an antibody domain and a cytokine trimer. Figure 18 shows the results of confirming the therapeutic effect of the fusion protein containing antibody domains and cytokine trimers of the present invention in an animal model of induced cancer. Implementation

[0030] The present invention will now be described in detail.

[0031] The present invention provides a fusion protein comprising: an antigen-binding domain and a cytokine trimer domain.

[0032] The aforementioned cytokine trimer domain is characterized by being formed by the combination of a dimer comprising a first monomer and a second monomer with a third monomer. The dimer is characterized by the first monomer and the second monomer being connected by a linker. In the dimer, the N- or C-terminus of the first monomer is interconnected with the N- or C-terminus of the second monomer via a linker.

[0033] The aforementioned adapters can be used without restriction as long as they are adapters known in the technical field of this invention that can connect the first monomer and the second monomer of a cytokine. In one embodiment of this invention, G1-G4S are connected by a short peptide adapter. The aforementioned G1 contains the sequence G, and the aforementioned G4S contains the sequence GGGGS (sequence 34).

[0034] In particular, in the cytokine trimer domain of the present invention, although the aforementioned cytokines can naturally form trimers, when using the naturally occurring trimer structure to prepare a fusion protein that binds to an antibody, mismatched disulfide bonds occur at the hinge portion of the Fab 3 portion of the antibody linked to each trimer, resulting in reduced purity and productivity. Therefore, to improve productivity with higher purity, it is preferable that one or more amino acid residues of the first or second monomer constituting the dimer form a club-and-mortar structure with one or more amino acid residues of the third monomer. Preferably, the club-and-mortar structure is formed in a region other than the cytokine-receptor binding region.

[0035] Furthermore, the aforementioned one or more amino acid residues are characterized by being located on the protein surface. In particular, the first monomer and the second monomer are connected in tandem via a linker, and the mortar can be generated in the first monomer or the second monomer, depending on the surface position of the pestle generated in the third monomer.

[0036] Through the aforementioned pestle-and-mortar structure, one of the first or second monomers constituting the dimer forming the mortar can induce selective interaction with the third monomer forming the pestle, while steric hindrance can be induced on the surfaces of the remaining first or second monomers and the third monomers that do not form the mortar. This induces interaction with the desired surface, thereby preventing mismatch induced when using the trimer in its natural state. That is, for example, in this invention, interactions can be induced on the surfaces of the mortar forming the first monomer and the pestle forming the third monomer, inducing steric hindrance between the second and third monomers; or interactions can be induced on the surfaces of the mortar forming the second monomer and the pestle forming the third monomer, inducing steric hindrance between the first and third monomers.

[0037] On the other hand, in protein-protein interactions, the three important interactions (a) hydrogen bond (2.4-3.2 Å), (b) hydrophobic interaction (3.2-3.9 Å), and (c) charge interaction (~4 Å) usually occur at distances within 4 Å.

[0038] Therefore, in the amino acid residues of the first monomer, second monomer, or third monomer of the present invention, one or more amino acid residues of the first monomer and an amino acid residue of the third monomer may be located at a distance of 6 Å or less, and one or more amino acid residues of the second monomer and one or more amino acid residues of the third monomer may be located at a distance of 6 Å or less. Preferably, one or more amino acid residues of the first monomer and an amino acid residue of the third monomer may be located at a distance of 4 Å or less, and one or more amino acid residues of the second monomer and one or more amino acid residues of the third monomer may be located at a distance of 4 Å or less. More preferably, one or more amino acid residues of the first monomer and an amino acid residue of the third monomer may be located at a distance of 2 Å to 4 Å, and one or more amino acid residues of the second monomer and one or more amino acid residues of the third monomer may be located at a distance of 2 Å to 4 Å.

[0039] Therefore, amino acid residues can be selected at a distance of less than 6 Å between the dimer and the monomer, preferably at a distance of less than 4 Å between the dimer and the monomer, and more preferably at a distance of 2 Å to 4 Å between the dimer and the monomer. For example, in the case of the dimer formed from 4-1BBL monomers, the cytokine can be one or more selected from the group consisting of F92, F238, Q94, F144, V234, Q146, E148, F199, Y142, L203, R202, Q200, and A180.

[0040] The mutation sites of the above amino acids were determined based on the amino acid sequence of TNFSF9 (TNF superfamily member 9) from NCBI (accession number: NM_003811.3).

[0041] More preferably, the club-and-mortar structure can be formed by inducing amino acid variations in each monomer. Therefore, the aforementioned amino acid residues can be one or more selected from the group consisting of F92W, F238V, Q94S, Q94Y, F144I, V234H, V234F, V234R, Q146S, E148G, F199L, Y142T, L203A, R202V, R202W, R202F, F199W, and A180E.

[0042] More preferably, the aforementioned amino acid residues can form a pestle-and-mortar structure by pairing two amino acid residues. Even more preferably, the aforementioned amino acid residues are characterized in that the pestle-and-mortar structure is formed by pairing R202W and Q94S.

[0043] In cytokines, such as 4-1BBL, a trimer can be prepared by combining the dimer (linking the first and second monomers via a linker) E148G (forming a mortise) and the monomer (the third monomer) A180E (forming a konb). Furthermore, a trimer can be prepared by combining the dimer (linking the first and second monomers via a linker) R202V (forming a mortise) and the monomer (the third monomer) V234R (forming a konb). Simultaneously, a trimer can be prepared by combining the dimer (linking the first and second monomers via a linker) Q94S (forming a mortise) and the monomer (the third monomer) R202W (forming a konb).

[0044] According to one embodiment of the present invention, in cytokines, for example in 4-1BBL, a trimer of a mortar and pestle is prepared, consisting of a dimer (Q94S, which forms a mortar) and a monomer (R202W, which forms a pestle) forming a mortar.

[0045] Meanwhile, as shown in Figure 2, two surfaces (Interface A and Interface B (KiH)) are formed between the dimer and monomers that form the mortar structure as described above. Therefore, the present invention can maintain the natural shape of the surface on Interface B (the surface opposite to the mortar (KiH), thereby achieving selective trimerization with minimal changes. Therefore, the trimer of the present invention is characterized by maintaining a parallel trimer structure with three monomers in parallel.

[0046] In the fusion protein of the present invention, the antigen-binding domain is an antibody or an antibody fragment.

[0047] In the fusion protein of the present invention, the antibody or antibody fragment thereof can be used without restriction as long as it is an antibody or fragment capable of binding to the target antigen. In one embodiment of the present invention, a fusion protein comprising an antibody selected from the group consisting of rituximab, cetuximab, trastuzumab, and avelumab, and a cytokine trimer domain is prepared.

[0048] The antibody fragments mentioned above may include light chain variable regions and heavy chain variable regions. Preferably, they may be one or more selected from the group consisting of Fab, Fab', F(ab')2, scFv, di-scFv and VHH (variable heavy chain domains of heavy chain antibody).

[0049] In particular, the antibody fragment is linked to a cytokine trimer via its N-terminus or C-terminus to form a fusion protein. More preferably, the antibody fragment can be F(ab')2 having the following structure: the first hinge of the F(ab')2 is linked to a first or second monomer connected via a linker; the second hinge of the F(ab')2 is linked to a third monomer.

[0050] In the fusion protein of the present invention, the aforementioned cytokines are characterized in that they bind to the receptor via a trimer in their natural state.

[0051] Therefore, preferably, the aforementioned cytokines can be the tumor necrosis factor superfamily capable of forming trimers under natural conditions. More preferably, they can be one or more selected from the group consisting of TNFα, Dif, Necrosin, TNFβ, TNFSF1B, TNFγ, CD252, Gp34, CD134L, CD154, TRAP, Gp39, T-BAM, CD178, APTL, CD95L, CD70, CD153, and 4-1BBL. More preferably, they can be 4-1BBL.

[0052] According to an embodiment of the present invention, in the fusion protein of the present invention, the globular protein domain (G90-T241) exposed to the cell exterior in the entire sequence of the above-mentioned 4-1BB (retrieval number: NM_003811.3) is used. The above-mentioned 4-1BBL is characterized in that it contains the amino acid sequence represented by sequence 17.

[0053] GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT (sequence 17).

[0054] More preferably, the trimer 4-1BBL contained in the above-mentioned fusion protein may have the following structure: the first 4-1BBL monomer and the second 4-1BBL monomer are linked by a linker; one or more amino acid residues of the first 4-1BBL monomer or the second 4-1BBL monomer and one or more amino acid residues of the third 4-1BBL monomer form a club-and-mortar structure.

[0055] The structure of a 4-1BBL-based fusion protein prepared according to an embodiment of the present invention is shown in Figure 1. The fusion protein of the present invention consists of a unit protein as shown in part A of Figure 1, having the structure shown in part B of Figure 1. More specifically, it consists of three components: 1) an antibody heavy chain containing a 4-1BB ligand dimer; 2) an antibody heavy chain containing a 4-1BB ligand monomer; and 3) a light chain of antibody. In part B of Figure 1, the antigen-binding fragment at the upper end is connected to the 4-1BB ligand trimer at the lower end via a hinge. The formation of the complex is achieved through two disulfide bonds present in the hinge and hydrophobic interactions present between the 4-1BB ligand trimer. In particular, the hydrophobic interactions between the ligand trimers are selectively formed between the 4-1BB ligand dimer and the 4-1BB ligand monomer via a pestle-and-mortar module, thereby significantly improving the physical properties and purity of the protein. The 4-1BBL monomer is known to have a pestle-containing structure, which is necessary to prevent homotrimerization of the heavy chain contained in the monomer. The 4-1BB ligand dimer is linked by a short amino acid linker between the two monomers, which has a mortar at the site where the "pestle" interacts with the corresponding monomer.

[0056] In a specific example of the cytokine trimer domain of the present invention based on the above, it may comprise a monomer and a dimer, wherein the monomer comprises an amino acid sequence represented by sequence 1, and the dimer comprises an amino acid sequence represented by sequence 2. In the dimer, the two monomers may be linked by a linker at amino acid sequence (G) at position 170. Alternatively, it may comprise a monomer and a dimer, wherein the monomer comprises a nucleotide sequence represented by sequence 3, and the dimer comprises a nucleotide sequence represented by sequence 4. In the dimer, the two monomers may be linked by a linker at GGA in the nucleotide sequence from position 508 to position 510.

[0057] In another specific example of the cytokine trimer domain of the present invention, a monomer and a dimer may be included, wherein the monomer comprises an amino acid sequence represented by sequence 5 (with a variation of R to W at amino acid position 202), and the dimer comprises an amino acid sequence represented by sequence 6 (with a variation of Q to S at amino acid position 94). In the dimer, the two monomers may be linked by a linker at amino acid position 170 (G). Simultaneously, the monomer and dimer may form a club-and-mortar structure at the position of the aforementioned amino acid variation. Alternatively, a monomer and a dimer may be included, wherein the monomer comprises a nucleotide sequence represented by sequence 7 (with a variation of AGA to TGG at amino acid position 202), and the dimer comprises a nucleotide sequence represented by sequence 8 (with a variation of CAG to AGC at amino acid position 94). In the dimer, the two monomers may be linked by a linker at GGA in the nucleotide sequence from position 508 to position 510.

[0058] In another specific example of the cytokine trimer domain of the present invention, it may comprise a monomer and a dimer, wherein the monomer comprises an amino acid sequence represented by sequence 9 (with a variation of A to E in the 180th amino acid sequence), and the dimer comprises an amino acid sequence represented by sequence 10 (with a variation of E to G in the 148th amino acid sequence). In the dimer, the two monomers may be linked by a linker at the 170th amino acid sequence (G). Simultaneously, the monomer and dimer may form a club-and-mortar structure at the position of the aforementioned amino acid variation. Alternatively, it may comprise a monomer and a dimer, wherein the monomer comprises a nucleotide sequence represented by sequence 11 (with a variation of GCT to GAA in the 180th amino acid sequence), and the dimer comprises a nucleotide sequence represented by sequence 12 (with a variation of CAA to GGA in the 148th amino acid sequence). In the dimer, the two monomers may be linked by a linker at the GGA of the nucleotide sequence from position 508 to position 510.

[0059] In another specific embodiment of the cytokine trimer domain of the present invention, a monomer and a dimer may be included, wherein the monomer comprises an amino acid sequence represented by sequence 13 (with a variation of V to R at amino acid position 234), and the dimer comprises an amino acid sequence represented by sequence 14 (with a variation of R to V at amino acid position 202). In the dimer, the two monomers may be linked by a linker at amino acid position 170 (G). Simultaneously, the monomer and dimer may form a club-and-mortar structure at the position of the aforementioned amino acid variation. Alternatively, a monomer and a dimer may be included, wherein the monomer comprises a nucleotide sequence represented by sequence 15 (with a variation of GTG to AGA at amino acid position 234), and the dimer comprises a nucleotide sequence represented by sequence 16 (with a variation of AGA to GTG at amino acid position 202). In the dimer, the two monomers may be linked by a linker at the GGA of the nucleotide sequence from position 508 to position 510.

[0060] Therefore, the fusion protein of the present invention may include a cytokine trimer domain selected from the group consisting of a monomer containing an amino acid sequence represented by sequence 1 and a dimer containing an amino acid sequence represented by sequence 2, a monomer containing an amino acid sequence represented by sequence 5 and a dimer containing an amino acid sequence represented by sequence 6, a monomer containing an amino acid sequence represented by sequence 9 and a dimer containing an amino acid sequence represented by sequence 10, and a monomer containing an amino acid sequence represented by sequence 13 and a dimer containing an amino acid sequence represented by sequence 14.

[0061] Those skilled in the art to which this invention pertains should understand that the amino acid sequences represented by sequences 1, 2, 5, 6, 9, 10, 13, and 14 are limited in terms of variation in amino acid sequences that exhibit equivalent biological activity. Such amino acid variation is based on the relative similarity of amino acid side chain substitutes, for example, based on hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the substitutions, shapes, and types of amino acid side chain substitutes reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically equivalent.

[0062] In terms of introducing variations, the hydrophobic index of amino acids can be considered. Hydrophobic indices are assigned to each amino acid based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); aspartamine (-3.5); lysine (-3.9); and arginine (-4.5).

[0063] The hydrophobic index of amino acids is crucial in imparting interactive biological functions to proteins or peptides. It is a well-known fact that similar biological activity can be maintained by substituting amino acids with similar hydrophobic indices. When introducing variations with reference hydrophobic indices, substitutions are preferably made between amino acids with a hydrophobic index difference within ±2, more preferably between amino acids with a hydrophobic index difference within ±1, and even more preferably between amino acids with a hydrophobic index difference within ±0.5.

[0064] On the other hand, it is well known that substitutions between amino acids with similar hydrophilicity values ​​also result in proteins or peptides with equivalent biological activities. The following hydrophilicity values ​​are assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); aspartamine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0065] When introducing variations based on hydrophilicity values, it is preferable to substitute between amino acids with a hydrophilicity value difference within ±2, more preferably, between amino acids with a hydrophilicity value difference within ±1, and even more preferably, between amino acids with a hydrophilicity value difference within ±0.5.

[0066] Amino acid exchanges in peptides that do not alter their overall molecular activity are well known in the art to which this invention pertains (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0067] Considering the aforementioned variations with biologically equivalent activity, the amino acid sequences represented by sequences 1, 2, 5, 6, 9, 10, 13, and 14 of this invention should be interpreted as containing sequences with substantial identity to those listed in the sequence listing. Substantial identity means that when the amino acid sequences represented by sequences 1, 2, 5, 6, 9, 10, 13, and 14 of this invention are aligned with any other sequence, and the aligned sequences are analyzed using algorithms commonly used in the art to which this invention pertains, at least 80% homology is shown, and more preferably, at least 90% homology is shown. The alignment method used for sequence comparison can be any method known in the art to which this invention pertains, without limitation.

[0068] Simultaneously, the nucleotide sequences represented by sequences 3, 4, 7, 8, 11, 12, 15, and 16 may include nucleotide sequences having more than 70% sequence homology with the nucleotide sequences represented by sequences 3, 4, 7, 8, 11, 12, 15, and 16, more preferably, they may include nucleotide sequences with more than 80% sequence homology, even more preferably, they may include nucleotide sequences with more than 90% sequence homology, and most preferably, they may include nucleotide sequences with more than 95% sequence homology. The "sequence homology" of the nucleotide sequences is confirmed by comparing two optimally arranged comparison regions. A portion of the nucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) relative to a reference sequence (excluding additions or deletions) of the optimal arrangement of the two sequences.

[0069] As previously described, in the cytokine trimer domain of the fusion protein of the present invention, the mortar and pestle structure is characterized by being generated through amino acid variations occurring separately in the monomer and dimer. This is to prevent the generation of mismatched disulfides during the misassembly of the hinge portion of the antibody (Fab) during the trimer assembly of the tumor necrosis factor superfamily, which exists as a trimer in its natural state.

[0070] Furthermore, the present invention provides a polynucleotide encoding the fusion protein of the present invention.

[0071] Meanwhile, the present invention provides a vector containing a polynucleotide encoding the fusion protein of the present invention.

[0072] A schematic diagram of the above-mentioned vector is shown in Figure 17. As shown in Figure 17, the present invention is characterized in that the gene site of the vector contains light chain, heavy chain dimer and / or heavy chain monomer.

[0073] Furthermore, the present invention provides a carrier composition for producing fusion proteins, comprising: a first carrier, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a first monomer and a second monomer linked by a linker; a second carrier, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a third monomer; and a third carrier comprising a polynucleotide encoding the light chain of Fab.

[0074] When using the above-described carrier composition for producing fusion proteins, the formation of trimers can be induced after separately preparing the dimers and Fab heavy chains in the trimer, and the monomers and Fab heavy chains in the trimer.

[0075] In this invention, the term "vector" refers to a gene preparation containing a gene nucleotide sequence operably linked to a suitable regulatory sequence for expressing a target gene within a suitable host. The regulatory sequence may include a promoter to initiate transcription, an operator sequence to regulate the transcription, and a termination sequence to regulate transcription and interpretation. The vector of this invention is not particularly limited as long as it can replicate within a cell; any vector known in the art can be used, such as plasmids, cosmids, phage particles, or viral vectors.

[0076] Meanwhile, the present invention provides a method for producing fusion proteins, including the step of transfecting a vector containing a polynucleotide encoding the fusion protein of the present invention into a host cell.

[0077] Furthermore, the present invention also provides a method for producing a fusion protein, comprising the step of transfecting a host cell with a vector composition for producing the fusion protein, the vector composition comprising: a first vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a first monomer and a second monomer linked by a linker; a second vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding a third monomer; and a third vector, comprising a polynucleotide encoding the light chain of Fab.

[0078] In this invention, the "recombinant vector," when operably linked to the coding gene of the target polypeptide to be expressed, can be used as an expression vector for the target polypeptide capable of efficiently expressing the aforementioned target polypeptide in a suitable host cell. The recombinant vector can be expressed in the host cell. Preferably, the host cell can be a eukaryotic cell, and expression regulatory sequences such as promoters, terminators, and enhancers, as well as sequences for membrane targeting or secretion, can be appropriately selected and combined in various ways according to the type of host cell, depending on the purpose.

[0079] According to an embodiment of the present invention, it has been confirmed that the fusion protein of the present invention, which includes an antigen-binding domain and a cytokine trimer domain, has a better binding capacity to antigens than the parent antibody and a better binding capacity to cytokine target receptors than the parent antibody. It has been confirmed that the protein can bind simultaneously and can successfully induce binding in cells expressing each target (protein).

[0080] Simultaneously, this invention provides a method for producing a fusion protein of an antibody and a tumor necrosis factor superfamily trimer, wherein the tumor necrosis factor superfamily forms a trimer comprising a dimer and a third monomer of the tumor necrosis factor superfamily, the dimer comprising a first monomer and a second monomer of the tumor necrosis factor superfamily, and the production method comprising: step 1) selecting one of two surfaces present between the dimer and the third monomer; step 2) selecting an amino acid pair in each of the first monomer or the second monomer and the third monomer at a distance of less than 6 Å between the first monomer or the second monomer and the third monomer on the selected surface; step 3) inducing a mutation at the third monomer site in the selected amino acid pair to form a club; step 4) screening for amino acid residues of the first monomer or the second monomer that pair with the club of the third monomer and cause structural collision; step 5) inducing a mutation in the amino acid residues of the screened first monomer or the second monomer to form a mortar; and step 6) inducing a club-mortar interaction between the club of the third monomer and the mortar of the first monomer or the second monomer. A Fab heavy chain is linked in the dimer and the third monomer.

[0081] In step 2) of the above-mentioned method for producing the fusion protein of antibody and tumor necrosis factor superfamily trimer, the amino acid pair may be located at a distance of less than 6 Å between the first monomer or the second monomer and the third monomer on a surface, preferably at a distance of less than 4 Å, and more preferably at a distance of 2 Å to 4 Å.

[0082] In the above-mentioned method for producing the fusion protein of antibody and tumor necrosis factor superfamily trimer, the amino acid residues that cause structural conflicts related to the relevant residues can be replaced by simulation with any amino acid residue selected from the group consisting of glycine (G), valine (V), serine (S), and alanine (A) as candidates.

[0083] Furthermore, the present invention is characterized in that a mortise and tenon joint is formed in the selected surface in step 1) above, inducing a steric hindrance effect between monomers on the remaining unselected surfaces. This steric hindrance effect can induce interactions with the desired surface. This prevents the formation of mismatched disulfides in the hinge portion of the connecting antibody (Fab) that may be induced when producing fusion proteins using trimers in their natural state.

[0084] According to one embodiment of the present invention, it was confirmed that in the case of fusion proteins in which cytokines and antibodies bind without employing a mortise and tenon structure, unpaired cysteine ​​residues were found near the hinge region where cysteine ​​residues pair to form disulfide bonds, or that all three hinges asymmetrically formed paired disulfide bonds.

[0085] Therefore, the production method of the present invention is the best method to prevent inhomogeneity caused by abnormal disulfide bonds in the hinge portion of the produced antibody-tumor necrosis factor superfamily trimer fusion protein, which can increase the productivity and purity of the fusion protein and provide the above-mentioned fusion protein as a more stable therapeutic protein.

[0086] The dimer described above is characterized by a first monomer and a second monomer connected by a linker. Furthermore, the N- or C-terminus of the first monomer and the N- or C-terminus of the second monomer are interconnected by the linker. The linker can be used without restriction as long as it is a linker known in the art capable of connecting the first and second monomers of a cytokine.

[0087] Furthermore, any antibody fragment (Fab) that can bind to the target antigen can be used without restriction.

[0088] Meanwhile, the present invention provides a fusion protein of antibody and tumor necrosis factor superfamily trimer produced by the above-described production method.

[0089] The aforementioned description of fusion proteins can also be applied to the fusion proteins of antibodies and tumor necrosis factor superfamily trimers produced by the above-described production method.

[0090] Furthermore, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention for the prevention or treatment of diseases caused by the inactivation or damage of host immune cells.

[0091] Furthermore, the present invention provides a method for treating diseases caused by the inactivation or damage of host immune cells, comprising the step of administering the fusion protein of the present invention to a subject.

[0092] Preferably, the subjects are mammals, including humans, including patients who need treatment for diseases caused by the inactivation or damage of host immune cells, patients who are being treated for diseases caused by the inactivation or damage of host immune cells, and patients who have been treated for diseases caused by the inactivation or damage of host immune cells. It may also include patients who have received surgical treatment for diseases caused by the inactivation or damage of host immune cells.

[0093] Furthermore, the fusion protein of the present invention can be combined with existing drugs or treatments to treat diseases caused by the inactivation or damage of host immune cells. When used in combination with the fusion protein of the present invention, it can be used in conjunction with or sequentially with other drugs or treatments to treat diseases caused by the inactivation or damage of host immune cells.

[0094] The diseases caused by the inactivation or damage of host immune cells can be one or more of the following groups: cancer, immune diseases, autoimmune diseases, central nervous system diseases, degenerative neurological diseases, autoimmune diseases, and inflammatory diseases. The cancers mentioned above can be one or more of the following groups: breast cancer, lung cancer, colorectal cancer, and colorectal cancer.

[0095] In this invention, the pharmaceutical composition is characterized in that it can be in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and is intended for human use. The pharmaceutical composition can be formulated using conventional methods into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as external applications, suppositories, and sterile injectable solutions, but is not limited thereto.

[0096] The pharmaceutical compositions of the present invention can contain pharmaceutically acceptable carriers. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. In the case of injections, they may be mixed with buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. For topical administration, they may include bases, excipients, lubricants, preservatives, etc. The dosage forms of the pharmaceutical compositions of the present invention can be prepared in a variety of ways by mixing them with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, they can be prepared as tablets, coated tablets, capsules, liquids (elixir), sustained-release tablets, syrups, round tablets, etc. For injections, they can be prepared as single-dose ampoules or multiple-dose formulations.

[0097] On the other hand, suitable carriers, excipients, and diluents for formulation include lactose, sucrose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil. Furthermore, they may also contain fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, and preservatives.

[0098] The pharmaceutical compositions of the present invention can be administered via oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, lesion, sublingual, or rectal routes, but are not limited thereto. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, lesion-related, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration.

[0099] The pharmaceutical compositions of the present invention can be varied in many ways depending on factors including the activity of the specific active ingredient used, age, weight, general health condition, sex, diet, administration time, route of administration, metabolic rate, drug combination, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical compositions can be appropriately selected by a practitioner based on the patient's condition, weight, disease severity, drug form, route of administration, and duration. Preferably, the dosage can be administered at the minimum amount that does not cause side effects while taking all the above factors into account, to achieve the maximum effect. More preferably, an effective dose of 1 μg / kg / day to 10000 μg / kg / day can be administered several times a day. Even more preferably, an effective dose of 10 mg / kg / day to 1000 mg / kg / day can be administered several times a day. The above dosages do not limit the scope of the invention in any way.

[0100] The contents of this invention described above can be used interchangeably as long as they do not contradict each other. Appropriate modifications made by those skilled in the art to which this invention pertains should also be included within the scope of this invention.

[0101] The present invention will be described in detail below through embodiments, but the scope of the present invention is not limited to the following embodiments.

[0102] Experimental Example 1. Preparation of Cell Lines

[0103] The Freestyle293F cell line (Gibco™, R79007) was cultured in Freestyle 293F expression medium (Gibco™, 12338018) at 37°C and 8% CO2 at a rotation speed of 120 RPM. Meanwhile, Raji-B cell lines (CCL-86™, ATCC), MDA-MB-231 cell lines (HTB-26™, ATCC), and SW-480 cell lines (10228, KCLB) were cultured in RPMI1640 (Welgene) medium supplemented with 10% (v / v) fetal bovine serum (FBS) at 37°C and 8% CO2. PC-9 cell lines (CVCL_B260) and HT-29 cell lines (HTB-38™, ATCC) were cultured in DMEM (Welgene) medium supplemented with 10% (v / v) fetal bovine serum at 37°C and 5% CO2.

[0104] Example 1. Preparation of a fusion protein linking cytokine trimer domains and antibody domains.

[0105] 1-1. Preparing genes

[0106] To construct the fusion protein of the present invention, the following three coding sequences were designed: 1) the light chain of the antibody, 2) the sequence of the C-terminus of the antigen-binding site of the antibody heavy chain linked to a 4-1BB ligand monomer, and 3) the sequence of the C-terminus of the antigen-binding site of the antibody heavy chain linked to a 4-1BB ligand dimer.

[0107] In this invention, the human 4-1BB ligand protein was used, and the related gene (NM_003811.3) was synthesized using Geneart. The invention utilizes the globular protein domains (G90-T241) exposed to the extracellular space within the entire sequence of the 4-1BB ligand protein. Gene cloning and mutation generation were performed using gateway cloning.

[0108] 1-2. Preparation of carriers for the production of fusion proteins

[0109] In order to prepare a fusion protein in which the Fc site of an antibody is replaced with a cytokine trimer (hereinafter referred to as "RACE" (receptor-antibody conjugated (cell) adaptor)), a first vector relating to the Fab heavy chain and the cytokine dimer, a second vector relating to the Fab heavy chain and the cytokine monomer, and a third vector relating to the light chain were prepared.

[0110] In this case, amino acid residues for forming mutations were selected in the trimeric domain of the 4-1BB ligand, which is a cytokine, by using simulations with Pymol software and the Dynamut server (http: / / biosig.unimelb.edu.au / dynamut / ).

[0111] A trimeric domain is formed using a portion of the 4-1BB ligand sequence (G90-T241) as the unit. Specifically, to significantly improve the physical properties and purity of the prepared trimer and prevent homotrimerization of the heavy chain containing the monomer, a mortar-and-groove structure is used to form the trimer. For this purpose, using 1) an antibody heavy chain containing a 4-1BB ligand dimer and 2) an antibody seed heavy chain containing a 4-1BB ligand monomer, a mortar-and-groove structure is formed only on one of the two surfaces between the dimer and the monomer, thus achieving selective trimerization with minimal variation. The mortar is formed in the 4-1BB ligand monomer, and the groove is formed in one of the 4-1BB ligand dimers. The units of the trimer and their binding morphology are shown in Figure 1, and the mortar-and-groove structures that can be formed on the surfaces of each unit are shown in Figure 2.

[0112] Based on the structure of the human 4-1BBL protein complex (PDB code: 6A3V, 2X29), Pymol software (https: / / pymol.org / 2 / ) was used to perform structural analysis of the trimer domain formed as described above.

[0113] The interaction sites between the 4-1BB ligand dimer and the monomer, as confirmed by this method, are shown in Figure 3.

[0114] As shown in Figure 3, amino acids located at distances less than 4 Å were identified on the interacting surfaces (part A of Figure 3). Meanwhile, in part B of Figure 3, the interactions formed by the amino acids in part A of Figure 3 are shown by dashed lines (hydrogen bonds) or solid lines (hydrophobic interactions).

[0115] Mutations were induced in the amino acid residues derived as described above to form a mortar structure between the dimer and the monomer. A schematic diagram of the wild type and the formation of the mortar of each variant, predicted by mutation induction, is shown in Figure 4.

[0116] The aforementioned 4-1BB ligand exists in its natural state as a trimer. During the assembly of this trimer, misaligned disulfides are generated due to misalignment of the sequence at the hinge portion connecting the antibody (Fab). To address this issue, this invention utilizes a pestle-and-mortar structure to produce the trimer.

[0117] As shown in Figure 4, if the amino acid residues of the monomers are not mutated, a pestle-and-mortar structure will not be formed (left side of Figure 4). If the amino acid residues of the monomers are mutated only, and the pestle is applied only to the mutated surface, the interaction with the natural surface will be subject to steric hindrance (center of Figure 4). Therefore, when the amino acids of both monomers are mutated as in this invention, the pestle and mortar are applied separately, thereby forming a stable complementary structure (see right side of Figure 4).

[0118] Considering the above, a first vector containing the heavy chain-cytokine (dimer) of the inserted antibody and wild-type cytokine sequence, a second vector containing the heavy chain-cytokine (monomer), and a third vector containing the antibody light chain were prepared. Mutations were then induced in the dimers and monomers to induce the formation of a club-and-socket structure.

[0119] More specifically, in terms of the formation of the above-mentioned pestle and mortar structure, it is prepared by forming a pestle and mortar by using a dimer (the first monomer and the second monomer are connected by a connector) Q94S (forming a mortar) and a monomer (the third monomer) R202W (forming a pestle).

[0120] First, a template (containing the antibody heavy chain, antibody light chain, 4-1BBL (WT) dimer, and monomer) for cloning was synthesized using Geneart (Thermofisher). In this case, a GGA (glycine) sequence, acting as a linker, was inserted into the dimer at nucleotide positions 508-510. Four antibodies were used: rituximab (Drugbank accession number (DB00073)), cetuximab (Drugbank accession number (DB00002)), trastuzumab (Drugbank accession number (DB00072)), or avelumab (Drugbank accession number (DB11945)). Then, each clone was performed using Gateway cloning based on the pCMV vector. More specifically, a first vector was prepared into the pCMV vector containing an antibody heavy chain-cytokine dimer, into which an antibody heavy chain-linker-4-1BBL (dimer) was inserted at the multiple cloning site (MCS). For this purpose, gateway cloning was performed after amplifying the first fragment of the antibody heavy chain, the second fragment of the first monomer in the 4-1BBL dimer, and the third fragment of the second monomer in the 4-1BBL dimer using the primers shown in Table 1 below.

[0121] Table 1 direction Sequence (5'→3') SEQ ID NO First segment positive tacacgtacttagtcgctgaagctcttcTATGGGATGGAGCTATATC 18 First segment Reverse taggtacgaactcgattgacggctcttcATCCCCCCAGGAGTTCAGG 19 Second segment positive tacacgtacttagtcgctgaagctcttcAGGAGGCATGTTTGCCCAGC 20 Second segment Reverse taggtacgaactcgattgacggctcttcAGCCTCCTGTCACTCTGAACAGGCC twenty one The third segment positive tacacgtacttagtcgctgaagctcttcAGGCATGTTTGCCCAGC twenty two The third segment Reverse aggtacgaactcgattgacggctcttcAGAGTTTATGTCACTCTGAACAGGCC twenty three

[0122] Simultaneously, a second vector was prepared in which an antibody heavy chain-cytokine monomer linked to the 4-1BBL chain via the VH-CH1-hinge was inserted into the multiple cloning site (MCS) of the pCMV vector. For this purpose, gateway cloning was performed after amplifying the first fragment of the antibody heavy chain and the second fragment of the 4-1BBL monomer using the primers shown in Table 2 below.

[0123] Table 2 direction Sequence (5'→3') SEQ ID NO First segment positive tacacgtacttagtcgctgaagctcttcTATGGGATGGAGCTATATC twenty four Reverse taggtacgaactcgattgacggctcttATCCCCCCAGGAGTTCAGG 25 Second segment positive tacacgtacttagtcgctgaagctcttcAGGAGGCATGTTTGCCCAGC 26 Reverse aggtacgaactcgattgacggctcttcAGAGTTTATGTCACTCTGAACAGGCC 27

[0124] Furthermore, a third vector was prepared to insert the antibody light chain into the multiple cloning site of the pCMV vector. In this case, the fragment was amplified using the primers shown in Table 3 below, followed by Gateway cloning.

[0125] Table 3 direction Sequence (5'→3') SEQ ID NO single fragment positive TACACGTACTTAGTCGCTGAAGCTCTTCTAGATCTGTGGCTGCACCA 28 Reverse AGGTACGAACTCGATTGACGGCTCTTCATCTCTTCACTTCCACCTT 29

[0126] Then, Gateway cloning was performed to apply the mutations in the dimer of the first vector and the monomer of the second vector. In this case, the primers shown in Table 4 below were used. As a result, the Q94S mutation was applied to the dimer of the first vector, and the R202W mutation was applied to the monomer of the second vector.

[0127] Table 4 direction Sequence (5'→3') SEQ ID NO Dimer (Q94S) positive tacacgtacttagtcgctgaagctcttcTagcCTGGTGGCCCAGAATG 30 Reverse taggtacgaactcgattgacggctcttcAgctGGCAAACATGCCTCC 31 monomer (R202W) positive tacacgtacttagtcgctgaagctcttcTtggCTGCTGCACCTGTCTG 32 Reverse gtacgaactcgattgacggctcttcAccaGCCTTGGAAGCCAAAGGC 33

[0128] The insertion of each gene was confirmed and the sequence was verified through gene sequencing (commissioned to Cosmogenetech).

[0129] 1-3. Transfection and production of RACE using the first, second, and third carriers.

[0130] For RACE transfection and expression, Freestyle293F cells prepared in Experiment 1 above were used. Freestyle293F cell lines were prepared at a concentration of 1×10⁶ / ml 24 hours prior to transfection. Transfection was performed in these cells using Fectopro (Polyplus, 116-010) reagent and the first, second, and third vectors prepared above. More specifically, 1 μg of deoxyribonucleic acid (DNA) (mixed with the first, second, and third vectors in a 1:1:2 volume ratio) and 1 μl of Fectopro reagent were added to 1 ml of the cell line. After temporary transfection, the cells were cultured in suspension for 5 days (37°C, 8% CO₂, 120 RPM), and the supernatant containing secreted proteins was separated by centrifugation at 1500g for 30 minutes. For subsequent purification, the separated supernatant was refrigerated for up to three days.

[0131] Example 2. Purification and purity analysis of RACE

[0132] RACE was purified from the supernatants obtained in Examples 1-2-3 above using AKTA go (Cytiva) and affinity chromatography. Affinity chromatography was performed using a CH1-xL column (Thermo). Washing solutions (phosphate-buffered saline, pH 7.4) and elution solutions (100 mM sodium acetate, pH 4.0) used in the filtration chromatography were prepared before use, and the pH was determined. The protein-containing fraction was collected from the elution fraction and dialyzed against phosphate-buffered saline using an Amicon® Ultra (30 kDa, 15 mL) filter for storage.

[0133] To analyze the purity of the purified RACE, size exclusion chromatography (PL1580-3250, Agilent) was performed using high-performance liquid chromatography (HPLC, Agilent 1260). The approximate protein size predicted by size exclusion chromatography was determined experimentally using standard proteins of known size (5190-2242, Agilent). Purity analysis was performed using a 150 mM sodium phosphate (pH 7.0) buffer.

[0134] The results are shown in Figure 5. The purity of avirumab-41BBL (RACE-04L) of the present invention is 97.24% (refer to the PD-L1 targeting (4AL) curve in Figure 5), the purity of cetuximab-41BBL (RACE-02B) is 96.47% (refer to the EGFR targeting (Cetuximab) curve in Figure 5), and the purity of trastuzumab-41BBL (RACE-02C) is 96.91% (refer to the HER2 targeting (Herceptin) curve in Figure 5). The RACEs prepared according to the present invention all show excellent productivity and purity.

[0135] Meanwhile, in the rituximab-41BBL (RACE-01C) of the present invention, when using the mortar and pestle (Q94S / R202W), as shown in part A of FIG6, it was confirmed that the 4-1BB ligand dimer (mortar) and the 4-1BB ligand monomer (mortar) successfully formed a 4-1BB ligand trimer. This trimer was confirmed to consist of a heavy chain dimer containing the mortar (Q94S), a heavy chain monomer containing the pestle (R202W), and a light chain. Furthermore, the purification and purity analysis of the rituximab-41BBL (RACE-01C) of the present invention, performed using the same method as described above, showed a production yield similar to that of a normal antibody with an Fc dimer (part B of FIG6). After a single purification based on CH1, it was confirmed that the protein was isolated with a purity of over 96% (part C of FIG6).

[0136] Example 3. Structural Analysis of RACE

[0137] The structure of the 4-1BBL trimer domain was analyzed using Pymol software (https: / / pymol.org / 2 / ) based on the structure of the human 4-1BBL protein complex (PDB code: 6A3V, 2X29).

[0138] The results, as shown in Figures 7 and 8, confirmed that the C-terminus of 4-1BBL a and the N-terminus of 4-1BBL b, constituting the dimer, are linked by a short peptide linker between G1 and G4S, thereby contributing to the stable expression of the heavy chain containing the 4-1BB ligand dimer. Furthermore, it was confirmed that the trimer is formed by forming a mortise in the dimer and a club in the monomer.

[0139] Example 4. Confirmation of the impact of mismatch on trimer formation.

[0140] In the 4-1BB ligand bound to the RACE of this invention, the effect of the mortise and tenon structure on the protein hinge was confirmed by LC (Vanquish, Thermo) and MS (Q Exactive Plus, Thermo) analysis of disulfide bonds. To form protein slices, analytical and standard samples at concentrations of 1 mg / ml were used, such as endoproteinases (Endoproteinase Lys-C (Sigma, USA)), chymotrypsin (Promega), and Glu-C (Promega, Sequencing Grade).

[0141] The analytical samples were prepared as follows. In the second vector (antibody heavy chain-monomer) and third vector (antibody light chain) of Examples 1-2-1 and 1-2-2 above, recombinant vectors prepared using rituximab as the antibody and wild-type 4-1BBL as the monomer were simultaneously transfected into Freestyle293F cells using the same method as in 1-2-3 above, and the supernatant of cells overexpressing the relevant protein was obtained. The analytical sample was a protein prepared by using a RACE heavy chain monomer and light chain with a wild-type 4-1BBL sequence, and had a RACE trimer structure with three antibody-binding fragments (Fab) and hinge regions. In this case, the effect of dimerization of the hinge through the mortise and tenon structure on the stability and uniformity of the structure was confirmed by observing the state of abnormal disulfide bonds generated in the protein hinge.

[0142] To confirm the characteristics of proteins polymerized via disulfide bonds, electrophoresis was performed using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) (Mini-protein tetra, Bio-Rad). The size of each protein was compared under reducing and non-reducing conditions. The gels were stained with Coomassie blue, and the results were analyzed using a LAS-500 (Cytiva).

[0143] As shown in Figure 9, in the untreated 4-1BB ligand in its natural state, when the 4-1BB ligand dimer is represented as A, the 4-1BB ligand monomer as B, and the light chain as L, misassembled substances such as B3L3 and B2L2 were observed in cell lines overexpressing the untreated monomers B and L (SDS-PAGE results at the lower end of part A in Figure 9). Particularly in the case of B3L3 with three hinges, unpaired cysteine ​​residues were found near the hinge region where two cysteine ​​residues form disulfide bonds through pairing, or all three hinges were found to form asymmetrically paired disulfide bonds (refer to part B in Figure 9). This phenomenon was particularly pronounced in the experimental group (YK) simultaneously treated with chymotrypsin and Lys-C, but negligible in the experimental group (EK) simultaneously treated with Glu-C and Lys-C. Such heterogeneity is a characteristic that should never exist in therapeutic proteins, clearly confirming the necessity to improve the purity and productivity of the final trimer by modifying the monomer (converting the monomer).

[0144] Example 5. Fusion protein (RACE) prepared by linking antibody domains to cytokine trimers and its characterization.

[0145] 5-1. Size Analysis

[0146] The fusion protein (RACE) platform prepared in Example 1 can bind to a variety of antigen-binding fragments according to the purpose. A schematic diagram of the structure of RACE that binds to the antibody domain is shown in Figure 10.

[0147] Furthermore, Figure 10 illustrates the RACE domain structure by listing them from the N-terminus to the C-terminus. In the fusion protein of this invention, the monomers of the 4-1BB ligands, which are hinged together, have a pestle module, and the dimers of the 4-1BB ligands have a mortar module.

[0148] 5-2. Thermal shift assay (TSA) analysis

[0149] The stability and melting temperature (Tm) of RACE were determined using the standard test method with SYPRO™ Orange (S6650, Thermo) reagent (Curr Protoc Protein Sci. 2015;79: 28.9.1-28.9.14).

[0150] The thermodynamic stability of rituximab-41BBL (RACE-01C) was compared with that of rituximab as its parent antibody, and the thermodynamic stability of trastuzumab-41BBL (RACE-02C) was compared with that of trastuzumab as its parent antibody, as shown in Figure 11. The results confirmed that both rituximab-41BBL (RACE-01C) and trastuzumab-41BBL (RACE-02C) of the present invention have Tm values ​​above 65°C and exhibit characteristics similar to the parent antibody.

[0151] 5-3. Determine binding affinity

[0152] The binding of cetuximab-41BBL (RACE-02B) to the antigen (EGFR) of the present invention was confirmed using Biacore (Cytiva) with surface plasmon resonance (SPR). First, to determine the binding affinity, EGFR (10001-H08H, Sino) or 4-1BB (10041-H08H, Sino) as the antigen was immobilized on the surface of a CM5 chip (29149604, Cytiva) used as a sensor chip via amine coupling. Then, a predetermined concentration of cetuximab-41BBL (RACE-02B) was added. Binding for 180 seconds and dissociation for 300 seconds were confirmed using HEPES-buffered saline (HBS).

[0153] As shown in Figure 12, the cetuximab-41BBL (RACE-02B) of the present invention exhibited similar binding ability to cetuximab as the parent antibody.

[0154] Meanwhile, as shown in Figure 13, the cetuximab-41BBL (RACE-02B) of the present invention can bind to three sites, one for each monomer, showing a stable binding state to the 4-1BB receptor (top of Figure 13). This shows a stronger binding ability than utolumab (Pfizer, which competitively binds to the 4-1BB ligand) which is an agglutinating antibody against the 4-1BB receptor.

[0155] 5-4. Confirming the binding ability of the RACE

[0156] Simultaneously, the simultaneous binding ability of cetuximab-41BBL (RACE-02B) to the target was confirmed. As shown in the schematic diagram on the left side of Figure 14, after immobilizing the 4-1BB receptor on the surface of the CM5 chip, the binding with cetuximab-41BBL (RACE-02B) (AI, DI) was observed, and in this state, the additional binding with EGFR (A-II, D-II) was observed sequentially.

[0157] The results confirmed that the cetuximab-41BBL (RACE-02B) of the present invention can bind to both targets simultaneously, and confirmed that it can successfully induce binding in cells expressing each protein.

[0158] 5-5.4-1BB Reporter Gene Analysis

[0159] The degree of 4-1BB signal transduction by rituximab-41BBL (RACE-01C) of the present invention was assessed using a 4-1BB bioassay (J2332, Promega). Analysis was performed according to the manufacturer's instructions. To confirm signal dependence on the target antigen, the CD20-expressing Raji B cell line from Experiment 1 described above was used. The relative value of the signal was measured using a GloMax® Discover microplate reader (GM30000, Promega) with a GloMax® (Promega) microplate reader.

[0160] As shown in Figure 15, the rituximab-41BBL (RACE-01C) of the present invention was confirmed to amplify the NF-KB reporter gene signal in a concentration-dependent manner (refer to the right side of Figure 15).

[0161] Example 6. Confirmation of RACE signaling-mediated processes in solid cancer cells

[0162] The signal-mediated process of the fusion protein of the present invention was confirmed in solid cancer cells. Therefore, the extent of 4-1BB signal transduction was evaluated by treating the MDA-MB-231 cell line (breast cancer cell line) known to incompletely express Her2, the PC-9 cell line (lung adenocarcinoma cell line) known to overexpress EGFR, the HT-29 cell line (colorectal cancer cell line), and the SW480 cell line (colorectal cancer cell line) described in Experimental Example 1 with trastuzumab-41BBL (RACE-02C), cetuximab-41BBL (RACE-02B), or rituximab-41BBL (RACE-01C) of the present invention using the same method as in Examples 5-5 above.

[0163] Part A of Figure 16 illustrates the on-target and off-target signal transduction in the MDA-MB-231 cell line. In the MDA-MB-231 cell line, despite negligible expression of the target protein HER2, treatment with trastuzumab-41BBL (RACE-02C), which binds to the relevant protein, resulted in a concentration-sensitive increase in signal transduction, reaching levels classified as triple-negative breast cancer. Conversely, treatment with rituximab-41BBL (RACE-01C), which targets the antigen CD20, which is not expressed at all in the relevant cells, did not show an increase in off-target signal transduction. Meanwhile, Part B of Figure 16 shows the differences in EGFR expression levels, on-target signal transduction, and off-target signal transduction among the three tumor cell lines expressing EGFR protein. In section B of Figure 16 above, cell lines labeled "-on" (e.g., (Ex) SW480-on, HT-29-on, PC9-on) indicate the on-target effect, i.e., the result of the degree of signal transduction confirmed after treatment with cetuximab-41BBL (RACE-02b), which can be prepared by using cetuximab that binds to EGFR expressed in each target cell. Conversely, cell lines labeled "-off" (e.g., SW480-off, HT-29-off, PC9-off) indicate the off-target effect of the drug, i.e., the result of the degree of signal generation confirmed after treatment with rituximab-41BBL (RACE-01C), which can bind to CD20 that is not expressed in each target cell.

[0164] As a result, when treating three cell lines overexpressing EGFR with cetuximab-41BBL (RACE-02B), concentration-proportional signal transduction was confirmed. However, when treating rituximab-41BBL (RACE-01C)-target-off, which is associated with CD20 protein not expressed in any of the cell lines, no signal transduction was confirmed. This confirmed the complete absence of off-target toxicity, a problem that had previously plagued 4-1BBL targeted therapy agents.

[0165] Meanwhile, it was confirmed that the fusion protein cetuximab-41BBL (RACE-02B) of the present invention specifically induces 4-1BB signal transduction to a significantly better degree than utolumab, which is a 4-1BB agglutinating antibody (Figure 16B).

[0166] Example 7. Confirmation of efficacy in a dynamic model of cancer induction.

[0167] The anticancer efficacy of cetuximab-41BBL (RACE-02B) was confirmed in an animal model of induced cancer. First, to express human EGFR on the surface of spontaneously growing mouse colon cancer cells (CT26) in Balb-c mice, relevant hEGFR-CT26 cells (5 × 10⁵ / 100 μL) were transplanted subcutaneously into humanized mice (Balb-c) expressing the extracellular domain of human 4-1BB (contracted to Gempharmatech). Starting when the average size of the transplanted tumor reached 100 mm³, the following groups were administered antibodies via tail vein at a rate of Q3D (once every three days) for a total of six doses: control group (5 mg / kg, human IgG (purchased from Sigma (14506))), cetuximab-41BBL (RACE-02B) (0.5 mg / kg, 2 mg / kg). The size of the tumors in each experimental group was measured twice a week using calipers (TV = 0.5a × b²), where a is the long axis of the tumor and b is the short axis of the tumor.

[0168] As shown in Figure 18, cetuximab-41BBL (ARCE-02B) exhibited significant tumor-suppressive activity compared to the human IgG antibody in the control group.

[0169] In summary, this invention relates to a cytokine trimer domain formed by the connection of a first monomer and a second monomer via a linker and the third monomer via a mortise and tenon joint, and to a novel form of fusion protein (RACE) prepared by replacing the constant region (Fc) of an antibody with the aforementioned cytokine trimer domain. The mortise and tenon joint-bound cytokine trimer domain of this invention exhibits a stronger binding ability to the 4-1BB receptor than agglutinating antibodies, and the fusion protein containing it has been confirmed to exhibit excellent binding ability to both targets, and it has been confirmed that it can successfully induce binding in cells expressing each target (protein).

[0170] none

[0171] none

[0172] TW202342551A_112104310_SEQL.xml

Claims

1. A fusion protein comprising: an antibody or antibody fragment; and a cytokine trimer domain, wherein, The cytokine trimer has a club-and-mortar structure formed by at least one amino acid residue of a first monomer or a second monomer and at least one amino acid residue of a third monomer, wherein the cytokine is a tumor necrosis factor superfamily, wherein the cytokine trimer domain comprises a dimer of the first monomer and the second monomer connected through a linker and the third monomer, wherein the N-terminus or C-terminus of the first monomer is interconnected with the N-terminus or C-terminus of the second monomer through the linker, and wherein the antibody fragment comprises at least one selected from the group consisting of Fab, Fab', F(ab')2, scFv, di-scFv and the heavy chain variable region (VHH) of a heavy chain antibody.

2. The fusion protein as described in claim 1, wherein, A mortar structure is formed in the dimer containing the first and second monomers, and a pestle structure is formed in the third monomer.

3. The fusion protein as described in claim 1, wherein, The aforementioned mortar-and-groove structure forms in the region outside the binding region of cytokines and receptors.

4. The fusion protein as described in claim 1, wherein, One or more of the above-mentioned amino acid residues are located on the protein surface.

5. The fusion protein as described in claim 1, wherein, One or more amino acid residues of the first monomer and one or more amino acid residues of the third monomer are located at a distance of less than 6 Å.

6. The fusion protein as described in claim 1, wherein, The amino acid residues of the second monomer and the amino acid residues of the third monomer are located at a distance of less than 6 Å.

7. The fusion protein as described in claim 1, wherein, The tumor necrosis factor superfamily is selected from the group consisting of TNFα (Dif, Necrosin), TNFβ (TNFSF1B), TNFγ, CD252 (Gp34, CD134L), CD154 (TRAP, Gp39, T-BAM), CD178 (APTL, CD95L), CD70, CD153, and 4-1BBL.

8. The fusion protein as described in claim 7, wherein, The tumor necrosis factor superfamily is 4-1BBL.

9. The fusion protein as described in claim 7, wherein, The 4-1BBL contains an amino acid sequence represented by sequence 17 (SEQ ID NO: 17).

10. The fusion protein as described in claim 8, wherein, The aforementioned amino acid residues are selected from one or more of the group consisting of F92, F238, Q94, F144, V234, Q146, E148, F199, Y142, L203, R202, Q200 and A180 of the amino acid sequence shown in sequence 35 (SEQ ID NO: 35), wherein 4-1BBL contains the globular protein domain G90-T241 of 4-1BBL represented by sequence 35 (SEQ ID NO: 35).

11. The fusion protein as claimed in claim 10, wherein, The aforementioned amino acid residues are selected from one or more of the group consisting of F92W, F238V, Q94S, Q94Y, F144I, V234H, V234F, V234R, Q146S, E148G, F199L, Y142T, L203A, R202V, R202W, R202F, F199W, and A180E.

12. The fusion protein as claimed in claim 11, wherein, The aforementioned amino acid residues form a pestle-and-mortar structure by selecting one or more pairs from the group consisting of pairs of A180E and E148G, pairs of V234R and R202V, and pairs of R202W and Q94S.

13. The fusion protein as claimed in claim 1, wherein, The N-terminus or C-terminus of the antibody fragment is linked to a cytokine trimer.

14. The fusion protein as claimed in claim 1, wherein, The antibody fragment described above is F(ab')2 with the following structure: the first hinge of the F(ab')2 is connected to a first monomer or a second monomer; the second hinge of the F(ab')2 is connected to a third monomer.

15. The fusion protein as claimed in claim 1, wherein, The aforementioned cytokines bind to their receptors in a trimer state under natural conditions.

16. The fusion protein as described in claim 8, wherein, The above-mentioned 4-1BBL has the following structure: the first 4-1BBL monomer and the second 4-1BBL monomer are connected by a linker; one or more amino acid residues of the first 4-1BBL monomer or the second 4-1BBL monomer and one or more amino acid residues of the third 4-1BBL monomer form a mortar and pestle structure.

17. A polynucleotide, wherein, The fusion protein as described in any one of the encoding request items 1 to 16.

18. A carrier, wherein, A polynucleotide comprising any one of the encoding requests 1 to 16.

19. A method for producing a fusion protein, wherein, The step includes transfecting a vector containing a polynucleotide encoding any one of claims 1 to 16 into a host cell.

20. A carrier composition for producing the fusion protein of claim 1, wherein, The device comprises: a first vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding the first monomer and the second monomer connected by the linker; a second vector, wherein a polynucleotide encoding the heavy chain of Fab is operatively linked to a polynucleotide encoding the third monomer; and a third vector comprising a polynucleotide encoding the light chain of Fab.

21. A method for producing the fusion protein described in claim 1, wherein, The steps include transfecting the vector composition described in request item 20 into host cells.

22. A method for producing a fusion protein of an antibody and a tumor necrosis factor superfamily trimer, wherein the tumor necrosis factor superfamily trimer forms a trimer comprising a dimer and a third monomer of the tumor necrosis factor superfamily, wherein the dimer comprises a first monomer and a second monomer of the tumor necrosis factor superfamily, wherein... The N-terminus or C-terminus of the first monomer is connected to the N-terminus or C-terminus of the second monomer via a connector, respectively. The method includes: step 1), selecting one of two surfaces present between the dimer and the third monomer; step 2), selecting an amino acid pair in the first monomer or the second monomer and the third monomer at a distance of less than 6 Å between the first monomer or the second monomer and the third monomer in the selected surface; step 3), inducing a mutation at the third monomer site in the selected amino acid pair to form a club; step 4), screening for wild-type amino acid residues of the first monomer or the second monomer that pair with the club of the third monomer and cause structural collision; step 5), inducing a mutation in the amino acid residues of the screened first monomer or the second monomer to form a mortar; and step 6), inducing the club of the third monomer to interact with the mortar of the first monomer or the second monomer, wherein a heavy chain of Fab is attached in the dimer and the third monomer.

23. The production method as described in claim 22, wherein, In step 1) above, a mortise and tenon joint is formed on the selected surface, and a steric hindrance effect is induced between the monomers on the remaining unselected surfaces.

24. A pharmaceutical composition for preventing or treating diseases caused by the inactivation or damage of host immune cells, wherein, The fusion protein comprising any one of claims 1 to 16.

25. The pharmaceutical composition for preventing or treating diseases caused by the inactivation or damage of host immune cells as described in claim 24, wherein, The diseases caused by the inactivation or damage of host immune cells are selected from one or more of the following groups: cancer, immune diseases, autoimmune diseases, central nervous system diseases, degenerative neurological diseases, autoimmune diseases, and inflammatory diseases.

26. Use of a medicament prepared for treating diseases caused by the inactivation or damage of host immune cells, wherein, The drug comprises a fusion protein as claimed in any one of claims 1 to 16, and the disease comprises at least one selected from the group consisting of cancer, immune diseases, autoimmune diseases, central nervous system diseases, degenerative neurological diseases, autoimmune diseases, and inflammatory diseases.

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