Fusion protein containing antigen-binding domain and cytokine trimer domain

Abstract

The present invention relates to a cytokine trimer domain in which a first monomer, a second monomer (dimer) and a third monomer linked by a linker are bound by a knob-into-hole system, and an antibody prepared by replacing the constant region (Fc) of an antibody with the trimer domain, and a new type of fusion protein (Receptor-Antibody Conjugated (cell) Engager (RACE)) in which the cytokine trimer domain is bound. It has been confirmed that the RACE according to the present invention not only exhibits a binding ability to the target receptor superior to that of the parent antibody, but also exhibits excellent simultaneous binding ability to the antigen and the target receptor, and can be usefully used as a bispecific pharmaceutical composition.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising an antigen-binding domain and a cytokine trimer domain, a method for producing the same, and its use. [Background technology]

[0002] The approval of blinatumomab (blincyto, Amgen), a CD19 / CD3 ligator designed using anti-CD19 and anti-CD3 antibodies, has accelerated the development of bispecific antineoplastic agents that induce tumor treatment through immune cell homing.

[0003] Anti-CD3 antibodies are molecules that primarily target the CD3 epsilon (CD3e) molecule present on the surface of T cells. They induce T cell activation and target cells expressing the target antigen bound by the bispecific antibody, thereby inducing apoptosis. In the case of such CD3-targeting bispecific antibodies, T cell activity is rapidly promoted in a short time, accelerating the death of target cells, and this process may also induce rapid cytokine secretion. This is thought to be due to the inherent properties of the CD3 molecule, which resides on the T cell surface and induces the primary activity of cells.

[0004] The activity of immune cells, including T cells, is regulated by the balance between costimulatory and inhibitory factors. Costimulatory factors that assist in the activation of immune cells can be broadly divided into two family proteins: the B7 receptor family, which includes CD28 and ICOS (Inducible Costimulatory Molecule), and the TNF receptor family, which includes 4-1BB and OX40. These costimulatory factor proteins are particularly well-known as inducible molecules expressed in activated T cells, and are thought to mitigate the toxic aspects of the aforementioned CD3 antibodies.

[0005] The 4-1BB ligand (CD137L) is known to be expressed in antigen-presenting cells and to signal to the 4-1BB receptor. The 4-1BB ligand exists in two forms: a membrane protein embedded in the cell membrane and a soluble form with the upper part of the membrane-binding site cleaved off. While the soluble 4-1BB ligand does not contribute to 4-1BB receptor signaling, the membrane protein 4-1BB ligand strongly induces the activation of T cells or NK cells expressing the 4-1BB receptor. This has led to reports that promoting 4-1BB signaling can suppress tumor growth. Therefore, various types of antibody molecules capable of functionally activating 4-1BB have been developed for the purpose of treating tumors.

[0006] Interestingly, in the case of activated antibodies that inhibit the binding of 4-1BB ligand and 4-1BB receptor and assist in the aggregation of 4-1BB receptors (e.g., utomilumab, Pfizer), it was observed that they did not have sufficient activity on their own, but exhibited some activity when bound to the Fc- receptor. On the other hand, in the case of activated antibodies that further bind to the 4-1BB ligand and 4-1BB receptor while they are already bound and assist in the aggregation of the 4-1BB receptor-ligand complex (e.g., urelumab, BMS-663513, BMS), it was observed that they had sufficient activity without the assistance of the Fc- receptor, but exhibited strong toxicity in the body due to the Fc receptor. In fact, urelumab was discontinued from clinical trials due to such hepatotoxicity (NCT00612664).

[0007] In addition, there have been attempts to design molecules with tumor-suppressing efficacy using the 4-1BB ligand itself, but significant results have not been achieved in designing bivalent molecules that maintain the ligand's unique structure with minimal changes and act specifically on tumors. [Overview of the Initiative]

[0008] The inventors of this invention were studying molecular design that could maintain the unique structure of ligands and act specifically on tumors, and in the process developed a cytokine trimer domain. In particular, they derived a method for producing fusion proteins containing an antigen-binding domain and a cytokine trimer domain linked to a knob-into-hole structure, and confirmed the binding ability of these fusion proteins to various targets, thus completing the present invention.

[0009] Therefore, the purpose of this disclosure is to provide a fusion protein.

[0010] Another objective of this disclosure is to provide polynucleotides that encode fusion proteins.

[0011] Another objective of this disclosure is to provide a vector containing a polynucleotide encoding a fusion protein.

[0012] Another objective of this disclosure is to provide a vector containing a polynucleotide encoding a fusion protein.

[0013] Another objective of this disclosure is to provide a method for producing fusion proteins.

[0014] Another objective of this disclosure is to provide a method for producing antibodies and tumor necrosis factor superfamily trimer fusion proteins.

[0015] Another purpose of this disclosure is to provide pharmaceutical compositions for the prevention or treatment of diseases caused by functional inactivation or impairment of host immune cells.

[0016] Another purpose of this disclosure is to provide a method for treating diseases caused by functional inactivation or impairment of host immune cells.

[0017] To achieve the above object, the present invention provides a fusion protein comprising an antigen-binding domain; and a cytokine trimer domain.

[0018] To achieve another object as described above, the present invention provides a polynucleotide encoding the fusion protein according to the present invention.

[0019] To achieve another object as described above, the present invention provides a vector comprising a polynucleotide encoding the fusion protein according to the present invention.

[0020] Furthermore, the present invention provides a first vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to polynucleotides encoding a first monomer and a second monomer linked by a linker; a second vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to a polynucleotide encoding a third monomer; and a third vector comprising a polynucleotide encoding the light chain of Fab; and provides a vector composition for producing a fusion protein.

[0021] To achieve another object as described above, the present invention provides a method for producing a fusion protein, comprising the step of infecting a host cell with a vector comprising a polynucleotide encoding the fusion protein according to the present invention.

[0022] Furthermore, the present invention provides a method for producing a fusion protein, comprising the step of infecting a host cell with the vector composition for producing a fusion protein according to claim 1, the vector composition comprising a first vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to polynucleotides encoding a first monomer and a second monomer linked by a linker; a second vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to a polynucleotide encoding a third monomer; and a third vector comprising a polynucleotide encoding the light chain of Fab.

[0023] To achieve yet another object as described above, the present invention provides a method for producing an antibody and a tumor necrosis factor superfamily trimeric fusion protein, which comprises: a dimer comprising the first and second monomers of the tumor necrosis factor superfamily (TNFSF); and a trimer that forms a trimer comprising the third monomer of the tumor necrosis factor superfamily (TNFSF), wherein: 1) a step of selecting one of two interfaces existing between the dimer and the third monomer; 2) a step of selecting an amino acid pair located at a distance of 6 Å or less between the first or second monomer located at the selected one interface and the third monomer, one from each of the first or second monomer; and the third monomer; 3) a step of inducing a mutation at the site of the third monomer of the selected amino acid pair to form a knob; 4) a step of selecting an amino acid residue of the wild type (WT) first or second monomer that pairs with the knob of the third monomer and causes steric hindrance; 5) a step of inducing a mutation at the selected amino acid residue of the first or second monomer to form a hole; and 6) a step of inducing a knob-into-hole interaction between the knob of the third monomer and the hole of the first or second monomer; wherein the Fab heavy chain is linked to the dimer and the third monomer.

[0024] To achieve yet another object as described above, the present invention provides a pharmaceutical composition for improving the function of immune cells for preventing or treating a disease caused by functional inactivation or impairment of host immune cells, which comprises the fusion protein according to the present invention.

[0025] To achieve another object as described above, the present invention provides a method for treating a disease caused by functional inactivation or impairment of host immune cells, which comprises the step of administering the fusion protein according to the present invention to an individual.

[0026] The present invention relates to a novel form of fusion protein, RACE (Receptor-Antibody Conjugated (cell)Engager), in which an antibody and a cytokine trimer domain are bound. This is prepared by substituting the constant region (Fc) of an antibody with the cytokine trimer domain, which is linked by a linker and comprises a first monomer, a second monomer (dimer), and a third monomer (monomer), preferably linked by a linker. The method for preparing RACE according to the present invention enhances the purity and productivity of the trimer. The RACE prepared thereby not only exhibits superior binding ability to the target receptor compared to the parent antibody, but also shows excellent co-binding ability to the antigen and the target receptor. This allows for its useful application as a bispecific pharmaceutical composition. [Brief explanation of the drawing]

[0027] [Figure 1] This figure shows a fusion protein according to the present invention, specifically a unit protein (A) contained therein and a fusion protein (B) containing the unit protein. [Figure 2] This is a diagram illustrating the knob-into-hole structure in the cytokine trimer domain according to the present invention. [Figure 3] This shows the interaction site and its distance (A) between the dimer and monomer in the cytokine trimer domain according to the present invention, and the respective interactions (B). [Figure 4] This figure compares the cases in the cytokine trimer domain according to the present invention where a knob is formed in only one direction or where a knob and a hole are formed simultaneously. [Figure 5] This is the result of confirming the purity of fusion proteins containing three different antibody domains and cytokine trimer domains. [Figure 6]This document presents the results of confirming the ability to form a knob-into-hole structure (A), the production volume (B), and the purity (C) of a fusion protein containing an antibody domain and a cytokine trimer domain according to the present invention. [Figure 7] This is the result of confirming the structure of the cytokine trimer domain according to the present invention and confirming that the dimer and monomer formed a knob-into-hole structure. [Figure 8] This figure shows the structure of the cytokine trimer domain and the linker that connects the dimer according to the present invention. [Figure 9] This report presents the results of identifying proteins with mismatched disulfide bonds in the protein hinge when a knob-into-hole structure is not formed in the cytokine trimer domain according to the present invention (A), and confirming the type and incidence of the mismatched hinge (B). [Figure 10] This result shows the structure and size of the fusion protein containing the antibody domain and cytokine trimer domain according to the present invention. [Figure 11] This is the result of confirming the thermodynamic stability of the fusion protein containing the antibody domain and cytokine trimer according to the present invention. [Figure 12] This is the result of confirming the binding affinity of the fusion protein containing the antibody domain and cytokine trimer according to the present invention to the antigen. [Figure 13] This is the result of confirming the binding affinity of the fusion protein containing the antibody domain and cytokine trimer according to the present invention to the target receptor. [Figure 14] This is the result of confirming the simultaneous binding ability of the fusion protein containing the antibody domain and cytokine trimer according to the present invention to the antigen and target receptor. [Figure 15] This is the result of evaluating the degree of 4-1BB signaling by a fusion protein containing an antibody domain and a cytokine trimer according to the present invention. [Figure 16]This report evaluates the degree of on-target and off-target signaling of fusion proteins containing an antibody domain and a cytokine trimer according to the present invention, and presents the results for RACE-02C(A) and RACE-02B(B). [Figure 17] This is a schematic diagram of a vector containing a polynucleotide encoding a fusion protein comprising an antibody domain and a cytokine trimer according to the present invention. [Figure 18] This is a result of confirming the therapeutic effect of the fusion protein containing the antibody domain and cytokine trimer according to the present invention in an animal model in which cancer was induced. [Modes for carrying out the invention]

[0028] The present invention will be described in detail below.

[0029] The present invention provides a fusion protein comprising an antigen-binding domain and a cytokine trimer domain.

[0030] The cytokine trimer domain is characterized by the binding of a dimer containing a first monomer and a second monomer to a third monomer, wherein the dimer is characterized by the linkage between the first monomer and the second monomer. In the dimer, the N-terminus or C-terminus of the first monomer is linked to the N-terminus or C-terminus of the second monomer by an interlinker.

[0031] The linker can be any linker known in the art that is capable of linking the first monomer and the second monomer of a cytokine. In one embodiment of the present invention, the linkers are connected by short peptide linkers between G1 and G4S. G1 contains sequence G, and G4S contains sequence GGGGS (SEQ ID NO: 34).

[0032] In particular, in the cytokine trimer domain of the present invention, the cytokine can spontaneously form a trimer. However, when producing a fusion protein by binding an antibody to the spontaneously formed trimer structure, mismatched disulfide bonds occur at the hinge portion connecting the Fab3 portion of the antibody to each trimer, resulting in a decrease in purity and productivity. Therefore, in order to increase productivity with higher purity, it is preferable that one or more amino acid residues of the first or second monomer constituting the dimer and one or more amino acid residues of the third monomer form a knob-into-hole structure. It is preferable that the knob-into-hole structure is formed in a region other than the binding region between the cytokine and the receptor.

[0033] Furthermore, the one or more amino acid residues are characterized by being located at the protein interface. In particular, the first monomer and the second monomer are linked in tandem via a linker, but depending on the interface position of the knob generated by the third monomer, a hole may be formed in the first monomer or in the second monomer.

[0034] Due to the knob-into-hole structure described above, a selective interaction is induced between the third monomer with the knob formed on either the first or second monomer constituting the hole-formed dimer, and steric hinderance can be induced at the interface between the remaining first or second monomer (where no hole is formed) and the third monomer, thereby inducing interaction at a desired interface and preventing the mismatch that would occur when using a trimer in its natural state. That is, for example, in this invention, an interaction may be induced at the interface where the hole in the first monomer and the knob-formed third monomer are located, inducing steric hinderance between the second monomer and the third monomer; or an interaction may be induced at the interface where the hole in the second monomer and the knob-formed third monomer are located, inducing steric hinderance between the first monomer and the third monomer.

[0035] On the other hand, in protein-protein interactions, three important interactions (a) hydrogen bonds (2.4–3.2 Å), hydrophobic interactions (3.2–3.9 Å), and charge interactions (~4 Å) usually occur within a distance of 4 Å or less.

[0036] 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 one or more amino acid residues of the third monomer can 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 can be located at a distance of 6 Å or less. Preferably, one or more amino acid residues of the first monomer and one or more amino acid residues of the third monomer can 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 can be located at a distance of 4 Å or less. More preferably, one or more amino acid residues of the first monomer and one or more amino acid residues of the third monomer can be located at a distance of 2 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 can be located at a distance of 2 to 4 Å.

[0037] Therefore, the amino acid residue can be selected from among amino acid residues located at a distance of 6 Å or less, preferably 4 Å or less, and more preferably 2 to 4 Å, between the dimer and monomer. For example, if the cytokine consists of a 4-1BBL monomer and a dimer, it may be one or more selected from the group consisting of F92, F238, Q94, F144, V234, Q146, E148, F199, Y142, L203, R202, Q200, and A180.

[0038] The mutation site of the aforementioned amino acid was determined based on the amino acid sequence of TNFSF9 (TNF superfamily member 9) from NCBI (accession number: NM_003811.3).

[0039] More preferably, amino acid mutations are induced in each monomer, thereby forming a knob-into-hole structure. Therefore, the amino acid residue may 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.

[0040] More preferably, the amino acid residues can form a knob-into-hole structure when paired together. Even more preferably, the amino acid residues are characterized by R202W and Q94S pairing together to form a knob-into-hole structure.

[0041] In cytokines such as 4-1BBL, trimers can be produced in which a dimer (E148G, where the first and second monomers are linked by a linker) forms a hole and a monomer (A180E, where the third monomer forms a knob) forms a knob-into-hole. In addition, trimers can be produced in which a dimer (R202V, where the first and second monomers are linked by a linker) forms a hole and a monomer (V234R, where the third monomer forms a knob) forms a knob-into-hole. Furthermore, trimers can be produced in which a dimer (Q94S, where the first and second monomers are linked by a linker) forms a hole and a monomer (R202W, where the third monomer forms a knob) forms a knob-into-hole.

[0042] According to one embodiment of the present invention, in cytokines such as 4-1BBL, a trimer was produced in which the dimer (a first monomer and a second monomer linked by a linker) Q94S (hole formation) and the monomer (a third monomer) R202W (knob formation) form a knob-into-hole structure.

[0043] Furthermore, as shown in Figure 2, two interfaces are formed between the dimer and monomer that have formed the knob-into-hole structure as described above (interface A, interface B (knob-into-hole (KiH))). Therefore, the present invention is for achieving selective trimerization with minimal change, and the interface opposite to interface B (knob-into-hole (KiH)) can maintain its natural form. Thus, the trimer according to the present invention is characterized by the three monomers maintaining a parallel trimer structure.

[0044] In the fusion protein of the present invention, the antigen-binding domain is an antibody or a fragment of an antibody.

[0045] In the fusion protein of the present invention, any antibody or antibody fragment that can bind to a desired antigen can be used without limitation. 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 was prepared.

[0046] The antibody fragment may include a light chain variable region and a heavy chain variable region, and preferably 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).

[0047] In particular, the antibody fragment is linked at its N-terminus or C-terminus to a cytokine trimer to form a fusion protein. More preferably, the antibody fragment may be F(ab')2 having the following structure: the first hinge of F(ab')2 is linked to a first or second monomer linked by a linker; the second hinge of F(ab')2' is linked to a third monomer.

[0048] In the fusion protein of the present invention, the cytokine is characterized in that it binds to the receptor as a trimer in its natural state.

[0049] Therefore, the cytokine may preferably be a tumor necrosis factor superfamily (TNFSF) capable of trimer formation in its natural state, and may preferably 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, and more preferably 4-1BBL.

[0050] According to one embodiment of the present invention, in the fusion protein of the present invention, when the extracellularly exposed globular protein domain of the entire sequence of 4-1BB (Accession number: NM_003811.3) is used (G90-T241), the 4-1BBL is characterized in that it contains the nucleotide sequence represented by SEQ ID NO: 17.

[0051] GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT (Sequence ID 17).

[0052] More preferably, the trimer 4-1BBL contained in the fusion protein may have the following structure: the first 4-1BBL monomer, at least one amino acid residue of the second 4-1BBL monomer and one or more amino acid residues of the third 4-1BBL monomer form a knob-into-hole structure.

[0053] Figure 1 shows the structure of a 4-1BBL-based fusion protein produced according to one embodiment of the present invention. The fusion protein of the present invention is composed of unit proteins as shown in Figure 1A and has the structure shown in Figure 1B. More specifically, it consists of three types: 1) an antibody heavy chain containing a 4-1BB ligand dimer, 2) an antibody heavy chain containing a 4-1BB ligand monomer, and 3) an antibody light chain. In Figure 1B, the antigen-binding fragment at the upper end is linked to the 4-1BB ligand trimer at the lower end via a hinge. The complex is formed by hydrophobic interactions between the two disulfide bonds present in the hinge and the 4-1BB ligand trimer. In particular, the hydrophobic interaction between the ligand trimers is selectively performed between the 4-1BB ligand dimer and the 4-1BB ligand monomer via a knob-into-hole module, which significantly improves the physical properties and purity of the protein. The 4-1BBL monomer always has a structure containing a knob, which is essential to prevent homotrimerization of the heavy chain containing the monomer. The 4-1BB ligand dimer is linked by a short amino acid linker between two monomers and has a "hole" at a site that interacts with the "knob" of the opposite monomer.

[0054] In a specific example of the cytokine trimer domain of the present invention based on the above, a monomer containing the amino acid sequence represented by SEQ ID NO: 1 and a dimer containing the amino acid sequence represented by SEQ ID NO: 2 may be included. In the dimer, two monomers may be linked by a linker at the 170th amino acid sequence (G). Alternatively, a monomer containing the nucleotide sequence represented by SEQ ID NO: 3 and a dimer containing the nucleotide sequence represented by SEQ ID NO: 4 may be included. In the dimer, two monomers may be linked by a linker at the 508-510th nucleotide sequence GGA.

[0055] In other specific examples of the cytokine trimer domain of the present invention, a monomer containing the amino acid sequence represented by SEQ ID NO: 5 (with a mutation from R to W at the 202nd amino acid sequence) and a dimer containing the amino acid sequence represented by SEQ ID NO: 6 (with a mutation from Q to S at the 94th amino acid sequence) may be included. In the dimer, the two monomers may be linked by a linker at the 170th amino acid sequence (G). Furthermore, the monomer and dimer can form a knob-into-hole structure at the amino acid mutation site. Alternatively, a monomer containing the nucleotide sequence represented by SEQ ID NO: 7 (with a mutation from AGA to TGG at the 202nd nucleotide sequence) and a dimer containing the nucleotide sequence represented by SEQ ID NO: 8 (with a mutation from CAG to AGC at the 94th nucleotide sequence) may be included. In the above dimer, the two monomers may be linked by a linker at GGA in the 508th to 510th nucleotide sequences.

[0056] In yet another embodiment of the cytokine trimer domain of the present invention, it may include a monomer containing the amino acid sequence represented by SEQ ID NO: 9 (mutated to A-E at amino acid position 180) and a dimer containing the amino acid sequence represented by SEQ ID NO: 10 (mutated to E-G at amino acid position 148). In the dimer, the two monomers may be linked by a linker at amino acid position 170 (G). Furthermore, the monomer and dimer can form a knob-into-hole structure at the amino acid mutation site. Alternatively, it may include a monomer containing the nucleotide sequence represented by SEQ ID NO: 11 (mutated to GCT-GAA at nucleotide position 180) and a dimer containing the nucleotide sequence represented by SEQ ID NO: 12 (mutated to CAA-GGA at nucleotide position 148). In the above dimer, the two monomers may be linked by a linker at GGA in nucleotide positions 508-510.

[0057] In yet another specific example of the cytokine trimer domain of the present invention, it may include a monomer containing the amino acid sequence represented by SEQ ID NO: 13 (mutated from V to R at the 234th amino acid sequence) and a dimer containing the amino acid sequence represented by SEQ ID NO: 14 (mutated from R to V at the 202nd amino acid sequence). In the dimer, the two monomers may be linked by a linker at the 170th amino acid sequence (G). Furthermore, the monomer and dimer can form a knob-into-hole structure at the amino acid mutation site. Alternatively, it may include a monomer containing the nucleotide sequence represented by SEQ ID NO: 15 (mutated from GTG to AGA at the 234th nucleotide sequence) and a dimer containing the nucleotide sequence represented by SEQ ID NO: 16 (AGA mutated to GTG at the 202nd nucleotide sequence). In the above dimer, the two monomers may be linked by a linker at the GGA of the nucleotide sequences 508 to 510.

[0058] Therefore, the fusion protein of the present invention may preferably include any cytokine trimer domain selected from the group consisting of: a cytokine trimer comprising a monomer containing the amino acid sequence represented by SEQ ID NO: 1 and a dimer containing the amino acid sequence represented by SEQ ID NO: 2; a cytokine trimer domain comprising a monomer containing the amino acid sequence represented by SEQ ID NO: 5 and a dimer containing the amino acid sequence represented by SEQ ID NO: 6; a cytokine trimer domain comprising a monomer containing the amino acid sequence represented by SEQ ID NO: 9 and a dimer containing the amino acid sequence represented by SEQ ID NO: 10; and a cytokine trimer domain comprising a monomer containing the amino acid sequence represented by SEQ ID NO: 13 and a dimer containing the amino acid sequence represented by SEQ ID NO: 14.

[0059] It will be apparent to those skilled in the art that the amino acid sequences represented by SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, and 14 are limited to mutations in amino acid sequences that exhibit equivalent biological activity. Such amino acid mutations are made based on the relative similarities of the amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side-chain substituents 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 said to be biologically functional equivalents.

[0060] When introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); lyucine (+3.8); phenylalanine (+2.8); cysteine / cystaine (+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); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0061] Hydrophobic amino acid indicators are extremely important in conferring the co-biological functions of proteins or peptides. It is a known fact that similar biological activity can be maintained as long as substitution is not performed with amino acids that have similar hydrophobic indicators. When introducing mutations by referring to hydrophobic indicators, it is preferable to substitute between amino acids that show a difference in hydrophobic indicators of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0062] On the other hand, it is well known that substitutions between amino acids with similar hydrophilicity values ​​can result in proteins or peptides with uniform biological activity. The following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+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).

[0063] When introducing mutations by referring to hydrophilicity values, substitutions are preferably made between amino acids that exhibit a difference in hydrophilicity values ​​of ±2, more preferably within ±1, and even more preferably within ±0.5.

[0064] Amino acid substitutions in peptides that do not alter the overall molecular activity are known in this art. The most common substitutions are exchanges 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.

[0065] Considering the above-mentioned mutations having bioequivalent activity, the amino acid sequences represented by SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, and 14 of the present invention are interpreted to include sequences that exhibit substantial identity with sequences listed in sequence listings. Substantial identity means sequences that exhibit at least 80% homology, more preferably 90% homology, when the amino acid sequences represented by SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, and 14 of the present invention are aligned to the maximum extent possible with any other sequence, and the aligned sequences are analyzed using algorithms commonly used in the industry. Any method known in the industry may be used without limitation for the alignment method for sequence comparison.

[0066] Furthermore, the nucleotide sequences represented by SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, and 16 may include nucleotide sequences having 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more sequence homology with the nucleotide sequences represented by SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, and 16. The "percentage of sequence homology" for a nucleotide sequence is identified by comparing two optimally sequenced sequences with a comparison region, and a portion of the nucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) for the optimal sequences of the two sequences.

[0067] As described above, in the cytokine trimer domain of the fusion protein according to the present invention, the knob-into-hole structure is characterized by being generated by amino acid mutations that occur in the monomer and dimer, respectively. This is to prevent the generation of mismatched disulfides due to sequence misassembly in the hinge portion that links the antibody (Fab) in the trimer assembly of the tumor necrosis factor superfamily (TNFSF), which exists as a trimer in its natural state.

[0068] Furthermore, the present invention provides a polynucleotide encoding the fusion protein according to the present invention.

[0069] Furthermore, the present invention provides a vector comprising a polynucleotide encoding a fusion protein according to the present invention.

[0070] A schematic diagram of the above vector is shown in Figure 17. In Figure 17, the vector is characterized by containing a light chain, a heavy chain dimer, and / or a heavy chain monomer in its gene region.

[0071] The present invention also provides a vector composition for fusion protein production comprising: a first vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to polynucleotides encoding a first monomer and a second monomer linked by a linker; a second vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to a polynucleotide encoding a third monomer; and a third vector comprising a polynucleotide encoding the light chain of Fab.

[0072] When using the above-mentioned vector composition for fusion protein production, the dimer and Fab heavy chain can be produced in the case of a trimer, and the monomer and Fab heavy chain can be produced in the case of a trimer, after which trimer formation can be induced.

[0073] In the present invention, the term "vector" means a gene construct comprising a gene sequence functionally linked to a suitable regulatory sequence for expressing a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, and sequences for regulating the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it is replicable within a cell, and any vector known in the art may be used, for example, a plasmid, cosmid, phage particle, or viral vector.

[0074] Furthermore, the present invention provides a method for producing a fusion protein, which includes infecting a host cell with a vector containing a polynucleotide encoding the fusion protein according to the present invention.

[0075] The present invention also provides a method for producing a fusion protein, comprising the step of infecting a host cell with a vector composition for fusion protein production according to claim 1, which includes: a first vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to polynucleotides encoding a first monomer and a second monomer linked by a linker; a second vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to a polynucleotide encoding a third monomer; and a third vector containing a polynucleotide encoding the light chain of Fab.

[0076] In the present invention, the "recombinant vector" can be used as an expression vector for a target polypeptide that can express the target polypeptide with high efficiency in a suitable host cell when the coding gene of the target polypeptide to be expressed is operably ligated to it, and the recombinant vector may be expressible in a host cell. The host cell is preferably a eukaryotic cell, and depending on the type of host cell, expression regulatory sequences such as promoters, terminators, and enhancers, sequences for membrane targeting or secretion, etc., can be appropriately selected and combined in various ways according to the purpose.

[0077] According to one embodiment of the present invention, a fusion protein containing an antigen-binding domain and a cytokine trimer domain exhibits superior antigen-binding ability compared to the parent antibody, superior binding ability to cytokine target receptors compared to the parent antibody, and the ability to bind simultaneously. This confirms that it can effectively induce binding to cells expressing each target (protein).

[0078] Furthermore, the present invention relates to a tumor necrosis factor superfamily that forms a dimer containing the first and second monomers of the tumor necrosis factor superfamily (TNFSF) and a trimer containing the third monomer of the tumor necrosis factor superfamily (TNFSF), comprising the steps of: 1) selecting one of two interfaces existing between the dimer and the third monomer; 2) selecting one amino acid pair each from the first or second monomer and the third monomer located at a distance of 6 Å or less between the first or second monomer and the third monomer at the selected interface; 3) inducing a mutation in the third monomer site of the selected amino acid pair to form a knob; and 4) the aforementioned The present invention provides a method for producing an antibody and tumor necrosis factor superfamily trimer fusion protein, comprising the steps of: 5) selecting a wild-type amino acid residue of the first or second monomer to pair with a knob of the third monomer and cause a structural collision; 6) inducing a mutation in the selected amino acid residue of the first or second monomer to form a hole; and 7) inducing a knob-into-hole interaction between the knob of the third monomer and the hole of the first or second monomer, wherein the dimer and the third monomer are linked to a Fab heavy chain.

[0079] In the method for producing the antibody and tumor necrosis factor superfamily trimer fusion protein, in step 2), the amino acid pair can be located at a distance of 6 Å or less, preferably 4 Å or less, and more preferably 2 to 4 Å, between the first or second monomer located at the interface and the third monomer.

[0080] In the above method for producing the antibody and tumor necrosis factor superfamily trimer fusion protein, the amino acid residue causing steric hindrance of the knob can be changed and inferred through simulation to any amino acid residue selected from the candidate group consisting of glycine (G), valine (V), serine (S), and alanine (A).

[0081] Furthermore, a knob-into-hole bond is formed at the selected interface in step 1) above, and steric hindrance is induced between monomers at the remaining unselected interfaces. This steric hindrance allows for the induction of interactions with desired interfaces. Therefore, when producing fusion proteins using native trimers, it is possible to prevent the formation of mismatched disulfides at the hinge portion that links the antibody (Fab), which may be induced.

[0082] According to one embodiment of the present invention, in the case of a fusion protein formed by binding a cytokine and an antibody to which a knob-into-hole structure is not applied, it was confirmed that unpaired cysteine ​​residues were found near the hinge region where cysteine ​​residues pair to form a disulfide bond, or that disulfide bonds were found where all three hinges were asymmetrically paired.

[0083] Therefore, the production method according to the present invention is an optimal method that can prevent heterogeneity caused by abnormal disulfide bonds in the hinge portion of the produced antibody and tumor necrosis factor superfamily trimer fusion protein, thereby increasing the productivity and purity of the fusion protein and providing the fusion protein as a more stable therapeutic protein.

[0084] The dimer is characterized by being a first monomer and a second monomer linked by a linker. In the dimer, the N-terminus or C-terminus of the first monomer is linked to the N-terminus or C-terminus of the second monomer by a mutual linker. The linker can be any linker known in the art that is capable of linking the first monomer and the second monomer of a cytokine, and can be used without limitation.

[0085] Furthermore, any antibody fragment (Fab) capable of binding to the desired antigen can be used without restriction.

[0086] Furthermore, the present invention provides an antibody and a tumor necrosis factor superfamily trimer fusion protein produced by the above-described manufacturing method.

[0087] The same explanation for the fusion protein is applicable to the antibody and tumor necrosis factor superfamily trimer fusion protein produced by the above production method, as described above.

[0088] Furthermore, the present invention provides a pharmaceutical composition for functionally improving immune cells, comprising the fusion protein according to the present invention, for preventing or treating diseases caused by functional inactivation or impairment of host immune cells.

[0089] Furthermore, the present invention provides a method for treating diseases caused by functional inactivation or impairment of host immune cells, comprising the step of administering the fusion protein according to the present invention to an individual.

[0090] The aforementioned individuals are preferably mammals, including humans, and include all patients who require treatment for a disease caused by functional inactivation or impairment of host immune cells, including patients currently being treated for a disease caused by functional inactivation or impairment of host immune cells, patients who have previously received treatment for a disease caused by functional inactivation or impairment of host immune cells, and patients who require treatment for a disease caused by functional inactivation or impairment of host immune cells, and may also include patients who have undergone surgery to receive treatment for a disease caused by functional inactivation or impairment of host immune cells.

[0091] Furthermore, the fusion protein of the present invention can be used in combination with existing drugs or therapeutic methods for treating diseases caused by functional inactivation or impairment of host immune cells. When the fusion protein of the present invention is used in combination, it can be used simultaneously with or sequentially with other drugs or therapeutic methods for treating diseases caused by functional inactivation or impairment of host immune cells.

[0092] Diseases resulting from functional inactivation or impairment of host immune cells may be one or more selected from the group consisting of cancer, immune diseases, autoimmune diseases, central nervous system diseases, degenerative neurological diseases, autoimmune diseases, and inflammatory diseases, and the cancer may be one or more selected from the group consisting of breast cancer, lung cancer, colorectal cancer, and colorectal cancer.

[0093] In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human use. The pharmaceutical composition is not limited to these, but each can be formulated and used by conventional methods in the form of an oral preparation such as a powder, granule, capsule, tablet, aqueous suspension, topical preparation, suppository, or sterile injection solution.

[0094] The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc.; for injectable preparations, a mixture of buffers, preservatives, pain relievers, solubilizers, isotonic agents, stabilizers, etc.; and for topical administration, a base, excipients, lubricants, preservatives, etc. can be used. Formulations of the pharmaceutical composition according to the present invention can be prepared in various forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, they can be manufactured in the form of tablets, trotskyis, capsules, elixirs, suspensions, syrups, wafers, etc.; and for injectable preparations, they can be manufactured in the form of single-dose ampoules or multi-dose ampoules.

[0095] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. Furthermore, fillers, anti-agglutinating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be used.

[0096] The routes of administration of the pharmaceutical compositions according to the present invention are not limited to, but include, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. The “parenteral” routes include injection or infusion techniques such as subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intrafocal, and intracranial. The pharmaceutical compositions according to the present invention may also be administered in the form of suppositories for rectal administration.

[0097] The pharmaceutical composition according to the present invention can vary in many ways depending on several factors, including the activity of the specific active ingredient used, age, weight, general health, sex, diet, administration time, route of administration, excretion rate, drug formulation, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical composition will vary depending on the patient's condition, weight, disease severity, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art. Preferably, taking all of the above factors into consideration, an amount can be administered that provides the maximum effect with the minimum amount without side effects, more preferably 1 to 10,000 μg / kg body weight / day, and even more preferably 10 to 1,000 mg / kg body weight / day, which can be administered repeatedly several times a day at an effective dose. The above dosage does not limit the scope of the present invention in any way.

[0098] The contents of the present invention described above are applicable equally to each other, insofar as they do not contradict each other, and their implementation by ordinary articulators in the art with appropriate modifications is also within the scope of the present invention. [Examples]

[0099] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to the following examples.

[0100] Experimental Example 1. Preparation of Cell Lines Freestyle293F cell line (Gibco TM (R79007) Freestyle293F expression culture medium (Gibco TM The cells were cultured in suspension at 120 RPM under conditions of 37°C and 8% CO2 using the Raji-B cell line (CCL-86). TM , ATCC), MDA-MB-231 cell line (HTB-26 TM ,ATCC), SW-480 cell line (10228, KCLB) is RPMI1640 (Welgene) with 10% (v / v) FBS (Fetal bovine serum, Gibco). TM Using a culture medium to which ) was added, PC-9 cell line (CVCL_B260) and HT-29 cell line (HTB-38 TMATCC was cultured at 37°C under 5% CO2 conditions using a culture medium of DMEM (Welgene) supplemented with 10% (v / v) FBS.

[0101] Example 1. Preparation of a fusion protein by linking a cytokine trimer domain and an antibody domain. 1-1. Gene preparation To construct the fusion protein according to the present invention, the following three coding sequences were designed: 1) the light chain of the antibody, 2) a sequence in which a 4-1BB ligand monomer is linked to the C-terminus of the antigen-binding site of the antibody heavy chain, and 3) a sequence in which a 4-1BB ligand dimer is linked to the C-terminus of the antigen-binding site of the antibody heavy chain.

[0102] In this regard, human 4-1BB ligand protein was used, and the corresponding gene (NM_003811.3) was synthesized using Geneart. In this invention, the extracellularly exposed globular protein domain of the entire sequence of the 4-1BB ligand protein was used (G90-T241). Gene cloning and mutation generation were performed using gateway cloning.

[0103] 1-2. Construction of a vector for producing fusion proteins To prepare a fusion protein in which the Fc region of an antibody is replaced with a cytokine trimer (hereinafter referred to as RACE (Receptor-Antibody Conjugated (cell) Engager)), we constructed a first vector for the Fab heavy chain and cytokine dimer, a second vector for the Fab heavy chain and cytokine monomer, and a third vector for the light chain.

[0104] In this study, the amino acid residues for mutation formation in the trimer domain of the cytokine 4-1BB ligand were selected through simulations using Pymol software and the Dynamut server (http: / / biosig.unimelb.edu.au / dynamut / ).

[0105] A trimer domain was constructed using a partial sequence of the 4-1BB ligand (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 knob-into-hole structure was used to ensure trimer formation. To achieve this, a knob-into-hole structure was formed at only one of the two interfaces between the dimer and monomer using 1) an antibody heavy chain containing a 4-1BB ligand dimer and 2) an antibody heavy chain containing a 4-1BB ligand monomer, thereby achieving selective trimerization with minimal change. The knob was formed on the 4-1BB ligand monomer, and the hole was formed on one of the 4-1BB ligand dimers. Figure 1 shows each unit of the trimer and its binding morphology, and Figure 2 shows the knob-into-holes that can be formed at the interface of each unit.

[0106] The structural analysis of the trimer domain formed as described above was performed using Pymol software (https: / / pymol.org / 2 / ) based on the human 4-1BBL protein complex structure (PDB code: 6A3V, 2X29).

[0107] The interaction sites between the 4-1BB ligand dimer and monomer, which were identified by this process, are shown in Figure 3.

[0108] As shown in Figure 3, amino acids located at a distance of less than 4 Å at the interaction interface were identified (Figure 3A). The interactions between each amino acid in Figure 3A are shown in Figure 3B with dotted lines (hydrogen bonds) or solid lines (hydrophobic interactions).

[0109] Mutations were induced in the amino acid residues derived as described above to form a knob-into-hole structure between the dimer and monomer. Schematic diagrams showing the presence or absence of knob-into-hole formation in the wild type and each mutation were predicted via mutagenesis and are shown in Figure 4.

[0110] The 4-1BB ligand described above exists naturally as a trimer. During the assembly of this trimer, misassembly occurs in the sequence of the hinge portion that links the antibody (Fab), resulting in the generation of a mismatched disulfide. To solve this problem, the present invention uses a knob-into-hole structure to produce the trimer.

[0111] As shown in Figure 4, if no mutation is introduced into the amino acid residues of the monomer, a knob-into-hole structure is not formed (left side of Figure 4). If only one of the monomer's amino acid residues is mutated, the knob is applied only to one side of the interface where the mutation occurred, and the interaction with the native interface is subject to steric hindering (center of Figure 4). Therefore, as in the present invention, when all the amino acids of two monomers are mutated, the knob and hole can be applied to each to form a stable complementary structure (see right side of Figure 4).

[0112] Considering the above, we fabricated a first vector for heavy chain-cytokine (dimer) with inserted antibody and wild-type cytokine sequences, a second vector for heavy chain-cytokine (monomer), and a third vector for antibody light chain. Subsequently, we induced mutations in the dimer and monomer to induce the formation of a knob-into-hole structure.

[0113] More specifically, the knob-into-hole structure was formed by creating a Q94S (hole formation) dimer (first monomer and second monomer linked by a linker) and an R202W (knob formation) monomer (third monomer) knob-into-hole structure.

[0114] First, templates for cloning (antibody heavy chain, antibody light chain, 4-1BBL (WT) dimer, and monomer) were synthesized by Geneart (Thermofisher). At this time, the GGA (Glycine) sequence, which corresponds to the linker, was inserted into the dimer at base positions 508-510 during synthesis. Four types of antibodies were used: rituximab (Drugbank accession number (DB00073)), cetuximab (Drugbank accession number (DB00002)), trastuzumab (Drugbank accession number (DB00072)), or avelumab (Drugbank accession number (DB11945)). Subsequently, each antibody was cloned using gateway cloning based on the pCMV vector. More specifically, the pCMV vector underwent MCS (multiple cloning). A first vector was prepared by inserting an antibody heavy chain-cytokine dimer, in which a 4-1BBL-linker-4-1BBL (dimer) was linked to the end of the antibody heavy chain (VH-CH1-hinge) into the site. For this purpose, the first fragment for the antibody heavy chain, the second fragment for the first monomer of the 4-1BBL dimer, and the third fragment for the second monomer of the 4-1BBL dimer were amplified using the primers shown in Table 1 below, and then gateway cloning was performed.

[0115] [Table 1]

[0116] Furthermore, a second vector was prepared by inserting an antibody heavy chain-cytokine monomer, in which 4-1BBL was ligated to the terminal end of the antibody heavy chain (VH-CH1-hinge), into the MCS of the pCMV vector. For this purpose, the first fragment for the antibody heavy chain and the second fragment for the 4-1BBL monomer were amplified using the primers shown in Table 2 below, and then gateway cloning was performed.

[0117] [Table 2]

[0118] Furthermore, a third vector was constructed by inserting an antibody light chain into the MCS of the pCMV vector. At this time, the fragment was amplified using the primers shown in Table 3 below, followed by gateway cloning.

[0119] [Table 3]

[0120] Subsequently, gateway cloning was performed to apply mutations to the dimer of the first vector and the monomer of the second vector. 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.

[0121] [Table 4]

[0122] The presence or absence of insertions in each gene and the verification of their sequences were confirmed by gene sequencing (commissioned to Cosmogenetech).

[0123] 1-3. Transfection and Production of RACE Using the First, Second, and Third Vectors For RACE transfection and expression, Freestyle293F cells prepared in Experimental Example 1 above were used. 24 hours before transfection, the Freestyle293F cell line was divided into 1 × 10⁶ cells. 6It was prepared at a concentration of / ml. The cells were transfected using Fectopro (Polyplus, 116 - 010) reagent and the first vector, the second vector, and the third vector prepared above. More specifically, it was performed by adding 1 μg of DNA per 1 ml of cell line (the first vector, the second vector, and the third vector were mixed at a volume ratio of 1:1:2) and 1 μl of Fectopro reagent. The temporarily transfected cells were cultured in suspension for an additional 5 days (37 °C, 8% CO 2、 120 RPM), centrifuged at 1500 g for 30 minutes to separate the supernatant containing the secreted protein. The separated supernatant was stored refrigerated for up to 3 days for subsequent purification.

[0124] Example 2. Purification and purity analysis of RACE RACE was purified from the supernatant obtained from Examples 1 - 3 above using AKTA go (Cytiva) and affinity chromatography. Affinity chromatography used a CH1 - xL column (Thermo). The washing solution (PBS, pH 7.4) and elution solution (100 mM sodium acetate pH 4.0) used for chromatography were prepared immediately before use, filtered, and used after pH measurement. Among the eluted fractions, the fractions containing protein were collected together and dialyzed using a PBS solution with an Amicon (R) Ultra (30 kda, 15 ml) filter and stored.

[0125] For purity analysis of the purified RACE, size exclusion chromatography (PL1580 - 3250, Agilent) was performed using high - performance liquid chromatography (HPLC, Agilent 1260). The approximate protein size prediction by size exclusion chromatography was determined by tests using standard proteins (5190 - 2242, Agilent) with known sizes. A buffer solution of 150 mM sodium phosphate pH 7.0 was used for purity analysis.

[0126] As a result, as shown in Figure 5, the purity of avelumab-41BBL (RACE-04L) according to the present invention was 97.24% (see PD-L1 targeting (4AL) graph in Figure 5), the purity of cetuximab-41BBL (RACE-02B) was 96.47% (see EGFR targeting (Cetuximab) graph in Figure 5), and the purity of trastuzumab-41BBL (RACE-02C) was 96.91% (see HER2 targeting (Herceptin) graph in Figure 5). All RACE products manufactured according to the present invention showed excellent productivity and purity.

[0127] Furthermore, in the case of rituximab-41BBL (RACE-01C) according to the present invention, when the knob-into-hole (Q94S / R202W) was applied, it was confirmed that the 4-1BB ligand dimer (hole) and the 4-1BB ligand monomer (knob) successfully formed a 4-1BB ligand trimer, similar to Figure 6A. It was confirmed that this trimer consisted of a heavy chain dimer containing the hole (Q94S), a heavy chain monomer containing the knob (R202W), and a light chain. In addition, the purification and purity analysis of rituximab-41BBL (RACE-01C) according to the present invention using the same method as above showed a yield comparable to that of a normal antibody containing an Fc dimer (Figure 6B). After primary purification based on CH1, it was confirmed that the protein was separated into high-purity proteins of 96% or more (Figure 6C).

[0128] Example 3. Structural analysis of RACE The structure of the 4-1BBL trimer domain was analyzed using Pymol software (https: / / pymol.org / 2 / ) based on the human 4-1BBL protein complex structure (PDB code: 6A3V, 2X29).

[0129] As a result, as shown in Figures 7 and 8, the C-terminus of 4-1BBLa constituting the dimer is 4-1BBL bWe confirmed that the N-terminus of the molecule is linked to a short peptide linker between G1-G4S, contributing to the stable expression of the heavy chain containing the 4-1BB ligand dimer. We also confirmed that a hole is formed in the dimer and a knob is formed in the monomer, forming a trimer.

[0130] Example 4. Confirmation of the effect of mismatch on trimer formation. The effect of applying a knob-into-hole structure to the protein hinge of the RACE-bound 4-1BB ligand according to the present invention was confirmed by disulfide bond analysis via LC (Vanquish, Thermo) and MS (Q Exactive Plus, Thermo). To form protein fragments, analytical and standard samples at concentrations of 1 mg / ml, such as endoproteinase (Endoproteinase Lys-C (Sigma, USA), chymotrypsin (Promega)), and Glu-C (Promega, Sequencing Grade), were used.

[0131] The analytical sample was prepared as follows. In the second vector (antibody heavy chain-monomer) and the third vector (antibody light chain) of Example 1-2, rituximab was used as the antibody and wild-type 4-1BBL was used as the monomer. The prepared recombinant vector was simultaneously transfected into Frestyle293F cells in the same manner as in Example 1-2-3, and the supernatant of cells overexpressing the protein was obtained. The analytical sample has a RACE trimer structure with a total of three antibody-binding fragments (Fabs) and a hinge region, produced via a RACE heavy chain monomer and light chain having a wild-type 4-1BBL sequence. At this time, the abnormal disulfide bond formation in the protein hinge was observed, and the effect of hinge dimerization due to the knob-into-hole structure on structural stability and homogeneity was confirmed.

[0132] Furthermore, to confirm the characteristics of proteins that polymerize via disulfide bonds, electrophoresis (Mini-protein tetra, Bio-Rad) was performed using SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel). The size of each protein was compared under reducing and non-reducing conditions. The gels after electrophoresis were stained with Coomassie-blue reagent, and the results were analyzed using LAS-500 (cytiva).

[0133] As shown in Figure 9, in the native 4-1BB ligand where the knob-into-hole technique was not applied, if the 4-1BB ligand dimer is denoted as A, the 4-1BB ligand monomer as B, and the light chain as L, then in cell lines overexpressing native monomers B and L without the knob application, substances such as misassembled B3L3 and B2L2 were observed (Figure 9A, middle and lower panel, SDS-PAGE results). In particular, in the case of B3L3, which has three hinges, unpaired cysteine ​​residues were found near the hinge region where two cysteine ​​residues pair to form a disulfide bond, or disulfide bonds with all three hinges asymmetrically paired were found (see Figure 9B). This phenomenon was particularly pronounced in the experimental group treated with chymotrypsin and Lys-C simultaneously (YK), and was also observed to a small extent in the experimental group treated with Glu-C and Lys-C simultaneously (EK). Such heterogeneity is an unacceptable characteristic for therapeutic proteins, and we clearly confirmed that monomer modification (knob addition) is absolutely necessary to improve the purity and productivity of the final trimer.

[0134] Example 5. Fusion protein (RACE) prepared by linking an antibody domain and a cytokine trimer, and its characterization. 5-1. Size Analysis The fusion protein (RACE) platform prepared in Example 1 can be used to bind various types of antigen-binding fragments as needed, and a schematic diagram of the RACE structure that binds to the antibody domain is shown in Figure 10.

[0135] Furthermore, the domain structure of RACE is enumerated from the N-terminus to the C-terminus and shown in Figure 10 above. In the fusion protein according to the present invention, the monomer of the hinge-linked 4-1BB ligand has a knob module, and the dimer of the 4-1BB ligand has a hole module.

[0136] 5-2. Thermal Shift Assay Analysis (TSA) The stability and melting temperature (Tm) values ​​of RACE were measured by SYPRO TM The test was performed using Orange (S6650, Thermo) reagents, following the standard test procedure.

[0137] The thermodynamic stability of rituximab-41BBL(RACE-01C) and its parent antibody, rituximab, was compared, and the thermodynamic stability of trastuzumab-41BBL(RACE-02C) and its parent antibody, trastuzumab, is shown in Figure 11. As a result, it was confirmed that both rituximab-41BBL(RACE-01C) and trastuzumab-41BBL(RACE-02C) according to the present invention have a Tm value of 65°C or higher, mimicking the characteristics of the parent antibody.

[0138] 5-3. Binding affinity measurement The binding of cetuximab-41BBL (RACE-02B) according to the present invention to the antigen (EGFR) was confirmed using Biacore (cytiva) with surface plasmon resonance (SPPR). First, to measure the binding strength, the antigen EGFR (10001-H08H,Sino) or 4-1BB (10041-H08H,Sino) was immobilized on the surface of the sensor chip CM5 chip (29149604,cytiva) via amine coupling, and then cetuximab-41BBL (RACE-02B) at a predetermined concentration was added. Binding for 180 seconds and dissociation for 300 seconds were confirmed using HBS (HEPES-buffered saline) buffer.

[0139] As a result, as shown in Figure 12, it was confirmed that cetuximab-41BBL (RACE-02B) according to the present invention exhibits similar binding ability to the parent antibody cetuximab.

[0140] Furthermore, as shown in Figure 13, cetuximab-41BBL (RACE-02B) according to the present invention has a total of three binding sites, one between each monomer, and exhibits a stable binding mode to the 4-1BB receptor (top of Figure 13). This shows a much stronger binding ability compared to utomirumab (Pfizer, (competitive binding of 4-1BB ligand)), which is an agglutinating antibody against the 4-1BB receptor.

[0141] 5-4. Confirmation of simultaneous binding ability of RACE Furthermore, the co-binding ability of cetuximab-41BBL (RACE-02B) to its target according to the present invention was confirmed. As shown in the schematic diagram on the left of Figure 14, after immobilizing the 4-1BB receptor on the surface of the CM5 chip, binding with cetuximab-41BBL (RACE-02B) was observed (AI, DI), and then binding to additional EGFR was sequentially observed (A-II, D-II).

[0142] As a result, it was confirmed that cetuximab-41BBL (RACE-02B) according to the present invention can simultaneously bind to both targets, thus confirming that it can successfully induce binding to cells expressing each protein.

[0143] 5-5.4-1 BB Reporter Assay The degree of 4-1BB signaling by rituximab-41BBL (RACE-01C) according to the present invention was evaluated using the 4-1BB bioassay (J2332, Promega). The assay was performed according to the manufacturer's instructions, and the CD20-expressing Raji B cell line from Experimental Example 1 was used to confirm the signal dependence to the target antigen. The relative signal values ​​were expressed using GloMax. (R) Using (Promega), GloMax (R) Measurements were taken using a Discover Microplate Reader (GM MF100, promega).

[0144] As a result, as shown in Figure 15, it was confirmed that rituximab-41BBL (RACE-01C) according to the present invention amplifies the NF-KB reporter signal in a concentration-dependent manner (see the right side of Figure 15).

[0145] Example 6. Confirmation of RACE signaling pathways in solid tumor cells. The signal-mediated pathway of the fusion protein according to the present invention in solid tumor cells was confirmed. To this end, as described in Experimental Example 1 above, trastuzumab-41BBL (RACE-02C), cetuximab-41BBL (RACE-02B), or rituximab-41BBL (RACE-01C) according to the present invention were treated with trastuzumab-41BBL (RACE-02C), cetuximab-41BBL (RACE-02B), or rituximab-41BBL (RACE-01C) according to the present invention in the same manner as in Example 5-5 above, and the degree of 4-1BB signaling was evaluated for each.

[0146] Figure 16A shows the degree of on-target and off-target signaling in the MDA-MB-231 cell line. In the case of the MDA-MB-231 cell line, despite expressing the target protein HER2 to such an extent that it is classified as triple-negative breast cancer, the degree of signaling was observed to increase sensitively depending on the concentration of trastuzumab-41BBL (RACE-02C) that can bind to HER2. In contrast, when treated with rituximab-41BBL (RACE-01C), which targets CD20, an antigen not expressed at all in these cells, no concentration-dependent increase in off-target signaling was observed. Figure 16B also shows the degree of EGFR expression and the difference in the degree of on-target and off-target signaling in three tumor cell lines in which the EGFR protein is expressed on the surface. In Figure 16B above, the entries labeled "cell line-on" (Ex. SW480-on, HT-29-on, PC9-on) represent the on-target effect, i.e., the results of checking the degree of signal transduction after treatment with cetuximab-41BBL (RACE-02B), which was produced using cetuximab that can bind to EGFR expressed in each target cell. On the other hand, the entries labeled "-off" for the same cell line (Ex. SW480-off, HT-29-off, PC9-off) represent the off-target effect of the drug, i.e., the results of checking the degree of signal generated after treatment with rituximab-41BBL (RACE-01C), which can bind to CD20, which is not expressed at all in each target cell.

[0147] As a result, when three types of cells overexpressing EGFR were treated with cetuximab-41BBL(RACE-02B), signal transduction proportional to the concentration was observed. Furthermore, when rituximab-41BBL(RACE-01C)-target-off was applied to the CD20 protein, which is not expressed at all in each cell line, no signal transduction was observed at all. This confirmed that there was absolutely no off-target toxicity, which has traditionally been a problem with 4-1BBL targeted therapies.

[0148] Furthermore, it was confirmed that the fusion protein cetuximab-41BBL (RACE-02B) according to the present invention induces 4-1BB signaling specifically to the target antigen and to a far greater degree than utomirumab, a 4-1BB agglutinating antibody (Figure 16B).

[0149] Example 7. Efficacy confirmation in an animal model in which cancer was induced. The anticancer efficacy of cetuximab-41BBL (RACE-02B) was confirmed in an animal model in which cancer was induced. First, human EGFR was expressed on the surface of spontaneously growing mouse colorectal cancer cells (CT26) in Balb-c mice, and these hEGFR-CT26 cells (5 × 10⁻¹⁴) were used. 5 The extracellular domain of human 4-1BB was subcutaneously transplanted (100 μL) into humanized mice (Balb-c) expressing the extracellular domain of human 4-1BB (contracted to Gempharmatech). The average size of the transplanted tumors was 100 mm. 3 Based on the point at which the following antibody levels were reached, the following antibody groups were administered intravenously via Q3D (once every 3 days) for a total of 6 doses: control group (5 mg / kg, purchased from human IgG (Sigma) (14506)), cetuximab-41BBL (RACE-02B): 0.5 mg / kg, 2 mg / kg. Tumor size (TV = 0.5a × b) in each experimental group. 2 The tumor was measured twice a week using a caliper. In the above, a is the long axis of the tumor and b is the short axis of the tumor.

[0150] As a result, as shown in Figure 18, cetuximab-41BBL (ARCE-02B) showed significantly greater tumor-suppressing activity compared to the control group, which was human IgG antibody.

[0151] Overall, the present invention relates to a novel form of fusion protein (RACE) in which a first monomer and a second monomer are linked by a linker to form a dimer, and a third monomer is bound to a cytokine trimer domain by a knob-into-hole mechanism, and the constant region (Fc) of the antibody is replaced with the cytokine trimer domain, and the cytokine trimer domain is bound to the antibody. The knob-into-hole bound cytokine trimer domain according to the present invention exhibits a much stronger binding ability to agglutinating antibodies against the 4-1BB receptor, and it has been confirmed that the fusion protein containing this domain exhibits excellent binding ability to both targets, thus confirming that it can successfully induce binding to cells expressing each target (protein).

Claims

1. antigen-binding domain; and A fusion protein containing a 4-1BBL trimer domain, The 4-1BBL trimer domain consists of a dimer containing a first monomer and a second monomer, and a third monomer, and each 4-1BBL trimer domain has two interfaces between one monomer of the dimer and the third monomer. The 4-1BBL trimer domain has a knob-into-hole structure formed by one or more mutant amino acid residues of the first or second monomer and one or more mutant amino acid residues of the third monomer, with one of the two interfaces selected. At least one amino acid residue selected from the group consisting of F92, F238, Q94, F144, V234, Q146, E148, F199, Y142, L203, R202, Q200, and A180 in the amino acid sequence represented by Sequence ID No. 35 is mutated to form a knob-into-hole structure. The 4-1BBL comprises the globular protein domain G90-T241 of the 4-1BBL, as represented by Sequence ID No. 35, and The aforementioned knob-into-hole structure is characterized by protein interactions between mutant amino acid residues located at the selected interface, and steric hindrance between monomers at the remaining unselected interfaces. Fusion protein.

2. The fusion protein according to claim 1, wherein the dimer is a first monomer and a second monomer linked by a linker, and the N-terminus or C-terminus of the first monomer is interconnected with the N-terminus or C-terminus of the second monomer by their respective linkers.

3. The fusion protein according to claim 1, characterized in that a hole structure is formed in the dimer comprising the first monomer and the second monomer, and a knob structure is formed in the third monomer.

4. The fusion protein according to claim 1, wherein the knob-into-hole structure is formed in a region other than the cytokine-receptor binding region.

5. The fusion protein according to claim 1, characterized in that 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 6 Å or less.

6. The fusion protein according to claim 1, characterized in that one or more amino acid residues of the second monomer and one or more amino acid residues of the third monomer are located at a distance of 6 Å or less.

7. The fusion protein according to claim 1, wherein the 4-1BBL comprises the amino acid sequence represented by Sequence ID No.

17.

8. The fusion protein according to claim 1, characterized in that the mutant amino acid residue is 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.

9. The fusion protein according to claim 8, characterized in that the mutant amino acid residues form a knob-into-hole structure with one or more pairs selected from the group consisting of a pair of A180E and E148G, a pair of V234R and R202V, and a pair of R202W and Q94S.

10. The fusion protein according to claim 1, wherein the antigen-binding domain is an antibody or a fragment of an antibody.

11. The fusion protein according to claim 10, wherein the antibody fragment includes a light chain variable region and a heavy chain variable region.

12. The fusion protein according to claim 10, wherein the antibody fragment is 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).

13. The fusion protein according to claim 10, characterized in that the antibody fragment has its N-terminus or C-terminus linked to a cytokine trimer.

14. The fusion protein according to claim 10, characterized in that the antibody fragment is F(ab')2 having the following structure: The first hinge of F(ab')2 is connected to the first monomer or the second monomer; The second hinge of F(ab')2 is a structure connected to the third monomer.

15. A polynucleotide encoding the fusion protein according to any one of claims 1 to 14.

16. A vector comprising a polynucleotide encoding a fusion protein according to any one of claims 1 to 14.

17. A method for producing a fusion protein, comprising the step of infecting a host cell with a vector containing a polynucleotide encoding the fusion protein according to any one of claims 1 to 14.

18. A first vector in which a polynucleotide encoding the heavy chain of Fab is operably linked to polynucleotides encoding the first monomer and the second monomer, linked by a linker; A second vector in which a polynucleotide encoding the heavy chain of Fab and a polynucleotide encoding a third monomer are operably linked; and A third vector containing a polynucleotide encoding Fab's light chain; A vector composition for producing the fusion protein according to claim 1, comprising:

19. A method for producing a fusion protein according to claim 1, comprising the step of infecting a host cell with the vector composition according to claim 18.

20. In a 4-1BBL forming a trimer comprising a dimer containing first and second monomers; and a third monomer, 1) A step of selecting one of the two interfaces existing between the dimer and the third monomer; 2) A step of selecting one amino acid pair each from the first or second monomer and the third monomer, which are located at a distance of 6 Å or less between the first or second monomer and the third monomer at the selected interface; 3) A step of inducing a mutation in the third monomeric site of the selected amino acid pair to form a knob; 4) Select a wild-type first or second monomer amino acid residue that pairs with the knob of the third monomer and causes a structural collision; and 5) A step of inducing a mutation in the amino acid residue of the selected first or second monomer to form a hole; and 6) A step of inducing a knob-into-hole interaction between the knob of the third monomer and a hole of the first or second monomer; The dimer and the third monomer are linked to a heavy chain of Fab. A method for producing antibodies and 4-1BBL, The position of the selected amino acid pair in step 2) is at least one selected from the group consisting of F92, F238, Q94, F144, V234, Q146, E148, F199, Y142, L203, R202, Q200, and A180 of the amino acid sequence represented by Sequence ID No.

35. A method for producing the aforementioned knob-into-hole structure, characterized by protein interactions between mutant amino acid residues located at the selected interface and steric hindrance between monomers at the remaining unselected interface.

21. A pharmaceutical composition for functional improvement of immune cells to prevent or treat diseases caused by functional inactivation or impairment of host immune cells, comprising the fusion protein described in any one of claims 1 to 14.

22. The pharmaceutical composition according to claim 21, characterized in that the disease resulting from the functional inactivation or impairment of host immune cells is one or more 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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