Novel TCTP-binding molecules

Abstract

The present invention relates to a novel binding molecule or a fragment thereof that specifically binds to extracellular TCTP (translationally controlled tumor protein). The present invention also relates to a binding molecule or a fragment thereof according to the present invention, and uses thereof. The binding molecule and a fragment thereof according to the present invention can be used to diagnose, treat, or prevent cancer. The binding molecule and a fragment thereof according to the present invention can be used to alleviate or prevent immunosuppression in the tumor immune microenvironment. The binding molecule and a fragment thereof according to the present invention can be used to diagnose, treat, or prevent cancer in patients who have failed to respond or have only responded to treatment using immunoanticancer drugs, or who are likely to do so.

Description

[Technical Field]

[0001] The present invention relates to novel binding molecules or fragments thereof that specifically bind to extracellular TCTP (translationally controlled tumor protein). The binding molecules and fragments thereof according to the present invention can be used to diagnose, treat, or prevent cancer. The binding molecules and fragments thereof according to the present invention can be used to alleviate or prevent immunosuppression in the tumor immune microenvironment. The binding molecules and fragments thereof according to the present invention can be used to diagnose, treat, or prevent cancer in patients who have failed or have only responded to treatment using immunoanticancer agents, or who are likely to do so. [Background technology]

[0002] The tumor immune microenvironment (TIM) promotes or suppresses tumor cell growth through various interactions between tumor cells and the various surrounding cells. The TIM contains various types of cells, including fibroblasts, endothelial cells, mast cells, and immune cells. Immune cells present in the TIM include T lymphocytes, natural killer (NK) cells, tumor-associated macrophages (TAMs), dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and neutrophils. The TIM contains mechanisms that suppress the immune response to tumor cells, which is known to be a major factor limiting the efficacy of immunotherapy.

[0003] TCTP is a highly conserved protein in eukaryotes. TCTP is located in both the cytoplasm and nucleus, is expressed in various tissues, and is regulated in response to a diverse range of extracellular stimuli. TCTP has also been found as a dimer and is known to interact with other proteins, including MCL1 and p53 (e.g., Acunzo et al. (2014) Cancer Treatment Reviews 40.6:760-769). It has been reported that TCTP dimers can be formed between intact TCTP or its N-terminal truncated forms (Kim et al. (2009): PloS one 4.7:e6464). Although therapeutic strategies have been proposed to suppress intracellular TCTP expression using nucleic acid molecules targeting TCTP-containing mRNA, such as antisense oligonucleotides, siRNA, and shRNA (e.g., Patent Document 1), the extracellular function of TCTP remains unclear. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2012 / 080509 Summary of the Invention [Problem to be solved by the invention]

[0005] Based on the discovery that TCTP is released from tumor cells and acts as an immunoregulatory factor that regulates the function and balance (aspects such as cell number and lifespan) of immune cells such as T cells, NK cells, and / or MDSCs in the tumor immune microenvironment, the present inventors aim to provide a novel binding molecule that specifically binds to TCTP. The novel binding molecule according to the present invention specifically binds to extracellular TCTP and inhibits its extracellular function, thereby suppressing / inhibiting the function of inhibitory immune cells in the tumor immune microenvironment or activating the function of anti-cancer immune cells. [Means for solving the problem]

[0006] The present invention provides novel binding molecules or fragments thereof that specifically bind to extracellular TCTP.

[0007] The present invention achieves a surface plasmon resonance measurement of approximately 1×10 -7 M or less, or 5 x 10 -8 The present invention provides a binding molecule or a fragment thereof that binds to human TCTP with a dissociation constant (Kd) of M or less. The binding molecule or a fragment thereof can bind to TCTP, its truncated form, or a multimer thereof and inhibit its function outside the cell, thereby suppressing / inhibiting the function of inhibitory immune cells or activating anti-cancer immune cells.

[0008] The present invention provides a binding molecule or fragment thereof that specifically binds to TCTP or its post-translational variant, truncated form, or multimer, comprising one or more CDR sequences selected from heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as defined in (a) to (f) below. (a) the heavy chain CDR1 is (a1) an amino acid sequence of X1-X2-X3-X4-H, where X1 is S, G, or V, X2 is Y, H, or N, X3 is A or Y, and X4 is M or V; (a2) an amino acid sequence of S-X5-X6-X7-X8-W-X9 (wherein X5 is S or N, X6 is N or S, X7 is A or W, X8 is A or absent, and X9 is N or S); (a3) the amino acid sequence GYTFTGYYMH, or (a4) the amino acid sequence of NARMGVS, or Containing conservative amino acid substitutions thereof; (b) the heavy chain CDR2 is (b1) an amino acid sequence of EI-X1-H-X2-G-X3-TTYNPSLKS, where X1 is D or Y, X2 is S or absent, and X3 is T or V; (b2) an amino acid sequence of WIN-X4-X5-X6-X7-X8-T-X9-Y-X10-QKFQ-X11 (wherein X4 is A or P, X5 is G, I, or N, X6 is K, N, S, or T, X7 is G or S, X8 is G, N, Y, D, or K, X9 is K, N, or E, X10 is A or S, and X11 is D or G); (b3) the amino acid sequence of RT-X12-Y-X13-SKWYNDYA-X14-SVKS, where X12 is F or Y, X13 is K or R, and X14 is E or V; or (b4) the amino acid sequence of HIFSNDEKSYSYSLKS, or Containing conservative amino acid substitutions thereof; (c) the heavy chain CDR3 is (c1) an amino acid sequence of G-X1-X2-X3-X4-FD-X5, where X1 is R or G, X2 is A or S, X3 is A or V, X4 is A or V, and X5 is P or Y; (c2) the amino acid sequence of DGSGSYEGY; (c3) the amino acid sequence of WVFDY; (c4) the amino acid sequence of X6-A-X7-RLR-X8-A-X9-DI, where X6 is L or P, X7 is P or W, X8 is G or M, and X9 is F or Y; (c5) the amino acid sequence of RLTIVRGVMRGGMDV; (c6) the amino acid sequence of GYYDILTGYYTTDAFDI; (c7) an amino acid sequence of X10-X11-X12-X13-X14-X15-X16-X17-X18-FDY (wherein X10 is V or absent, X11 is R, L or absent, X12 is G or L, X13 is Y, I, T, or W, X14 is F or T, X15 is D or G, X16 is W, T, Y, or E, X17 is W, T, Y, or L, and X18 is A, P, or Y), (c8) the amino acid sequence GGVLLYFGELSRFDY, or (c8) the amino acid sequence EGITVGRGVMLYFDL, or Containing conservative amino acid substitutions thereof; (d) the light chain CDR1 is (d1) an amino acid sequence of RASQ-X1-X2-X3-X4-X5-L-X6 (wherein X1 is A, D, or G, X2 is I or V, X3 is S, R, or G, X4 is N or S, X5 is H, Y, D, N, or S, and X6 is A, G, or H); (d2) an amino acid sequence of X7-G-X8-X9-S-X10-X11-G-X12-X13-X14-X15-VS (wherein X7 is S or T, X8 is S or T, X9 is N or S, X10 is D or N, X11 is I or V, X12 is G, N, or T, X13 is F, K, or Y, X14 is N, D, or absent, and X15 is K or Y); (d3) an amino acid sequence of KSSQ-X16-X17-LY-X18-X19-NNK-X20-Y-X21-A (wherein X16 is N or S, X17 is I, L, or V, X18 is N or S, X19 is A or S, X20 is D or N, and X21 is I or L), or (d4) the amino acid sequence of RSSQSLV-X22-X23-DGNT-X24-L-X25 (wherein X22 is H or Y, X23 is R or S, X24 is Y or H, and X25 is S, N, or K); or Containing conservative amino acid substitutions thereof, (e) the light chain CDR2 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is A, D, E, G, K, W, or Y, X2 is A, D, I, or V, X3 is N, S, or T, X4 is I, K, N, Q, S, or T, X5 is L, R, S, or W, X6 is A, D, F, G, K, P, Q, or Y, and X7 is F, S, or T, or a conservative amino acid substitution thereof; (f) the light chain CDR3 is (f1) an amino acid sequence of X1-Q-X2-X3-X4-X5-P-X6-T (wherein Xi is M or Q, X2 is A, G, or Y, X3 is F, H, N, T, or Y, X4 is G, H, Q, N, S, or Y, X5 is F, I, L, T, Y, or W, and X6 is H, I, L, P, R, W, or Y); (f2) an amino acid sequence of X7-X8-X9-X10-X11-X12-X13-X14-X15-V (wherein X7 is A, N, or V, X8 is S or T, X9 is W or Y, X10 is A or D, X11 is G or S, X12 is D, N, or S, X13 is L or N, X14 is N or R, and X15 is A or L); or (f3) an amino acid sequence of X16-Q-X17-X18-S-X19-P-X20-T (wherein X16 is L or H, X17 is H or S, X18 is N or S, X19 is Y or L, and X20 is Y, R, or Q); or Conservative amino acid substitutions thereof are included. [Brief explanation of the drawings]

[0009] [Figure 1a] FIG. 1 shows the results of an ELISA test for assaying the binding of anti-TCTP antibodies to human TCTP. [Figure 1b] FIG. 1 shows the results of an ELISA test for assaying the binding of anti-TCTP antibodies to human TCTP. [Figure 2] FIG. 1 shows the results of assaying the binding ability of anti-TCTP Ab10 antibody mutants to mouse TCTP and human TCTP by ELISA. [Figure 3a] 1 shows the results of an ELISA test showing that anti-TCTP antibodies inhibit TCTP-TLR2 binding. [Figure 3b] 1 shows the results of a HEK Blue™ hTLR2 reporter cell assay demonstrating that anti-TCTP antibodies suppress TLR2 signaling. [Figure 4a] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 4b] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 4c] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 4d] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 4e] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 4f] Representative plots showing FACS analysis of single cell suspensions obtained from HT29 tumors implanted subcutaneously in T cell-free BALB / c nude mice. [Figure 5] FIG. 1 shows the results of measuring the PMN-MDSC levels by FACS analysis when HT29 tumors subcutaneously transplanted into T cell-free BALB / c nude mice were treated with anti-TCTP antibody. [Figure 6a] FIG. 1 shows the change in tumor volume over time when SL4 cells into which human TCTP had been knocked in were treated with anti-TCTP antibody alone. [Figure 6b]FIG. 1 shows the change in tumor volume over time when cancer cells Pan02 were treated with anti-TCTP antibody or anti-PD-1 antibody. [Figure 6c] FIG. 1 shows changes in tumor volume over time when Hepa1-6 cancer cells were treated with anti-TCTP antibody or anti-PD-1 antibody. [Figure 6d] FIG. 1 shows the change in tumor volume over time when B16BL6 cancer cells were treated with anti-TCTP antibody or anti-PD-1 antibody. [Figure 6e] FIG. 1 shows changes in tumor volume over time when MC38 cancer cells were treated with anti-TCTP antibody or anti-PD-1 antibody. [Figure 7] FIG. 1 shows the change in tumor volume over time when B16F10 cells were treated with an anti-PD-1 antibody and an anti-TCTP antibody, either alone or in combination. [Figure 8] FIG. 1 shows the change in tumor volume over time when SL4 cells were treated with anti-CTLA-4 antibody and anti-TCTP antibody, either alone or in combination.

[0010] [Best Mode for Carrying Out the Invention] The present invention relates to a binding molecule or a fragment thereof that specifically binds to extracellular TCTP and inhibits its function outside the cell, thereby inhibiting / suppressing the function of inhibitory immune cells in the tumor immune microenvironment or activating the function of anti-cancer immune cells.

[0011] The "extracellular" preferably refers to the tumor immune microenvironment. According to the prior research of the present invention, it has been confirmed that TCTP is released from tumor cells, particularly from tumor cells that are damaged or dead under stress conditions. Therefore, the "extracellular" may mean that TCTP is released from such tumor cells, but the tumor cells are not necessarily damaged or dead cells.

[0012] Tumors are known to employ a variety of strategies to slow immune responses that could hinder their own growth, including activating inhibitory immune cells in the tumor immune microenvironment, suppressing anti-cancer immune cells, or upregulating immunosuppressive cytokines / chemokines. Examples of inhibitory immune cells include tumor-associated macrophages (TAMs), myeloid-derived suppressor immune cells (MDSCs), tumor-associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), and regulatory T cells (Tregs). Examples of anti-cancer immune cells in the tumor immune microenvironment include, but are not limited to, T cells and NK cells. Examples of immunosuppressive cytokines and chemokines secreted from the tumor immune microenvironment include CCL2, CCL3, CCL5, CCL17, CCL19, CCL21, CCL22, CCL28, CXCL5, CXCL8, CXCL12, CXCL15, CXCL17, MIF, HMGB1, CSF, CSF2, IL-4, IL-6, IL-10, IL-13, G-CSF, PGE2, and TGF-β. As used herein, alleviating or suppressing immunosuppression in the tumor immune microenvironment means reducing, suppressing, or inhibiting / inhibiting the activity of suppressive immune cells in the tumor immune microenvironment, increasing, enhancing, or activating / activating the activity of anti-cancer immune cells, or down-regulating the activity of immunosuppressive cytokines or chemokines.

[0013] The "extracellular function" of TCTP can be, for example, binding to an immunomodulatory receptor (e.g., TLR2) present on suppressive immune cells (e.g., myeloid-derived immunosuppressive cells) or inducing the secretion of cytokines (e.g., CXCL1 and CXCL2) from suppressive immune cells (e.g., myeloid-derived immunosuppressive cells). By inhibiting such extracellular function of TCTP, the function of suppressive immune cells can be inhibited. Inhibition of the function of suppressive immune cells means that the immunosuppressive activity of the suppressive immune cells is directly or indirectly reduced, suppressed, or inhibited. Such reduction, suppression, or inhibition of immunosuppressive activity can mean that the accumulation or recruitment of suppressive immune cells in the tumor immune microenvironment is reduced, suppressed, or inhibited. The immunosuppressive activity of suppressive immune cells is reduced, suppressed, or inhibited, which can be manifested, for example, by the induction of death of suppressive immune cells, a decrease in the survival rate of suppressive immune cells, a decrease in the secretion of anti-cancer immunosuppressive cytokines by suppressive immune cells, a decrease in the secretion of T cell inhibitors by suppressive immune cells, a decrease in differentiation into suppressive immune cells, or the suppression of immune checkpoint expression by suppressive immune cells.

[0014] As described above, the effect of suppressing / inhibiting the function of inhibitory immune cells in the tumor immune microenvironment or activating the function of anti-cancer immune cells by specifically binding to TCTP on cells and inhibiting its function extracellularly can be achieved by the TCTP-specific binding molecule or a fragment thereof according to the present invention, as described in detail below.

[0015] The binding molecules or fragments thereof according to the invention have a binding affinity of about 1×10 as measured by surface plasmon resonance. -7 M or less, or 5 x 10 -8 By binding to human TCTP or its truncated form or multimers with a dissociation constant (Kd) of M or less and inhibiting its function outside the cell, it can suppress / inhibit the function of inhibitory immune cells or activate anti-cancer immune cells.

[0016] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the detection of protein concentrations within a biosensor matrix and the analysis of interactions that occur, for example, using a BIAcore™ system.

[0017] A binding molecule or fragment thereof according to the invention may also comprise one or more CDR sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as defined in (a) to (f) below. (a) the heavy chain CDR1 is (a1) an amino acid sequence of X1-X2-X3-X4-H, where X1 is S, G, or V, X2 is Y, H, or N, X3 is A or Y, and X4 is M or V; (a2) an amino acid sequence of S-X5-X6-X7-X8-W-X9 (wherein X5 is S or N, X6 is N or S, X7 is A or W, X8 is A or absent, and X9 is N or S); (a3) the amino acid sequence GYTFTGYYMH, or (a4) the amino acid sequence of NARMGVS, or Containing conservative amino acid substitutions thereof; (b) the heavy chain CDR2 is (b1) an amino acid sequence of EI-X1-H-X2-G-X3-TTYNPSLKS, where X1 is D or Y, X2 is S or absent, and X3 is T or V; (b2) an amino acid sequence of WIN-X4-X5-X6-X7-X8-T-X9-Y-X10-QKFQ-X11 (wherein X4 is A or P, X5 is G, I, or N, X6 is K, N, S, or T, X7 is G or S, X8 is G, N, Y, D, or K, X9 is K, N, or E, X10 is A or S, and X11 is D or G); (b3) the amino acid sequence of RT-X12-Y-X13-SKWYNDYA-X14-SVKS, where X12 is F or Y, X13 is K or R, and X14 is E or V; or (b4) the amino acid sequence of HIFSNDEKSYSYSLKS, or Containing conservative amino acid substitutions thereof; (c) the heavy chain CDR3 is (c1) an amino acid sequence of G-X1-X2-X3-X4-FD-X5, where X1 is R or G, X2 is A or S, X3 is A or V, X4 is A or V, and X5 is P or Y; (c2) the amino acid sequence of DGSGSYEGY; (c3) the amino acid sequence of WVFDY; (c4) the amino acid sequence of X6-A-X7-RLR-X8-A-X9-DI, where X6 is L or P, X7 is P or W, X8 is G or M, and X9 is F or Y; (c5) the amino acid sequence of RLTIVRGVMRGGMDV; (c6) the amino acid sequence of GYYDILTGYYTTDAFDI; (c7) an amino acid sequence of X10-X11-X12-X13-X14-X15-X16-X17-X18-FDY (wherein X10 is V or absent, X11 is R, L or absent, X12 is G or L, X13 is Y, I, T, or W, X14 is F or T, X15 is D or G, X16 is W, T, Y, or E, X17 is W, T, Y, or L, and X18 is A, P, or Y), (c8) the amino acid sequence GGVLLYFGELSRFDY, or (c8) the amino acid sequence EGITVGRGVMLYFDL, or Containing conservative amino acid substitutions thereof; (d) the light chain CDR1 is (d1) an amino acid sequence of RASQ-X1-X2-X3-X4-X5-L-X6 (wherein X1 is A, D, or G, X2 is I or V, X3 is S, R, or G, X4 is N or S, X5 is H, Y, D, N, or S, and X6 is A, G, or H); (d2) an amino acid sequence of X7-G-X8-X9-S-X10-X11-G-X12-X13-X14-X15-VS (wherein X7 is S or T, X8 is S or T, X9 is N or S, X10 is D or N, X11 is I or V, X12 is G, N, or T, X13 is F, K, or Y, X14 is N, D, or absent, and X15 is K or Y); (d3) an amino acid sequence of KSSQ-X16-X17-LY-X18-X19-NNK-X20-Y-X21-A (wherein X16 is N or S, X17 is I, L, or V, X18 is N or S, X19 is A or S, X20 is D or N, and X21 is I or L), or (d4) the amino acid sequence of RSSQSLV-X22-X23-DGNT-X24-L-X25 (wherein X22 is H or Y, X23 is R or S, X24 is Y or H, and X25 is S, N, or K); or Containing conservative amino acid substitutions thereof, (e) the light chain CDR2 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is A, D, E, G, K, W, or Y, X2 is A, D, I, or V, X3 is N, S, or T, X4 is I, K, N, Q, S, or T, X5 is L, R, S, or W, X6 is A, D, F, G, K, P, Q, or Y, and X7 is F, S, or T, or a conservative amino acid substitution thereof; (f) the light chain CDR3 is (f1) an amino acid sequence of X1-Q-X2-X3-X4-X5-P-X6-T (wherein Xi is M or Q, X2 is A, G, or Y, X3 is F, H, N, T, or Y, X4 is G, H, Q, N, S, or Y, X5 is F, I, L, T, Y, or W, and X6 is H, I, L, P, R, W, or Y); (f2) an amino acid sequence of X7-X8-X9-X10-X11-X12-X13-X14-X15-V (wherein X7 is A, N, or V, X8 is S or T, X9 is W or Y, X10 is A or D, X11 is G or S, X12 is D, N, or S, X13 is L or N, X14 is N or R, and X15 is A or L); or (f3) an amino acid sequence of X16-Q-X17-X18-S-X19-P-X20-T (wherein X16 is L or H, X17 is H or S, X18 is N or S, X19 is Y or L, and X20 is Y, R, or Q); or Conservative amino acid substitutions thereof are included.

[0018] A "conservative amino acid substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art [Pearson (1994) Methods Mol. Biol. 24:307-331]. Examples of amino acid groups with chemically similar side chains include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are as follows: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution can be any change that has a positive value on the PAM250 log likelihood matrix as described in Gonnet et al. (1992) Science 256:1443-45.

[0019] A binding molecule of the present invention or a fragment thereof may comprise heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences as set forth below, or conservative amino acid substitutions thereof.

[0020] [Table 1]

[0021] [Table 2]

[0022]

Table 3

[0023]

Table 4

[0024]

Table 5

[0025]

Table 6

[0026]

Table 7

[0027]

Table 8

[0028]

Table 9

[0029]

Table 10

[0030]

Table 11

[0031]

Table 12

[0032]

Table 13

[0033]

Table 14

[0034]

Table 15

[0035] Table 16

[0036] Table 17

[0037] Table 18

[0038]

Table 19

[0039] Table 20

[0040] Table 21

[0041] Table 22

[0042] Table 23

[0043] [Table 24]

[0044] [Table 25]

[0045] [Table 26]

[0046] [Table 27]

[0047] [Table 28]

[0048] [Table 29]

[0049] [Table 30]

[0050] [Table 31]

[0051] A binding molecule of the present invention or a fragment thereof may also have a heavy chain variable region comprising an amino acid sequence as set forth below, or an amino acid sequence that is 70% or more, 80% or more, or 90% or more homologous thereto.

[0052] [Table 32] TIFF2025525492000034.tif216149 TIFF2025525492000035.tif191149 TIFF2025525492000036.tif65149

[0053] The underlined parts of the variable region sequences correspond, in order, to the heavy chain CDR1, CDR2, and CDR3 of the antibody.

[0054] A binding molecule of the invention or a fragment thereof may also have a light chain variable region comprising an amino acid sequence as set forth below, or an amino acid sequence that is 70% or more, 80% or more, or 90% or more homologous thereto.

[0055] [Table 33] TIFF2025525492000038.tif185149 TIFF2025525492000039.tif222149

[0056] The underlined parts of the variable region sequence correspond, in order, to the light chain CDR1, CDR2, and CDR3 of the antibody.

[0057] Sequence homology for polypeptides, sometimes referred to as percent similarity or sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to measure sequence homology or sequence identity between closely related polypeptides, e.g., homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein (GCG Version 6.1). Polypeptide sequences can also be compared using FASTA, using default or recommended parameters, or using the GCG Version 6.1 program. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity for the region of maximum overlap between the unknown and query sequences (Pearson 2000). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters [Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402].

[0058] The binding molecule or fragment thereof according to the present invention may comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 3, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 115, or 121, and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 85, 91, 97, 103, 109, 115, 121, or 131. 0, 86, 92, 98, 104, 110, 116, 122, 127, 135, or 136, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 128, 129, 130, or 131; and the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 128, 129, 130, or 131. DR1 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 132, or 133, and light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 10 The light chain CDR3 of the antibody may comprise the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134, or a fragment thereof, which competes for binding to human TCTP with an antibody comprising the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134.

[0059] As used herein, the term "compete for binding" refers to a binding molecule, particularly an antibody or antigen-binding fragment thereof, binding to an antigen and inhibiting or blocking the binding of another antibody or antigen-binding fragment thereof. This term also includes bidirectional competition between two antibodies, i.e., a first antibody binds and blocks the binding of a second antibody, or vice versa. In certain embodiments, the first and second antibodies may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that one antibody blocks or inhibits the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, such as real-time, label-free biolayer interferometry analysis.

[0060] TCTP is also known as histamine-releasing factor (HRF), Tpt1, p23, or portylin. The amino acid and nucleic acid sequences of TCTP can be found in NCBI under accession numbers CCDS9397.1 (cDNA) and NP_003286.1 (amino acid). As used herein, the term "TCTP" is intended to encompass not only the full-length form of TCTP (including variants), but also truncated forms or multimers (e.g., dimers) thereof. The term "variant" can refer to a form in which a variant has occurred in the polypeptide backbone (e.g., amino acid sequence) of TCTP, or a form in which the TCTP polypeptide has undergone post-translational modification (e.g., glycosylation or phosphorylation).

[0061] As used herein, the term "specifically binds" refers to binding that is measurably different from non-specific interactions. Specific binding can be determined by competition with a control molecule that is similar to the target and has no binding activity. "Specific binding" can be assayed by methods known in the art, including, but not limited to, Western blotting, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) testing, and peptide scanning.

[0062] As used herein, the terms "affinity" or "avidity" or "avidity" refer to the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, and are determined by the characteristics of the antigen, such as its size, pattern, and / or charge, and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining such affinity are known in the art.

[0063] The term "binding molecule" as used in the present invention means any molecule that can recognize or interact with TCTP and specifically bind to it. The binding molecule according to the present invention is preferably a polypeptide, which may contain a protein portion and a non-protein portion (e.g., a chemical linker, a cross-linking agent, a drug, etc.). Preferably, the polypeptide is an antibody.

[0064] The term "antibody" as used herein refers to an intact immunoglobulin of any isotype, or an antigen-binding fragment capable of competing with the intact antibody for binding to a target antigen. Examples include chimeric, humanized, fully human, or bispecific antibodies, or antigen-binding fragments thereof. An antibody is also a type of antigen-binding protein in itself. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some cases, such as those naturally found in camelids, antibodies may contain only heavy chains. An antibody or antigen-binding fragment thereof may be derived from a single source or may be chimeric. Chimeric antibodies contain portions derived from two different antibodies and are described in more detail below. Antibodies or antigen-binding fragments thereof may be produced by hybridomas (e.g., Kohler and Milstein 1975), recombinant DNA techniques (e.g., U.S. Pat. No. 4,816,567), or enzymatic or chemical cleavage of intact antibodies. Alternatively, antibodies may be isolated using methods such as phage antibody libraries (e.g., Clarkson et al., 1991; Marks et al., 1991). Antibodies may also be obtained by B cell screening or hybridoma screening using mice obtained by genetic engineering techniques, such as humanized mice (e.g., CAMouse®) (https: / / www.berkeleylights.com / systems / Beacon / , Rapid single B cell antibody discovery and structured light, mABs, https: / / doi.Org / 10.1080 / 19420862.2019.1624126). Unless otherwise specified, the term "antibody" as used herein includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof, examples of which are described below.

[0065] As used herein, "light chain" includes full-length light chains and fragments thereof that contain sufficient variable region sequence to provide binding specificity to an antigen or epitope. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is located at the amino terminus of the light chain polypeptide. Types of light chains include kappa and lambda chains.

[0066] As used herein, "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to provide binding specificity for an antigen or epitope. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is located at the amino terminus of the heavy chain polypeptide, the CH domain is located at the carboxy terminus, and CH3 is located closest to the carboxy terminus. Heavy chains include isotypes of IgG (including IgG1, IgG2a, IgG2b, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0067] As used herein, the term "fragment" refers to an "antigen-binding fragment." An "antigen-binding fragment" of a binding molecule, particularly an antibody or immunoglobulin chain (heavy or light chain), lacks some amino acids compared to the full-length chain but contains a portion of the antibody that is capable of specifically binding to an antigen. Such fragments are biologically active in that they can specifically bind to a target antigen or compete with other antibodies or antigen-binding fragments for binding to a particular epitope. In one embodiment, such fragments contain at least one CDR present in a full-length light or heavy chain, and in some embodiments, a shortened heavy and / or light chain, or portion thereof. Such biological fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, ab, Fab', F(ab')°2, Fv, domain antibodies, and short-chain antibodies. They can be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. Functional portions of the antibodies disclosed herein, such as one or more CDRs, can be covalently linked to a second protein or small molecule compound and used as targeted therapeutics against specific targets.

[0068] A "Fab fragment" consists of one light chain and one heavy chain containing only the CH1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0069] The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of an antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0070] The "Fab' fragment" further comprises a region located between the CH1 and CH2 domains of the heavy chain of the Fab fragment, and an interchain disulfide bond can be formed between the two heavy chains of the Fab' fragments of two molecules to form an F(ab')2 molecule.

[0071] As described above, an "F(ab')2 fragment" comprises two light chains and two heavy chain molecules each containing a variable region and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chain molecules. Thus, an F(ab')2 fragment is composed of two Fab' fragments, which are associated with each other by disulfide bonds between them.

[0072] The "Fv region" is an antibody that comprises the variable regions of the heavy and light chains, but does not include the constant regions.

[0073] A "single-chain antibody" is a single polypeptide chain in which the antigen-binding domains of the heavy and light chain variable regions are connected by a flexible linker. See, for example, U.S. Patent No. 5,260,203.

[0074] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody can target the same or different antigens.

[0075] A "bivalent antigen-binding protein" or "bivalent antibody" contains two antigen-binding sites. The two antigen-binding sites in such a bivalent antibody may have the same antigen specificity, or may be bispecific antibodies that bind to different antigens.

[0076] A "multispecific antigen binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[0077] A "bispecific" or "dual-specific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Such bispecific antibodies are a type of multispecific antigen-binding protein or antibody and can be produced by a variety of known methods, such as hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 1990, 79:315-321; Kostelny et al. J. Immunol. 1992, 148:1547-1553. The two distinct epitopes bound by the two antigen-binding sites of a bispecific antigen-binding protein or antibody can be located on the same or different protein targets.

[0078] The TCTP-specific binding molecule or a fragment thereof according to the present invention binds to TCTP released from tumor cells and inhibits the function of TCTP extracellularly, preferably in the tumor immune microenvironment, thereby suppressing the function of suppressive immune cells in the tumor immune microenvironment. The suppressive immune cells may preferably be myeloid-derived suppressor cells (MDSCs). The myeloid-derived suppressor cells (MDSCs) may be polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) or monocytic myeloid-derived suppressor cells (M-MDSCs).

[0079] The TCTP-specific binding molecule or a fragment thereof according to the present invention can activate the function of anti-cancer immune cells and suppress tumor growth by suppressing the function of TCTP extracellularly, preferably in the tumor immune microenvironment. It can also activate or enhance T cells and / or NK cells. The anti-cancer immune cells can be preferably T cells or NK cells.

[0080] The TCTP-specific binding molecule or a fragment thereof according to the present invention can suppress the function of TCTP extracellularly, preferably in the tumor immune microenvironment, thereby acting as an antagonist against suppressor immune cells in the tumor immune microenvironment and suppressing tumor growth. The suppressor immune cells can be preferably myeloid-derived suppressor cells (MDSCs). The myeloid-derived suppressor cells (MDSCs) can be PMN-MDSCs or M-MDSCs.

[0081] The TCTP-specific binding molecule or a fragment thereof according to the present invention can inhibit the extracellular function of TCTP, preferably in the tumor immune microenvironment, thereby inhibiting the accumulation or recruitment of suppressive immune cells in the tumor immune microenvironment and suppressing tumor growth. The suppressive immune cells can be preferably myeloid-derived suppressor cells (MDSCs). The myeloid-derived suppressor cells (MDSCs) can be PMN-MDSCs or M-MDSCs.

[0082] The TCTP-specific binding molecule or a fragment thereof according to the present invention can inhibit the function of TCTP extracellularly, preferably in the tumor immune microenvironment, thereby preventing the binding of TCTP to immunoregulatory receptors present on inhibitory immune cells in the tumor immune microenvironment and suppressing tumor growth. The inhibitory immune cells can be myeloid-derived suppressor cells (MDSCs), and the immunoregulatory receptor can be Toll-like receptor 2 (TLR2). The myeloid-derived suppressor cells (MDSCs) can be PMN-MDSCs or M-MDSCs.

[0083] The TCTP-specific binding molecule or a fragment thereof according to the present invention can inhibit the binding of TCTP to immunoregulatory receptors present on inhibitory immune cells extracellularly, preferably in the tumor immune microenvironment, thereby suppressing the activation of the immunoregulatory receptors and thereby suppressing tumor growth. The inhibitory immune cells can preferably be myeloid-derived suppressor cells (MDSCs), and the immunoregulatory receptor can be Toll-like receptor 2 (TLR2). The myeloid-derived suppressor cells (MDSCs) can be PMN-MDSCs or M-MDSCs.

[0084] The TCTP-specific binding molecule or a fragment thereof according to the present invention may be useful for treating or preventing cancer. In this aspect, the present invention provides a method for treating or preventing cancer using the TCTP-specific binding molecule or a fragment thereof.

[0085] As used herein, "treatment" means preventing or alleviating the progression or worsening of the disease condition caused in a mammal with cancer, and "prevention" means preventing the onset of cancer in a mammalian patient at risk of developing the disease.

[0086] The TCTP-specific binding molecule or a fragment thereof according to the present invention may also be useful for enhancing the responsiveness of an individual in need of cancer treatment who has not responded, is likely to not respond, or has responded only partially or is likely to respond only partially to treatment using an anticancer immunotherapy. In this respect, the present invention provides a method for enhancing the responsiveness of an anticancer immunotherapy using a TCTP-specific binding molecule or a fragment thereof.

[0087] The term "immune anticancer agent" as used in the present invention collectively refers to drugs used to treat patients suffering from cancer or at risk of cancer recurrence by methods that involve inducing, enhancing, suppressing, or modifying the immune response, and may have the ability to restore or enhance the immune system's tumor recognition or destruction ability in order to overcome the immune suppression or immune evasion mechanisms acquired by cancer cells.

[0088] Immune checkpoint inhibitors are one example of such immune anticancer drugs. The term "immune checkpoint inhibitor" refers to drugs that block the activity of immune checkpoint proteins involved in suppressing immune cells, activating immune cells to attack cancer cells. Known immune checkpoints that can be targeted by immune checkpoint inhibitors include CTLA-4, PD-1, PD-L1, TIM3, LAG3, BTLA, TIGIT, VISTA, GITR, CD47, and KIR.

[0089] The TCTP-specific binding molecule or a fragment thereof according to the present invention may also be useful for removing, alleviating, or preventing immunosuppression in the tumor microenvironment of cancer patients. In this aspect, the present invention provides a method for removing, alleviating, or preventing immunosuppression using the TCTP-specific binding molecule or a fragment thereof.

[0090] The TCTP-specific binding molecule or a fragment thereof according to the present invention may also be useful for suppressing suppressive immune cells in the tumor microenvironment of cancer patients. In this respect, the present invention provides a method for suppressing suppressive immune cells using the TCTP-specific binding molecule or a fragment thereof. The suppressive immune cells may preferably be myeloid-derived suppressor cells (MDSCs). The myeloid-derived suppressor cells (MDSCs) may be PMN-MDSCs or M-MDSCs.

[0091] As used herein, cancer may include, but is not limited to, hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, metastatic cell carcinoma, adenocarcinoma, gastrinoma, melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, meningioma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, brain tumor, head and neck cancer, epithelial cell derived neoplasms (epithelial cell carcinoma), lip cancer, oral cancer, esophageal cancer, gastrointestinal cancer, e.g., small intestine cancer and stomach cancer, colorectal cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, breast cancer, thyroid cancer, skin cancer, e.g., squamous cell carcinoma and basal cell carcinoma, and prostate cancer. Alternatively, cancer may include other cancers known to affect blood cells. Preferably, the cancer is colorectal cancer, melanoma fibrosarcoma, pancreatic cancer, liver cancer, or skin cancer.

[0092] The method of the present invention using the TCTP-specific binding molecule or a fragment thereof may comprise a step of administering a therapeutically effective amount of the TCTP-specific binding molecule or a fragment thereof to an individual. As used herein, the term "mammal" as a target of prevention or treatment means any animal classified as a mammal, including, but not limited to, pets such as dogs, cats, rabbits, and ferrets, as well as livestock such as cows, pigs, sheep, and horses, in addition to humans. A particularly preferred "mammal" is human.

[0093] The present invention also provides pharmaceutical compositions containing a TCTP-specific binding molecule or a fragment thereof as an active ingredient for therapeutic or prophylactic use.

[0094] The composition may contain pharmaceutically acceptable inactive ingredients (see Handbook of Pharmaceutical Excipients, etc.). The composition may be prepared, for example, in the form of a freeze-dried powder, a slurry, an aqueous solution, or a suspension by mixing with a physiologically acceptable carrier, excipient, or stabilizer.

[0095] The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in pharmaceutical formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, hydroxymethylbenzoic acid, hydroxypropylbenzoic acid, talc, magnesium stearate, and mineral oil.

[0096] In addition to the above ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Commonly used pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0097] The pharmaceutical compositions of the present invention may be administered orally or parenterally, for example, but not limited to, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, topical administration, intranasal administration, intracerebral administration, intracranial administration, intrapulmonary injection, and rectal administration.

[0098] The appropriate dosage of the pharmaceutical composition of the present invention can be adjusted depending on various factors, including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the pharmaceutical composition, the type of dosage form, the method of administration, the patient's age, weight, sex, and diet, the time of administration, the route of administration, the duration of treatment, and any concomitant medications. A physician of ordinary skill can easily determine and prescribe an effective dosage for the desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.001 to 1,000 mg (weight of active ingredient), preferably administered continuously or intermittently. As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease or condition depending on the context.

[0099] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-volume containers by formulating them using pharmaceutically acceptable carriers and / or excipients in a manner easily practiced by those skilled in the art to which the invention pertains. In this case, the dosage form may be a solution, suspension, or emulsion in an oily or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may further contain a dispersing agent or stabilizer.

[0100] The pharmaceutical composition of the present invention may contain an additional therapeutically active ingredient other than the TCTP-specific binding molecule or a fragment thereof, such as other anti-cancer drugs or immune checkpoint inhibitors, for example, anti-PD-1 antibodies or anti-CTLA-4 antibodies.

[0101] The present invention also provides an isolated polynucleotide encoding the TCTP-specific binding molecule or a fragment thereof according to the present invention, a vector containing the polynucleotide, and an isolated cell containing the polynucleotide or vector. The present invention also provides a method for producing the TCTP-specific binding molecule or a fragment thereof according to the present invention, comprising the steps of culturing the isolated cells to express the TCTP-specific binding molecule or a fragment thereof according to the present invention, and isolating the expressed binding molecule or a fragment thereof from the cells. Such methods are well known to those of ordinary skill in the field of biotechnology.

[0102] Based on the above, the present invention may relate to the following (1) to (21), but is not limited thereto.

[0103] (1) When measuring by surface plasmon resonance, 1 × 10 -7 A binding molecule or fragment thereof that binds to human TCTP or its cleavage carrier or its multimer with the following dissociation constant (Kd) and inhibits its function outside the cell, thereby suppressing / inhibiting the function of suppressive immune cells or activating anti-cancer immune cells.

[0104] (2) A binding molecule or fragment thereof that specifically binds to TCTP or its post-translational variant, truncation, or multimer, comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as defined in (a) to (f) below, (a) the heavy chain CDR1 is (a1) an amino acid sequence of X1-X2-X3-X4-H, where X1 is S, G, or V, X2 is Y, H, or N, X3 is A or Y, and X4 is M or V; (a2) an amino acid sequence of S-X5-X6-X7-X8-W-X9 (wherein X5 is S or N, X6 is N or S, X7 is A or W, X8 is A or absent, and X9 is N or S); (a3) the amino acid sequence GYTFTGYYMH, or (a4) the amino acid sequence of NARMGVS, or Containing conservative amino acid substitutions thereof; (b) the heavy chain CDR2 is (b1) an amino acid sequence of EI-X1-H-X2-G-X3-TTYNPSLKS, where X1 is D or Y, X2 is S or absent, and X3 is T or V; (b2) an amino acid sequence of WIN-X4-X5-X6-X7-X8-T-X9-Y-X10-QKFQ-X11 (wherein X4 is A or P, X5 is G, I, or N, X6 is K, N, S, or T, X7 is G or S, X8 is G, N, Y, D, or K, X9 is K, N, or E, X10 is A or S, and X11 is D or G); (b3) the amino acid sequence of RT-X12-Y-X13-SKWYNDYA-X14-SVKS, where X12 is F or Y, X13 is K or R, and X14 is E or V; or (b4) the amino acid sequence of HIFSNDEKSYSYSLKS, or Containing conservative amino acid substitutions thereof; (c) the heavy chain CDR3 is (c1) an amino acid sequence of G-X1-X2-X3-X4-FD-X5, where X1 is R or G, X2 is A or S, X3 is A or V, X4 is A or V, and X5 is P or Y; (c2) the amino acid sequence of DGSGSYEGY; (c3) the amino acid sequence of WVFDY; (c4) the amino acid sequence of X6-A-X7-RLR-X8-A-X9-DI, where X6 is L or P, X7 is P or W, X8 is G or M, and X9 is F or Y; (c5) the amino acid sequence of RLTIVRGVMRGGMDV; (c6) the amino acid sequence of GYYDILTGYYTTDAFDI; (c7) an amino acid sequence of X10-X11-X12-X13-X14-X15-X16-X17-X18-FDY (wherein X10 is V or absent, X11 is R, L or absent, X12 is G or L, X13 is Y, I, T, or W, X14 is F or T, X15 is D or G, X16 is W, T, Y, or E, X17 is W, T, Y, or L, and X18 is A, P, or Y), (c8) the amino acid sequence GGVLLYFGELSRFDY, or (c8) the amino acid sequence EGITVGRGVMLYFDL, or Containing conservative amino acid substitutions thereof; (d) the light chain CDR1 is (d1) an amino acid sequence of RASQ-X1-X2-X3-X4-X5-L-X6 (wherein X1 is A, D, or G, X2 is I or V, X3 is S, R, or G, X4 is N or S, X5 is H, Y, D, N, or S, and X6 is A, G, or H); (d2) an amino acid sequence of X7-G-X8-X9-S-X10-X11-G-X12-X13-X14-X15-VS (wherein X7 is S or T, X8 is S or T, X9 is N or S, X10 is D or N, X11 is I or V, X12 is G, N, or T, X13 is F, K, or Y, X14 is N, D, or absent, and X15 is K or Y); (d3) an amino acid sequence of KSSQ-X16-X17-LY-X18-X19-NNK-X20-Y-X21-A (wherein X16 is N or S, X17 is I, L, or V, X18 is N or S, X19 is A or S, X20 is D or N, and X21 is I or L), or (d4) the amino acid sequence of RSSQSLV-X22-X23-DGNT-X24-L-X25 (wherein X22 is H or Y, X23 is R or S, X24 is Y or H, and X25 is S, N, or K); or Containing conservative amino acid substitutions thereof, (e) the light chain CDR2 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is A, D, E, G, K, W, or Y, X2 is A, D, I, or V, X3 is N, S, or T, X4 is I, K, N, Q, S, or T, X5 is L, R, S, or W, X6 is A, D, F, G, K, P, Q, or Y, and X7 is F, S, or T, or a conservative amino acid substitution thereof; (f) the light chain CDR3 is (f1) an amino acid sequence of X1-Q-X2-X3-X4-X5-P-X6-T (wherein Xi is M or Q, X2 is A, G, or Y, X3 is F, H, N, T, or Y, X4 is G, H, Q, N, S, or Y, X5 is F, I, L, T, Y, or W, and X6 is H, I, L, P, R, W, or Y); (f2) an amino acid sequence of X7-X8-X9-X10-X11-X12-X13-X14-X15-V (wherein X7 is A, N, or V, X8 is S or T, X9 is W or Y, X10 is A or D, X11 is G or S, X12 is D, N, or S, X13 is L or N, X14 is N or R, and X15 is A or L); or (f3) an amino acid sequence of X16-Q-X17-X18-S-X19-P-X20-T (wherein X16 is L or H, X17 is H or S, X18 is N or S, X19 is Y or L, and X20 is Y, R, or Q); or including conservative amino acid substitutions thereof, Binding molecules or fragments thereof.

[0105] (3) In the above (1) or (2), the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 3, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 115, or 121 or a conservative amino acid substitution thereof, and the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 , 86, 92, 98, 104, 110, 116, 122, 127, 135, or 136, or conservative amino acid substitutions thereof, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 128, 129, 130, or 131, or conservative amino acid substitutions thereof. and a light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 132, or 133 or a conservative amino acid substitution thereof, and a light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83 , 89, 95, 101, 107, 113, 119, or 125 amino acid sequence or a conservative amino acid substitute thereof, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134 amino acid sequence or a conservative amino acid substitute thereof.

[0106] (4) In any one of (1) to (3), the heavy chain variable region is an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, and 177, or an amino acid sequence having 90% or more homology thereto. 170, 172, 174, 176, and 178, or an amino acid sequence that is 90% or more identical thereto.

[0107] (5) In any one of (1) to (4), the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 3, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 115, or 121, and the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 127, 135, or 136, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 128, 129, 130, or 131. and wherein the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 132, or 133, and the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, A binding molecule or fragment thereof that competes for binding to human TCTP with an antibody comprising the amino acid sequence of SEQ ID NO: 1, 101, 107, 113, 119, or 125, and whose light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134.

[0108] (6) In any one of (1) to (5), the binding molecule or fragment thereof is an antibody or antigen-binding fragment thereof, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 43, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 44, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 45, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 46, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 48.

[0109] (7) In any one of (1) to (5), the binding molecule or fragment thereof is an antibody or antigen-binding fragment thereof, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 55, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 56, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 57, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 58, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 60.

[0110] (8) In any one of (1) to (5), the binding molecule or fragment thereof is an antibody or antigen-binding fragment thereof, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 61, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 62, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 63, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 64, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 65, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 66.

[0111] (9) In any one of (1) to (5), the binding molecule or fragment thereof is an antibody or antigen-binding fragment thereof, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 109, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 110, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 111, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 112, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 113, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 114.

[0112] (10) A binding molecule or a fragment thereof according to any one of (1) to (9) above, wherein the binding molecule is an antibody and the fragment thereof is an antigen-binding fragment.

[0113] (11) An isolated polynucleotide encoding any one of the binding molecules (1) to (9) above, or a fragment thereof.

[0114] (12) A method for producing a binding molecule or a fragment thereof according to any one of claims 1 to 9 (preferably claim 2), comprising the steps of expressing in a host cell an isolated polynucleotide encoding any one of the binding molecules or fragments thereof according to (1) to (9), and isolating the expressed binding molecule or fragment thereof.

[0115] (13) A binding molecule or a fragment thereof according to any one of (1) to (9) above for use in treating or preventing cancer; a method for treating or preventing cancer, comprising administering the binding molecule or a fragment thereof to an individual in need thereof; or a pharmaceutical composition for treating or preventing cancer, comprising the binding molecule or a fragment thereof.

[0116] (14) A binding molecule or fragment thereof according to any one of (1) to (9) above for enhancing responsiveness to an immunoanticancer drug in an individual in need of cancer treatment who has not responded, is likely to not respond, or has responded or is likely to respond only partially to a treatment using an immunoanticancer drug; a method for enhancing responsiveness to an immunoanticancer drug, comprising administering the binding molecule or fragment thereof to an individual who has not responded, is likely to not respond, or has responded or is likely to respond only partially to a treatment using an immunoanticancer drug; or a pharmaceutical composition for enhancing responsiveness to an immunoanticancer drug, comprising the binding molecule or fragment thereof.

[0117] (15) A binding molecule or fragment thereof according to any one of (1) to (9) for use in removing, alleviating, or preventing immunosuppression in the tumor microenvironment of a cancer patient; a method for removing, alleviating, or preventing immunosuppression, comprising administering the binding molecule or fragment thereof to a cancer patient in need thereof; or a pharmaceutical composition for removing, alleviating, or preventing immunosuppression, comprising the binding molecule or fragment thereof.

[0118] (16) A binding molecule or fragment thereof according to any one of (1) to (9) for use in suppressing inhibitory immune cells in the tumor microenvironment of a cancer patient; a method for suppressing inhibitory immune cells in the tumor microenvironment, comprising administering the binding molecule or fragment thereof to a cancer patient; or a pharmaceutical composition for suppressing inhibitory immune cells in the tumor microenvironment, comprising the binding molecule or fragment thereof.

[0119] (17) The binding molecule or fragment thereof, method, or composition according to (16), wherein the suppressive immune cells are bone marrow-derived suppressive immune cells (MDSCs).

[0120] (18) The binding molecule or fragment thereof, method, or composition according to any one of (13) to (17), wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, fibrosarcoma, pancreatic cancer, liver cancer, and skin cancer.

[0121] (19) In any one of (13) to (17), the method or composition is used in combination with an immune checkpoint inhibitor.

[0122] The present invention will be described in detail below with reference to the following examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0123] Example 1: Production of antibodies against TCTP Example 1-1: Humanized mouse monoclonal antibody immunization To generate antibodies that bind to TCTP, humanized mice (CAMouse®) that produce human antibodies were immunized. Human TCTP with the following amino acid sequence was used as the antigen. Human TCTP: MIIYRDLISHDEMFSDIYKIREIADGLCLEVEGKMVSRTEGNIDDSLIGGNASAEGPEGEGTESTVITGVDIVMNHHLQETSFTKEAYKKYIKDYMKSIKGKLEEQRPERVKPFMTGAAEQIKHILANFKNYQFFIGENMNPDGMVALLDYREDGVTPYMIFFKDGLEMEKC

[0124] Male humanized mice were subcutaneously injected with 50 μg of C-terminally tagged MYC / DDK-labeled human TCTP, followed by six booster injections of 25 μg of human TCTP at 2-week intervals. One week after boosting, blood was collected and immunization titration was performed using a standard ELISA method to assay binding to the administered antigen.

[0125] Hybridoma Hybridomas producing monoclonal antibodies against human TCTP were obtained by fusing SP2 / 0 mouse myeloma cells with spleen cells isolated from mice immunized with human TCTP using a standardized protocol. After fusion, fusion with antibody-producing mouse B cells was confirmed using ELISA assays against antigens administered to mice using the cell culture medium. Hybridomas were then subjected to single-cell cloning to select hybridomas producing monoclonal antibodies. Antibodies were then produced and purified from the hybridomas, and antibodies against human TCTP were screened using a standard ELISA assay to assay for binding to TCTP. Antibodies Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, and Ab22 were obtained and their sequences were analyzed. The sequences of these antibodies are disclosed herein.

[0126] Beacon Screening In addition to the hybridoma method, we also used the Beacon screening method (Beacon® Optofluidic System) to screen antibodies against TCTP from mice immunized with human TCTP. Spleen cells producing antibodies against TCTP were selected from isolated mouse spleen cells using Beacon screening, and the variable region sequences of the selected antibodies were analyzed. Human IgG1 constant regions were added to the analyzed variable region sequences, and antibodies were produced and purified. To test binding to TCTP, we used the ELISA method to screen antibodies that bind to human TCTP. As a result, antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, and Ab9 were obtained, and their sequences were analyzed. The sequences of these antibodies are as disclosed in the applicant's specification.

[0127] Antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, and Ab22 were procured from CAMouse® in collaboration with CAMAB (China) and GenScript (China).

[0128] Example 1-2: Phage library screening In addition to the immunization method, we also used biopanning to screen single-chain antibodies against human TCTP from a phage library. We used a diverse human-derived single-chain antibody library (Wuxi Biologics). A total of four rounds of biopanning were performed to screen for single-chain antibodies against TCTP from the phage library. Specifically, human TCTP protein at a concentration of 10 μg / ml was adsorbed onto the surface of an immunoassay tube, and bovine serum albumin (BSA) buffer solution was added to the tube to protect the surface from TCTP adsorption. The phage library was placed in the immunoassay tube and incubated at room temperature to extract phages that bound to TCTP. The extracted phages were used in the next round of panning. This process was repeated four times to amplify antigen-specific phages. The polyclonal phages extracted from each round were confirmed to bind to the antigen via ELISA.

[0129] To select a single clone phage that specifically binds to human TCTP from the phage pool obtained by panning, a single colony was isolated from the phage pool. Single clone phages were generated from the isolated colony, and phages that bind to human TCTP were selected using an ELISA method to assay binding to human TCTP. The sequences of the selected phages were analyzed to remove duplicate clones, and then a human IgG constant region was added to produce and purify the antibody. An antibody that binds to human TCTP was selected using an ELISA method to assay binding to TCTP. As a result, antibody Ab10 was obtained and its sequence was analyzed. The sequence of this antibody is as disclosed in the present specification.

[0130] Example 2: Analysis of binding ability to TCTP Example 2-1: ELISA analysis using human TCTP antibody ELISA was performed to quantitatively analyze the binding affinity of the selected antibodies to the antigen. Human TCTP was applied to an ELISA plate at a concentration of 1 μg / ml, and various concentrations of antibodies were added to determine their relative binding affinity. The results showed that Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab12, Ab13, Ab14, Ab15, Ab17, Ab18, Ab19, Ab20, Ab21, and Ab22 all bound well to human TCTP (Figures 1a and 1b).

[0131] Example 2-2: SPR analysis using human TCTP antibody The antigen binding affinity of the antibodies selected in Example 1 was analyzed using SPR techniques. A Biacore 8K (GE Healthcare) instrument was used. Human TCTP was immobilized on the chip, and antibodies were injected at 6-7 concentrations. A CM5 chip (GE Healthcare, Cat. 29-1275-56), a regeneration buffer (10 mM glycine-HCl (GE Healthcare)), and a running buffer (HBS-EP+) were used. For kinetic analysis, the binding time in the binding phase was 100 or 120 seconds, and the flow rate was 30 or 50 μl / min. The dissociation time in the dissociation phase was 300 or 360 seconds, and the flow rate was 30 or 50 μl / min. A 1:1 binding model was used for fitting, and the evaluation software used was BIACore Evaluation software (GE Healthcare).

[0132] [Table 34]

[0133] Example 3: Development of mutant antibodies to improve the binding ability of TCTP antibodies and prevent post-translational modification Example 3-1: Confirmation of the difference in binding ability of TCTP antibodies to human TCTP and mouse TCTP To confirm the difference in binding ability between the selected antibodies to human TCTP and mouse TCTP, ELISA was performed in the same manner as in Example 2-1, and it was confirmed that antibody Ab10 had low binding ability to mouse TCTP.

[0134] Since the sequences of human and mouse TCTP differ by seven amino acids, we created a mutation in which the mouse TCTP sequence was replaced with the human TCTP sequence, and confirmed the binding ability of the resulting Ab10 mutant. As a result, we confirmed that the binding ability to mouse TCTP was restored by replacing the 13th leucine of mouse TCTP with a methionine like human TCTP.

[0135] Example 3-2: Post-translational modification of TCTP antibody (Ab10) After storing the selected antibody Ab10 at 40°C for 14 days, imaging capillary isoelectric focusing (iCIEF) testing confirmed that the peak percentage of acidic variants increased from 35.3% to 64.8%. This is presumed to be due to post-translational modifications of antibody Ab10, and it was confirmed that post-translational modifications may have occurred at the asparagine and serine positions 5 and 6 of the heavy chain CDR2 of antibody Ab10.

[0136] Example 3-3: Development of mutant antibodies to enhance the binding ability of TCTP antibody (Ab10) and prevent post-translational deformation To improve the binding ability of antibody Ab10 to mouse TCTP, a mutant was prepared by mutating one amino acid in the CDR sequence using a phage library as follows.

[0137] [Table 35]

[0138] The underlined amino acids correspond to residues mutated from the parent antibody Ab10. For a given mutant antibody, the CDR sequences not shown are identical to those of the parent antibody Ab10. For example, Ab10-1 antibody is mutated only in the heavy chain CDR2, and the sequences of the remaining CDRs (heavy chain CDR1, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3) are identical to those of Ab10. To prevent post-translational modification of Ab10, we also constructed a mutant (Ab10-9) in which the fifth asparagine residue in heavy chain CDR2 was replaced with isoleucine, and a mutant (Ab10-10) in which the sixth serine residue in the same CDR2 was replaced with threonine.

[0139] [Table 36]

[0140] The underlined amino acids correspond to residues mutated from the parent antibody Ab10. The CDR sequences not shown for the given mutant antibodies are identical to those of the parent antibody Ab10. The TCTP binding of these mutants was assayed by ELISA. It was confirmed that Ab10-1, Ab10-2, Ab10-3, Ab10-4, Ab10-5, Ab10-6, Ab10-7, and Ab10-8 had similar human TCTP binding as the parent antibody Ab10, but exhibited enhanced mouse TCTP binding compared to the parent antibody Ab10 (Figure 2). Furthermore, Ab10-9 and Ab10-10 maintained similar human TCTP binding, despite the low likelihood of being acidic variants (Figure 2). The Ab10 antibody mutants were obtained by mutating a single amino acid from the Ab10 antibody; however, two or more mutations may be performed simultaneously.

[0141] Example 4: Inhibition of binding between TCTP and TLR2 by TCTP antibody Example 4-1: ELISA TCTP is known to exert its biological function via its extracellular membrane receptor, TLR2. ELISA analysis was performed to confirm whether the selected TCTP antibodies could inhibit the binding between TCTP and TLR2.

[0142] To do this, human TCTP was applied to an ELISA plate at a concentration of 1 μg / ml, and then 150 mM TLR2 and various concentrations of Ab8, Ab10-3, Ab12, and Ab20 were added to each well. The ability to inhibit the binding between TCTP and TLR2 was confirmed by detecting TLR2 bound to human TCTP. The results confirmed that Ab10-3, Ab12, and Ab20 inhibited the binding between TCTP and TLR2 (Figure 3a).

[0143] Example 4-2: Reporter Cell assay TLR2 is known to regulate immune cells through NF-κB signaling via ligand binding. To confirm whether the antibodies tested in Example 4-1 for their ability to inhibit TCTP-TLR2 binding inhibit signaling at the cellular level, analysis was performed using HEK Blue™ hTLR2 Reporter cells.

[0144] To achieve this, HEK Blue™ hTLR2 Reporter cells were plated at 5 x 10 per well of a 96-well plate. 4 The cells were cultured in aliquots and treated simultaneously with 9 μg / ml human TCTP and various concentrations of Ab10-3, Ab12, and Ab20. The activity of secreted alkaline phosphatase (SEAP), which is regulated by NF-κB, was then measured. The results confirmed that Ab10, Ab12, and Ab20 suppressed TLR2 signaling (Figure 3b).

[0145] Example 5: PMN-MDSC inhibitory effect of TCTP antibody TCTP stimulates PMN-MDSCs extracellularly and has the effect of suppressing anti-cancer immunity in the tumor immune microenvironment. One of its effects is to attract PMN-MDSCs into the tumor immune microenvironment. Experiments were conducted to confirm whether the anti-TCTP antibody of the present invention has the effect of suppressing the action of TCTP and alleviating the anti-cancer immune suppressive environment by suppressing the accumulation of PMN-MDSCs in the tumor immune microenvironment.

[0146] To this end, immune cells within the tumor immune microenvironment were collected from HT29 cell line (human colon cancer cell line) tumors and analyzed by FACS. Because suppression of tumor growth by T cells may affect accurate PMN-MDSC measurement, we used HT29 cell line (2 × 10 6 (100 cells) were subcutaneously implanted into T cell-free BALB / c nude mice. 13 days after implantation, tumors were harvested, dissociated into single cells, and subjected to FACS analysis (Figures 4a-f). PMN-MDSCs were assessed by counting the number of CD45+CD11b+Ly6G+ cells. As shown in Figure 5, PMN-MDSCs were reduced in HT29 tumors upon treatment with a CXCR2 inhibitor (AZD5069) or anti-TCTP antibodies 55F3 and A10. 55F3 is a known anti-TCTP antibody. CXCR2 inhibitors were used as a control to suppress PMN-MDSC accumulation.

[0147] Example 6: Inhibitory effect of TCTP antibody on tumor growth Example 6-1: Single administration To confirm the tumor growth inhibitory activity of anti-TCTP antibodies, we used the SL4 cell line (SL4 hTCTP KI) in which human TCTP was knocked in. SL4 hTCTP KI cells (2 × 10 5 C57BL / 6 mice were subcutaneously inoculated with 100 μg of TCTP antibody (Ab12 or Ab20, 100 μg, intraperitoneal administration) or a control isotype IgG antibody (BioXCell, 100 μg, intraperitoneal administration) were administered on days 1, 4, 8, 11, and 15, starting from the day of inoculation (Day 1). Tumor growth suppression was confirmed in the animal model administered Ab12 or Ab20 (Figure 6a).

[0148] Mouse tumor cell lines Pan02 (pancreas), Hepa1-6 (liver cancer), B16BL6 (skin cancer), and MC38 (colon cancer) were subcutaneously inoculated into C57BL / 6 mice (3 × 10 5 , 5×10 5 , 2 × 10 5 , 1×10 6 Starting from the day of inoculation (Day 1), Ab12, Ab20, or anti-PD-1 antibodies were administered intraperitoneally at 100 μg twice weekly for two or three weeks. The results confirmed that Ab12 or Ab20 reduced the tumor growth rate in all four cancer cell lines (Figures 6b-6e). In the experiment using MC38, an anti-PD-1 antibody treatment group was included, and Ab20 demonstrated greater tumor growth inhibition than the anti-PD-1 antibody.

[0149] Example 6-2: Combined administration To confirm the effect of the anti-TCTP antibody of the present invention on alleviating the immunosuppressive environment in the tumor immune microenvironment on tumor growth, the effect of the combination of anti-TCTP antibody and anti-PD-1 antibody on tumor suppression was investigated. 5 C57BL / 6 mice were subcutaneously inoculated with TCTP cells. Anti-TCTP antibody Ab8 (100 μg, intraperitoneal administration) and anti-PD-1 antibody (BioXCell; 100 μg, intravenous injection) were administered alone or in combination on days 1, 4, 8, 11, 15, 18, and 22, starting from the day of inoculation (Day 1). The results confirmed that tumor size was further reduced when anti-TCTP antibody was administered in addition to anti-PD-1 antibody compared to when anti-PD-1 antibody alone was administered (Figure 7).

[0150] The same effect was observed in a combined administration experiment with other immune checkpoint inhibitors using SL4 cells. 5C57BL / 6 mice were subcutaneously inoculated with 100 μg of the anti-TCTP antibody Ab8 (100 μg, intraperitoneal administration) and anti-CTLA-4 antibody (BioXCell; 100 μg, intravenous injection) on days 1, 4, 8, 11, 15, 18, and 22, starting from the day of inoculation (Day 1). The anti-TCTP antibody Ab8 (100 μg, intravenous administration) was administered alone or in combination with anti-CTLA-4 antibody Ab8. The results showed that tumor size was further reduced when anti-TCTP antibody was administered in addition to anti-CTLA-4 antibody compared to when anti-CTLA-4 antibody alone was administered (FIG. 8). These results indicate that the TCTP-specific antibody of the present invention not only effectively inhibits tumor growth by itself, but also synergistically improves the tumor growth inhibitory effect when used in combination with an immune checkpoint inhibitor.

Claims

1. When measuring using surface plasmon resonance, approximately 1 × 10⁻⁶ －７ An antibody or antigen-binding fragment that binds to human TCPP (translationally controlled tumor protein) or its cleaved form or multimer, with the following dissociation constant (Kd), thereby suppressing its extracellular function, thereby inhibiting the function of suppressive immune cells and / or activating anti-cancer immune cells.

2. An antibody or antigen-binding fragment according to claim 1, comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 as defined in (a) to (f) below, (a) The heavy chain CDR1 is (a1) The amino acid sequence X1-X2-X3-X4-H (where X1 is S, G, or V, X2 is Y, H, or N, X3 is A or Y, and X4 is M or V), (a2) Amino acid sequence S-X5-X6-X7-X8-W-X9 (where X5 is S or N, X6 is N or S, X7 is A or W, X8 is A or absent, and X9 is N or S), (a3) The amino acid sequence G-Y-T-F-T-G-Y-Y-M-H, or (a4) The amino acid sequence N-A-R-M-G-V-S, or Including its conserved amino acid substitution; (b) The heavy chain CDR2 is (b1) The amino acid sequence E-I-X1-H-X2-G-X3-T-T-Y-N-P-S-L-K-S (where X1 is D or Y, X2 is S or absent, and X3 is T or V), (b2) The amino acid sequence W-I-N-X4-X5-X6-X7-X8-T-X9-Y-X10-Q-K-F-Q-X11 (where X4 is A or P, X5 is G, I, or N, X6 is K, N, S, or T, X7 is G or S, X8 is G, N, Y, D, or K, X9 is K, N, or E, X10 is A or S, and X11 is D or G), (b3) The amino acid sequence R-T-X12-Y-X13-S-K-W-Y-N-D-Y-A-X14-S-V-K-S (where X12 is F or Y, X13 is K or R, and X14 is E or V), or (b4) The amino acid sequence H-I-F-S-N-D-E-K-S-Y-S-Y-S-L-K-S, or Including its conserved amino acid substitution; (c) The heavy chain CDR3 is (c1) Amino acid sequence of G-X1-X2-X3-X4-F-D-X5 (where X1 is R or G, X2 is A or S, X3 is A or V, X4 is A or V, and X5 is P or Y), (c2) The amino acid sequence D-G-S-G-S-Y-E-G-Y, (c3) Amino acid sequence of W-V-F-D-Y, (c4) Amino acid sequence of X6-A-X7-R-L-R-X8-A-X9-D-I (where X6 is L or P, X7 is P or W, X8 is G or M, and X9 is F or Y), (c5) Amino acid sequence of R-L-T-I-V-R-G-V-M-R-G-G-M-D-V, (c6) The amino acid sequence of G-Y-Y-D-I-L-T-G-Y-Y-T-T-D-A-F-D-I, (c7) Amino acid sequence X10-X11-X12-X13-X14-X15-X16-X17-X18-F-D-Y (where X10 is V or absent, X11 is R or L or absent, X12 is G or L, X13 is Y, I, T, or W, X14 is F or T, X15 is D or G, X16 is W, T, Y, or E, X17 is W, T, Y, or L, and X18 is A, P, or Y), (c8) The amino acid sequence G-G-V-L-L-Y-F-G-E-L-S-R-F-D-Y, or (c9) The amino acid sequence E-G-I-T-V-G-R-G-V-M-L-Y-F-D-L, or Including its conserved amino acid substitution; (d) The light chain CDR1 is (d1) Amino acid sequence R-A-S-Q-X1-X2-X3-X4-X5-L-X6 (where X1 is A, D, or G, X2 is I or V, X3 is S, R, or G, X4 is N or S, X5 is H, Y, D, N, or S, and X6 is A, G, or H), (d2) Amino acid sequence X7-G-X8-X9-S-X10-X11-G-X12-X13-X14-X15-V-S (where X7 is S or T, X8 is S or T, X9 is N or S, X10 is D or N, X11 is I or V, X12 is G, N, or T, X13 is F, K, or Y, X14 is N or D or absent, and X15 is K or Y), (d3) The amino acid sequence K-S-S-Q-X16-X17-L-Y-X18-X19-N-N-K-X20-Y-X21-A (where X16 is N or S, X17 is I, L, or V, X18 is N or S, X19 is A or S, X20 is D or N, and X21 is I or L), or (d4) The amino acid sequence R-S-S-Q-S-L-V-X22-X23-D-G-N-T-X24-L-X25 (where X22 is H or Y, X23 is R or S, X24 is Y or H, and X25 is S, N, or K), or Including the conserved amino acid substitution, (e) The light chain CDR2 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7 (wherein X1 is A, D, E, G, K, W, or Y; X2 is A, D, I, or V; X3 is N, S, or T; X4 is I, K, N, Q, S, or T; X5 is L, R, S, or W; X6 is A, D, F, G, K, P, Q, or Y; and X7 is F, S, or T), or a conservative amino acid substitution thereof. (f) The light chain CDR3 is (f1) Amino acid sequence X1-Q-X2-X3-X4-X5-P-X6-T (where X1 is M or Q, X2 is A, G, or Y, X3 is F, H, N, T, or Y, X4 is G, H, Q, N, S, or Y, X5 is F, I, L, T, Y, or W, and X6 is H, I, L, P, R, W, or Y), (f2) The amino acid sequence X7-X8-X9-X10-X11-X12-X13-X14-X15-V (where X7 is A, N, or V, X8 is S or T, X9 is W or Y, X10 is A or D, X11 is G or S, X12 is D, N, or S, X13 is L or N, X14 is N or R, and X15 is A or L), or (f3) The amino acid sequence X16-Q-X17-X18-S-X19-P-X20-T (where X16 is L or H, X17 is H or S, X18 is N or S, X19 is Y or L, and X20 is Y, R, or Q), or Including the conserved amino acid substitutions, Antibody or antigen-binding fragment.

3. Heavy chain CDR1 contains the amino acid sequence of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 115, or 121 or its conserved amino acid substitutions, and heavy chain CDR2 contains SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 1 The heavy chain CDR3 comprises the amino acid sequence 10, 116, 122, 127, 135, or 136 or its conserved amino acid substitution, and the light chain C DR1 contains the amino acid sequence of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 132, or 133 or its conserved amino acid substitutions, and light chain CDR2 contains SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, The antibody or antigen-binding fragment according to claim 2, comprising the amino acid sequence 107, 113, 119, or 125 or a conserved amino acid substitution thereof, wherein the light chain CDR3 comprises the amino acid sequence 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134 or a conserved amino acid substitution thereof.

4. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, and 177, or an amino acid sequence homologous to it by 90% or more, and the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, and 178, or an amino acid sequence homologous to it by 90% or more.

5. Heavy chain CDR1 contains the amino acid sequence of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 115, or 121, and heavy chain CDR2 contains the amino acid sequence of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104 The heavy chain CDR3 contains the amino acid sequence 110, 116, 122, 127, 135, or 136, and the light chain CDR1 contains the amino acid sequence 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 128, 129, 130, or 131, and the light chain CDR1 contains the amino acid sequence 4, 1 The amino acid sequence contains 0, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 132, or 133, and the light chain CDR2 contains SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 11 The antibody or antigen-binding fragment according to claim 2, comprising an amino acid sequence of 9 or 125, wherein the light chain CDR3 competes for binding to human TCP with an antibody comprising an amino acid sequence of SEQ ID NOs. 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, or 134.

6. The antibody or antigen-binding fragment according to claim 2, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 43, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 44, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 45, light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 46, light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 47, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

48.

7. The antibody or antigen-binding fragment according to claim 2, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 55, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 56, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 57, light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 58, light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 59, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

60.

8. The antibody or antigen-binding fragment according to claim 2, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 61, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 62, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 63, light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 64, light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 65, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

66.

9. The antibody or antigen-binding fragment according to claim 2, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 109, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 110, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 111, light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 112, light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 113, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

114.

10. A pharmaceutical composition for treating or preventing cancer, comprising the antibody or antigen-binding fragment described in Claim 2.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment described in Claim 2 for enhancing the responsiveness to an immunosuppressant in an individual in need of cancer treatment who has not responded to, may not respond to, or has responded restrictively to, or may respond restrictively to, an immunosuppressant.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment described in Claim 2, for eliminating, mitigating, or preventing immunosuppression in the tumor microenvironment of a cancer patient.

13. A pharmaceutical composition for suppressing inhibitory immune cells in the tumor microenvironment of a cancer patient, comprising the antibody or antigen-binding fragment described in Claim 2.

14. The pharmaceutical composition according to claim 10, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, fibrosarcoma, pancreatic cancer, liver cancer, and skin cancer.

15. The pharmaceutical composition according to claim 10, wherein an immune checkpoint inhibitor is used together with the pharmaceutical composition according to claim 10.

Images