USE OF TCTP AS BIOMARKER FOR PREDICTING EFFICACY, PROGNOSIS OF IMMUNOTHERAPY OR RESISTANCE THERETO, AND TARGET OF IMMUNOTHERAPY FOR ENHANCING EFFICACY

Abstract

Disclosed herein are a method and a biomarker for predicting efficacy and prognosis of or resistance to an immunotherapy. The use of the biomarkers (TCTP, EGFR, AKT, MCL1, and/or CXCL10) of the present disclosure allows the prediction of resistance to or prognosis of a cancer immunotherapeutic agent and the selection of a therapy guaranteeing therapeutic benefit, thereby finding advantageous applications in treating cancers or tumors resistant to cancer immunotherapeutic agents.

Claims

1. A method comprising: (a) measuring presence or expression of TCTP (translationally controlled tumor protein) at a protein or gene level in a sample isolated from an individual; and (b) providing information necessary for prediction of resistance to an immunotherapeutic agent for a cancer or a tumor.

2. The method of claim 1, wherein a higher measurement of the presence or expression of TCTP (translationally controlled tumor protein) at a protein or gene level in step (a) than that in a reference sample indicates the likelihood that the cancer or tumor is more resistant to the cancer immunotherapeutic agent.

3. The method of claim 1, wherein the step (a) further comprises measuring presence or expression of at least one selected from the group consisting of EGFR, AKT, MCL1, NANOG, and CXCL10 at a gene or protein level.

4. The method of claim 3, wherein a higher measurement of the presence or expression of one selected from the group consisting of EGFR, AKT, MCL-1, and NANOG at a protein or gene level than that in a reference sample or a lower measurement of the presence or expression of CXCL10 at a protein or gene level than that in a reference sample indicates that the cancer or tumor is more likely to exhibit resistance to the cancer immunotherapeutic agent.

5. The method of claim 1, wherein a higher measurement of the presence or expression of TCTP (translationally controlled tumor protein) at a protein or gene level in step (a) than that in a reference sample indicates the likelihood that the cancer or tumor is less responsive to the cancer immunotherapeutic agent or has poorer prognosis for the cancer immunotherapeutic agent.

6. The method of claim 3, wherein a higher measurement of the presence or expression of one selected from the group consisting of EGFR, AKT, MCL-1, and NANOG at a protein or gene level than that in a reference sample or a lower measurement of the presence or expression of CXCL10 at a protein or gene level than that in a reference sample indicates that the individual is more likely to exhibit resistance to the cancer immunotherapeutic agent or have poor prognosis for the cancer immunotherapeutic agent.

7. The method of claim 1, wherein a higher measurement of the presence or expression level of TCTP (translationally controlled tumor protein) in step (a) than that in a reference sample indicates the likelihood that a therapy using a TCTP protein or gene-targeting inhibitor is highly likely to impart high therapeutic benefit.

8. The method of claim 3, wherein a higher measurement of the presence or expression of one selected from the group consisting of EGFR, AKT, MCL-1, and NANOG at a protein or gene level than that in a reference sample or a lower measurement of the presence or expression of CXCL10 at a protein or gene level than that in a reference sample indicates that a therapy using at least one selected from the group consisting of i) an inhibitor targeting one or more selected from EGFR, AKT, MCL-1, and NANOG at a protein or gene level, ii) a CXCL10 activator at a protein or gene level, and iii) a combination thereof is more likely to impart high therapeutic benefit to the individual.

9. A method for treating an individual suffering from a cancer, comprising: (a) measuring presence or expression of TCTP (translationally controlled tumor protein) at a protein or gene level in a sample isolated from the individual; (b) identifying the individual whose sample having a higher presence or expression level of the TCTP protein or gene measured in step (a) compared to the presence or expression level of the TCTP protein or gene measured in the reference sample; and (c) administering a pharmaceutical composition comprising i) an immunotherapeutic agent, ii) a TCTP inhibitor, or iii) an immunotherapeutic agent and a TCTP inhibitor as an active ingredient to the individual identified in step (b).

10. The method of claim 9, wherein the cancer immunotherapeutic agent is an immune checkpoint blocker or an adoptive cell therapeutic agent.

11. The method of claim 9, wherein the TCTP inhibitor is an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a compound, a peptide, a fusion protein, or an aptamer, which all bind specifically to TCTP; or an siRNA, an shRNA, an miRNA, a ribozyme, or an antisense oligonucleotide, which all bind complementarily to a TCTP gene.

12. The method of claim 9, wherein the TCTP inhibitor is at least one selected from the group consisting of dihydroartemisinin (DHA), rapamycin, sertraline, and thioridazine.

13. The method of claim 9, wherein the cancer is selected from non-small cell lung cancer, small cell lung cancer, liver cancer, bone cancer, tongue cancer, laryngeal cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, Merkle cell cancer, and hematologic malignancies.

14. A composition for treatment of cancer in an individual suffering from cancer, the composition comprising: (a) a TCTP inhibitor and a cancer immunotherapeutic agent as active ingredients; and (b) a pharmaceutically acceptable carrier, excipient, or diluent

15. The composition of claim 14, wherein a sample isolated from the individual is measured to exhibit higher presence probability or a higher expression level of TCTP at a protein or gene level compared to a reference sample isolated from an individual suffering from no cancer or an individual suffering from cancer with no resistance to the cancer immunotherapeutic agent.

16. The composition of claim 14, wherein the cancer immunotherapeutic agent is an immune checkpoint blocker or an adoptive cell therapeutic agent.

17. The composition of claim 14, wherein the TCTP inhibitor is an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a compound, a peptide, a fusion protein, or an aptamer, which all bind specifically to TCTP; or an siRNA, an shRNA, an miRNA, a ribozyme, or an antisense oligonucleotide, which all bind complementarily to a TCTP gene.

18. The composition of claim 14, wherein the TCTP inhibitor is at least one selected from the group consisting of dihydroartemisinin (DHA), rapamycin, sertraline, and thioridazine.

19. The composition of claim 14, wherein the cancer is selected from non-small cell lung cancer, small cell lung cancer, liver cancer, bone cancer, tongue cancer, laryngeal cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, Merkle cell cancer, and hematologic malignancies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The above and other aspects, features and advantages of the present disclosure will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0094] FIGS. 1A, 1B, 1C and 1D illustrate that increased expression of TPT1 is associated with a non-responder phenotype to anti-PD-L1 therapy. FIG. 1A is a view showing comparison of TPT1 expression between responders (R, n=68) and non-responders (NR, n=230). FIG. 1B is a view showing Kaplan-Meier analysis of overall survival and median expression cutoff values for the expression level of TPT1 (TPT1.sup.high>median; TPT1.sup.low<median, p=0.00822). FIG. 1C is a view showing CD8.sup.+ T cell signature scores in TPT1.sup.low and TPT1.sup.high patients. FIG. 1D is a view showing anti-apoptosis signature scores in TPT1.sup.low and TPT1.sup.high patients.

[0095] FIG. 2A shows results of the establishment of CT26 P3-implanted mouse model resistant to PD-L1 antibody therapy (A and B) and lower levels of CD8+ T cell counts in tumor (C-E) and apoptosis of cancer cells (F), compared to mother cell line CT26 P0 in the cancer resistant model.

[0096] FIGS. 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K and 2L are views illustrating that silencing TCTP reverses tumor intrinsic resistance to CTL-mediated cell killing and the non-T cell inflamed tumor microenvironment of immune-refractory cancer. FIG. 2B shows a schematic of the therapy regimen in BALB/c mice implanted with CT26 P0 or CT26 P3 cells (upper panel). FIGS. 2B, 2C, 2D and 2E show CT26 P3 cells transfected with the indicated siRNAs. FIG. 2C shows TCTP protein levels determined by Western blot analysis. FIG. 2D is a view showing transwell-based T cell chemotaxis assay. SiGFP- or siTPT1 #1, 2, 3-treated CT26 P3 cell-derived conditioned media (CM) was added to the lower chamber, and T cells were plated in the upper chamber. The T cells that migrated into the lower chamber were counted after 6 hours. FIG. 2E is a view illustrating that carboxyfluorescein succinimidyl ester (CFSE)-labeled tumor cells were exposed to tumor-specific CTLs and the frequency of CFSE+ apoptotic tumor cells was determined by flow cytometric analysis of active-caspase-3.

[0097] FIG. 2F is a schematic of the therapy regimen in BALB/c mice implanted with CT26 P3 cells. FIGS. 2G, 2H, 2I, 2J, 2K and 2L shows results of experiments in which CT26 P3 tumor-bearing mice were administered siGFP- or siTPT1-loaded chitosan nanoparticles (CNPs) with PD-L1 antibody. FIG. 2G shows tumor growth and FIG. 2H survival of mice inoculated with CT26 P3 cells treated with the reagents. FIG. 2I shows flow cytometry profiles of tumor-infiltrating CD8+ T cells. FIG. 2J shows tumor-infiltrating CD8+ T cell to CD4+, Foxp3+ Treg cell ratios. FIG. 2K shows the percentage of granzyme B+ cells in CD8+ T cells. FIG. 2L shows the frequency of apoptotic cells in the tumors. For the in vivo experiments, 10 mice from each group were used.

[0098] FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K and 3L are views showing effects of the overexpression of TCTP on change in properties of tumor cells. CT26 cells were stably transfected with empty vector (No) or TCTP. FIG. 3A shows levels of TCTP, CXCL10, and MCL-1 proteins as analyzed by Western blots. FIG. 3B shows data of T cell chemotaxis assays performed using CT26 No or CT26 TCTP cell-derived CM in the lower chamber and plating CD8+ T cells in the upper chamber. The T cells migrated into the lower chamber media were counted. FIG. 3C shows counts of the migrated T cells after incubation with empty vector- or CXCL10-transfected CT26 TCTP cell-derived CM.

[0099] FIGS. 3D and 3E show frequencies of CFSE+ apoptotic tumor cells as measured by flow cytometric analysis of active-caspase-3 after CFSE-labeled tumor cells were incubated with tumor-specific CTLs.

[0100] FIG. 3F is a schematic of the therapy regimen in BALB/c mice implanted with CT26 No or CT26 TCTP cells. FIGS. 3G, 3H, 3I, 3J, 3K and 3L show tumor-bearing mice treated or not treated with the PD-L1 antibody. FIG. 3G shows tumor growth of mice inoculated with CT26 No or TCTP cells treated with or without PD-L1 antibody and FIG. 3H shows survival of the mice. FIG. 3I shows flow cytometry profiles of tumor-infiltrating CD8+ T cells. FIG. 3J shows tumor-infiltrating CD8+ T cell to CD4+, Foxp3+ Treg cells ratios. FIG. 3K shows the percentage of granzyme B+ to tumor-infiltrating CD8+ T cells. FIG. 3L shows the frequency of apoptotic cells in the tumors treated with the indicated reagents. For the in vivo experiments, 10 mice from each group were used. The p-values by one-way ANOVA (3B)-(3D) and (3I)-(3L), two-way ANOVA (3G), and the log-rank (Mantel-Cox) test (3H) are indicated. The data represent the mean±SD.

[0101] FIG. 4A is a view illustrating the establishment of A375 (NY-ESO1 tumor antigen expression) P3 cell line resistant to human-derived NY-ESO1-specific T cell therapy.

[0102] FIGS. 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4J, 4K, 4L, and 4M illustrate that CTL-mediated immune selection enriches TCTP+ immune-refractory tumor cells. FIG. 4B is a schematic of the therapy regimen in NOD/SCID mice implanted with A375 P0 or A375 P3 cells (upper panel) and shows TPT1 mRNA and TCTP protein levels in A375 cells at various stages of immune resistance as determined by qRT-PCR and Western blot analysis (lower panel). FIG. 4C shows the quantification of the frequency of TCTP+ tumor cells as analyzed by flow cytometry. FIG. 4D shows the protein levels of CXCL10 and MCL-1 as analyzed by Western blots. FIGS. 4E and 4G show data of experiments using A375 P3 cells transfected with the indicated siRNAs. In detail, FIG. 4E shows the protein levels of TCTP, CXCL10, and MCL-1 as determined by Western blot analysis. FIG. 4F shows data of transwell-based T cell chemotaxis assays performed by using SiGFP- or siTPT1-treated A375 P3 cell-derived conditioned media. FIG. 4G shows CFSE-labeled tumor cells exposed to tumor-specific CTLs and the frequency of CFSE+ apoptotic tumor cells (active-caspase-3) as determined by flow cytometry.

[0103] FIG. 4H is a schematic of the therapy regimen in NOD/SCID mice implanted with A375 P3 cells. FIGS. 4I, 4J, 4K, 4L and 4M show A375 P3 tumor-bearing mice administered siGFP- or siTPT1-loaded chitosan nanoparticles (CNPs) with or without NY-ESO1-specific T cell adoptive transfer treatment. FIG. 4I shows flow cytometry profiles of CFSE+ adoptively transferred NY-ESO1-specific T cells. FIG. 4J show the percentage of active-caspase-3+ apoptotic cells in the tumors treated with the indicated reagents. FIG. 4K show the frequency of apoptotic cells in the tumor relative to the NY-ESO1-specific T cells migrated to the tumor. FIG. 4L shows tumor growth of mice inoculated with A375 P3 cells treated with the indicated reagents and FIG. 4M shows survival of the mice. For the in vivo experiments, 10 mice from each group were used. The p-values by one-way ANOVA (FIGS. 4B, 4C, 4F, 4G, and 4I-4K), two-way ANOVA (FIG. 4L), and the log-rank (Mantel-Cox) test (FIG. 4M) are indicated. The data represent the mean±SD.

[0104] FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H illustrating that TCTP phosphorylation is crucial to activating the EGFR/NANOG signaling pathway via binding with Na, K ATPase. FIG. 5A shows the protein levels of EGFR, pEGFR, pAKT, AKT, MCL-1, and CXCL10 as measured by Western blot analysis. FIG. 5B shows the protein levels of pEGFR, EGFR, pAKT, AKT, MCL-1, and CXCL10 in SiGFP- or siEGFR-treated A375 TCTP cells as analyzed by Western blots. FIG. 5C shows data of experiments in which SiGFP- or siEGFR-treated A375 TCTP cell CM was added to the lower chamber, and CD8+ T cells were plated in the upper chamber. The T cells migrated into the lower chamber media were collected after 6 hours and counted. FIG. 5D shows the frequency of CFSE+ apoptotic tumor cells as determined by flow cytometric analysis of active-caspase-3 after CFSE-labeled tumor cells were exposed to NY-ESO1-specific CTLs.

[0105] FIGS. 5E, 5F, 5G and 5H illustrate A375 cells transfected with FLAG-TCTP wild type (TCTP), FLAG-TCTP S46A mutant, or FLAG-TCTP S46D mutant. FIG. 5E shows activation of EGFR, AKT signaling and expression of MCL-1 and CXCL10 as analyzed by Western blot assays. FIG. 5F shows data of T cell chemotaxis assays performed by using the indicated tumor cell-derived CM. FIG. 5G shows active caspase-3+ apoptotic tumor cells as analyzed by flow cytometry after incubation with CTLs. FIG. 5H shows cross-linked lysates immunoprecipitated with anti-Na, K ATPase antibody. The immunoprecipitated proteins were analyzed by Western blot assays. The numbers below the blot images indicate the expression as measured fold-change. The error bars represent mean±SD.

[0106] FIGS. 6A, 6B, 6C, 6D, 6E and 6F illustrate that inhibition of TCTP by DHA sensitizes TCTP.sup.high tumor cells to T cell-mediated killing and increases T cell migration. FIG. 6A shows data of the treatment of CT26 No and TCTP cells with the indicated concentrations of cisplatin, DHA, rapamycin, sertraline, and thioridazine for 24 hours, and IC.sub.50 values. FIG. 6B shows results in term of the percentage of active-caspase3+ apoptotic tumor cells after CT26 TCTP cells were treated with the indicated agents, and incubated with tumor-specific CTLs at the indicated tumor:T cell ratio. FIG. 6C shows the combination score calculated based on changes in the percentage of apoptosis in drug-treated tumor cells with or without CTLs. Combination score=(% of active-caspase 3+ tumor cells by drug and CTLs)/(% of active-caspase 3+ tumor cells by drug).

Combination score=(% of active-caspase 3+tumor cells by drug and CTLs)/(% of active-caspase 3+tumor cells by drug).

[0107] FIG. 6D shows results after SiGFP- or siTPT1-treated CT26 P3, MDA-MB231, 526Mel, and HCT116 cells were treated with PBS or DHA. The levels of TCTP, pEGFR, EGFR, pAKT, AKT, MCL-1, CXCL10, and β-ACTIN were analyzed by Western blots. FIG. 6E shows the percentage of CTL-mediated anti-apoptotic tumor cells as determined by flow cytometry. FIG. 6F shows data of T cell chemotaxis assays performed using PBS or DHA treated siGFP- or siTPT1-treated tumor cell CM. The numbers below the blot images indicate the expression as measured as fold change. The error bars represent mean±SD.

[0108] FIGS. 7A. 7B and 7C show results indicating that therapeutic efficacy was increased when TCTP targeting by treatment of CT26 P3 with DHA in vivo was used in combination with PD-L1 antibody therapy. FIGS. 7D, 7E and 7F show results indicating that TCTP targeting increased counts of T cells in tumors and the apoptosis of cancer cells.

[0109] FIGS. 7G, 7H and 7I show results indicating that therapeutic efficacy was increased when TCTP targeting by treatment of CT26 P3 with DHA in vivo was used in combination with PD-L1 antibody therapy. FIGS. 7J, 7K, 7L and 7M show results indicating that TCTP targeting increased counts of T cells in tumors and the apoptosis of cancer cells.

[0110] FIGS. 8A, 8B and 8C show results indicating that extracellular secretion of TCTP also increases in ACT-refractory A375 P3 and when a TCTP neutralizing antibody was used to target TCTP, phenotypes to anticancer immune resistance was decreased and the previously reported immune resistance and the expression of the cross resistance (cisplatin resistance) and multiple malignance (cancer metastasis and cancer stemness) regulator NANOG as well as the AKT signaling pathway were reduced. The data imply that the TCTP secreted outside tumor cells play a crucial role in the therapeutic resistance and multiple malignancy phenotypes of resistant cancer and the neutralization through an neutralizing antibody could reverse the phenotypes of resistant cancer.

[0111] FIG. 9 shows results indicating that in samples of patients with various carcinomas, there is a significant correlation between immune resistance and the expression of NANOG, a factor regulating cross resistance (cisplatin resistance) and multiple malignancy (cancer metastasis and cancer stemness).

[0112] FIG. 10 shows results indicating that in the TC-1 LP3 constructed as an immune checkpoint antibody therapy-refractory orthostatic lung cancer model, intracellular and extracellular TCTP secretion remarkably increases.

[0113] FIGS. 11A and 11B show results indicating that when TCTP was neutralized through an anti-TCTP neutralizing antibody, the previously reported immune resistance and cross resistance (cisplatin resistance) was decreased and multiple malignancy (cancer metastasis and cancer stemness) was also reduced.

DETAILED DESCRIPTION

[0114] A better understanding of the present disclosure may be obtained via the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

EXAMPLES

[0115] Unless stated otherwise, “%”, used to indicate concentrations of particular substances, stands for (wt./wt.) % for solid/solid, (wt./vol.) % for solid/liquid, and (vol./vol.) % for liquid/liquid throughout the specification.

[0116] Experimental Methods

[0117] Mice and Cell Lines

[0118] Female BALB/c and NOD/SCID mice at 6 to 8 weeks of age were purchased from Central Lab. Animal, Inc. (Seoul, Korea). All mice were handled and maintained under the protocol approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2014-175). All animal procedures were performed in accordance with the recommendations for the proper use and care of laboratory animals.

[0119] A375, CaSki, 526Mel, MDA-MB-231, and HCT116 cells were obtained commercially from the American Type Culture Collection (ATCC, Manassas, Va., USA). All cell lines were purchased between 2010 and 2014 and tested for mycoplasma using a Mycoplasma Detection Kit (Thermo Fisher Scientific, San Jose, Calif., USA). The identities of the cell lines were confirmed by short tandem repeat (STR) profiling by IDEXX Laboratories, Inc., and used within 6 months for testing.

[0120] To generate the A375/TCTP cells, pMSCV-TCTP plasm ids were first transfected along with viral packaging plasmid (VSVG and Gag-pol) into HEK293FT cells. After three days, the viral supernatant was filtered through a 0.45 μm filter and introduced into A375 cells. Then, the infected cells were selected with 1 μg/ml puromycin. For the generation of the A375/P3 tumor line, 1×10.sup.6 A375 cells were inoculated subcutaneously into NOD/SCID mice. After the initial tumor challenge, 2×10.sup.6 NY-ESO1-specific CD8+ T cells and 3000 U of IL-2 (Novartis, Basel, Switzerland) were injected intravenously. After T cell adoptive transfer, the explanted tumor was expanded in vitro. This escape variant cell line was designated A375/P1 and injected into a new group of mice and selected by adoptive T cell transfer again. This treatment regimen was repeated for three rounds. All cells were grown at 37° C. in a 5% CO.sub.2 humidified incubator chamber.

[0121] Chemical Reagents

[0122] The following chemical reagents were used in this study: BI2536 and cisplatin (Selleckchem, Houston, Tex., USA). Dihydroartemisinin, sertraline hydrochloride, and rapamycin (Sigma-Aldrich, USA), and thioridazine (Tocris, UK).

[0123] DNA Constructs

[0124] DNA fragments of the TCTP gene were generated with a PCR-based strategy from genomic DNA extracted from A549 cells using primers (5′-GGATCCATGATTATCTACCGGGAC-3′ and 5′-CTCCAGTTAACATTTTTCCATTTCT-3′) for the BamHI and XhoI sites. The BamHI and XhoI restriction fragments of the PCR product were subcloned into a pGEM-T vector (Promega, USA).

[0125] Site-Directed Mutagenesis

[0126] To generate mutations in the TCTP phosphorylation sites, the QuikChange Site-directed Mutagenesis Kit (Stratagene, San Diego, Calif., USA) was used according to the manufacturer's instructions. In detail, the following primers were used (Table 1).

TABLE-US-00001 TABLE 1 SEQ Sequence ID No. Primer (5′ to 3′) NO: 1 TCTP S46D GGTAACATTG 1 forward ATGACGACCT CATTGGTGGA AATGCCTCCG C 2 TCTP S46D GCGGAGGCAT 2 reverse TTCCACCAAT GqAGGTCGTC ATCAATGTTA CC 3 TCTP S46A CGAGGGCGAA 3 forward GGTACCGAAG CAACAGTAAT CACTGGTGTC G 4 TCTP S46A CGACACCAGT 4 reverse GATTACTGTT GCTTCGGTAC CTTCGCCCTC G

[0127] The PCR thermal cycling conditions were 95° C. for 5 minutes; 18 cycles of 95° C. for 1 minute, and 64° C. for 1 minute, and 68° C. for 15 minutes. The PCR products were digested with Dpn I at 37° C. for 1 hour and transformed into XL10-Gold ultracompetent bacterial cells. Mutations were confirmed through DNA sequencing.

[0128] Real-Time Quantitative RT-PCR

[0129] Total RNA from the cells was purified using a RNeasy Micro kit (Qiagen, Valencia, Calif., USA) and cDNA was synthesized by reverse transcriptase (RT) using an iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif., USA) according to the manufacturer's recommended protocol. Real-time PCR was performed using iQ SYBR Green Super mix (Bio-Rad) with the specific primers on a CFX96 real-time PCR detection system. All experiments were performed in triplicate and the quantification cycle (Cq) values were measured using Bio-Rad CFX 96 Manager 3.0 software.

[0130] Predesigned QPCR primers were purchased from Bioneer (South Korea).

TABLE-US-00002 TABLE 2 Sequence SEQ No. Primer (5′ to 3′) ID NO: 1 TPT1 ATGACGAGCT 5 forward GTTCTCCGAC 2 TPT1 AACACCGGTG 6 reverse ACTACTGTGC

[0131] Relative quantifications of the mRNA levels were performed using the comparative Ct method with beta-actin as the reference gene. Fold-change was calculated relative to the expression level of mRNA in the control cells.

[0132] siRNAs Constructs

[0133] Synthetic small interfering RNAs siGFP, siTPT1, and siEGFR were purchased from Bioneer (South Korea), and had the following sequences.

TABLE-US-00003 TABLE 2 Sequence SEQ siRNA Sequence ID No. name (5′ to 3′) NO: 1 GFP sense GCAUCAAGGUGAACUUCAA 7 2 GFP antisense UUGAAGUUCACCUUGAUGC 8 3 mouse TPT1 #1 GAAAUCACUCAAAGGCAAA 9 sense 4 mouse TPT1 #1 UUUGCCUUUGAGUGAUUUC 10 antisense 5 mouse TPT1 #2 CUGUUCUCCGACAUCUACA 11 sense 6 mouse TPT1 #2 UGUAGAUGUCGGAGAACAG 12 antisense 7 mouse TPT1 #3 AGCACAUCCUUGCUAAUUU 13 sense TT 8 mouse TPT1 #3 AAAUUAGCAAGGAUGUGCU 14 antisense TA 9 human TPT1 GCAUGGUUGCUCUAUUGGA 15 sense 10 human TPT1 UCCAAUAGAGCAACCAUGC 16 antisense 11 human EGFR AGGAAUUAAGAGAAGCAAC 17 sense AU 12 human EGFR AUGUUGCUUCUCUUAAUUC 18 reverse CU 13 mouse MCL-1 GGGCAGGAUUGUGACUCUU 19 sense AUUUCU 14 mouse MCL-1 AGAAAUAAGAGUCACAAUC 20 antisense CUGCCC

[0134] siRNA was delivered into 6-well plates at a dose of 200 pmol/well using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA) in vitro. siRNA was delivered into mice after formulation with chitosan nanoparticles. Briefly, siRNA (1 μg/ul) and tripolyphosphate (0.25% w/v) were combined in RGD-chitosan solution, and the mixture was incubated at 4° C. for 40 minutes. siRNA-loaded nanoparticles were purified by centrifugation and injected into the tail veins of tumor-bearing mice.

[0135] Granzyme B Apoptosis Assays

[0136] Granzyme B (Enzo Life Sciences, NY, USA) was delivered into cells by the BioPORTER QuikEasy Protein Delivery Kit (Sigma-Aldrich, St. Louis, Mo., USA). Tumor cells (5×10.sup.4) were plated in 12-well plates and cultured overnight at 37° C. The cells were washed and 200 ng of granzyme B with BioPORTER in Opti-MEM was added to each well. After incubation for 4 hours, the frequency of apoptotic cells was determined by staining with anti-active caspase-3 antibody and analyzed by flow cytometry.

[0137] In Vitro CTL Assays

[0138] The tumor cells were harvested by trypsinization and washed once with DMEN (Thermo Fisher, USA) containing 0.1% fetal bovine (FBS), resuspended, and labeled in 0.1% DMEN with 10 μM CFSE for 10 minutes in a 37° C. incubator with 5% CO.sub.2. Then, the CFSE-labeled (MCF-7, HCT116, CaSki, MDA-MB-231) tumor cells were resuspended in 10 μM MART-1 peptide containing 1 ml of DMEM. In the case of A375 and 526Mel, the peptide-pulsing process was not needed. After peptide-pulsing for 1 hour, the cells were incubated for 4 hours with MART-1- or the NY-ESO1-specific CD8+ T cell lines at an E/T ratio of 1:1. The frequency of apoptotic cells was analyzed by staining with anti-active caspase-3 antibody and performing flow cytometry. All analysis was performed using a Becton Dickinson FACSverse (BD Bioscience, USA).

[0139] In Vitro Transwell-Based T Cell Chemotaxis Assays

[0140] T cells were applied at 1×10.sup.5 cells/well to the upper wells of 3.0 μm 24-well cell culture inserts (Corning Lowell, Mass., USA). The wells were filled with tumor cell-derived conditioned media (CM). After 4 hours of incubation at 37° C., the migrated T cells were collected from the bottom wells and counted by flow cytometry.

[0141] Cell Viability Assays

[0142] CT26 tumor cells were treated with indicated concentrations of cisplatin, dihydroartemisinin, rapamycin, sertraline, and thioridazine for 24 hours. Cell viability was measured by the trypan blue exclusion assay, and the concentrations causing a 50% decrease in cell viability (IC.sub.50 values) were determined.

[0143] Gene Set Used for Signatures

[0144] Because KEGG pathways often include large numbers of genes with only loosely related functions, the present inventors constructed two core, refined gene sets. Specifically: [0145] CD8+ T-effector signature genes [0146] anti-apoptosis signature genes, the present inventors used the genes within the negative regulation of apoptotic processes.

[0147] Gene Set Used for Signatures

[0148] For gene expression analysis, the expression of each gene in a signature was first z-score-transformed. Then, a principal component analysis was performed for expression values of T cell signature genes (CD8A, CD8B, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, TBX21) and anti-apoptotic signature genes (IL1RAP, IRAK2, IRAK3, PPP3CA, PRKAR2B, CHP1, CHP2, TNFRSF10B, IKBKG, CFLAR, PIK3R1, FAS, XIAP, CYCS, BCL2), and PC1 (principal component 1) was extracted to serve as a gene signature score.

[0149] In Vivo Tumor Treatment Experiments

[0150] To characterize the in vivo resistance to anti-PD-L1 conferred by TCTP, BALB/C mice were inoculated subcutaneously with 1×10.sup.5 CT26 tumor cells per mouse. Seven days following tumor challenge, siGFP- or siTPT1-loaded chitosan nanoparticles (5 μg/animal) was administered via intravenous injection for a day before anti-PD-L1 (BioXcell, NH, USA) (200 μg/m ice).

[0151] In addition, to characterize the in vivo resistance to CTL killing conferred by TCTP, NOD/SCID mice were inoculated subcutaneously with 1×10.sup.6 A375 tumor cells per mouse. Seven days following tumor challenge, siGFP- or siTPT1-loaded chitosan nanoparticles (5 μg/animal) was administered via intravenous injection for a day before adoptive transfer with NY-ESO1-specific CTLs. This treatment protocol was repeated for 3 cycles. The mice were monitored for tumor burden and survival for 26 and 76 days after the challenges, respectively.

[0152] Statistics

[0153] All data shown are representative of at least three separate experiments. Comparisons between individual experimental data points were made using the 2-tailed Student's t test. All p-values of <0.05 were considered statistically significant.

Example 1: Correlation of TCTP with Poor Response to Anti-PD-L1 Therapy

[0154] To determine the clinical relevance of TCTP in response to outcomes of ICB (immune checkpoint blocker) therapy, the present inventors used the transcriptome data from metastatic urothelial cancer (mUC) patients classified as responders (R) or non-responders (NR) to anti-PD-L1 therapy. From the comparative transcriptome analysis of the differentially expressed genes (DEGs) in the two patient groups, it was found that the expression level of TPT1 (encoding TCTP) was significantly higher in the NR compared to the R (FIG. 1A).

[0155] In addition, patients with high TPT1 expression in their tumors (TPT1.sup.high) had poor prognosis compared to patients with low TPT1 expression (TPT1.sup.low) (p<0.02) (FIG. 1B), indicating that the expression of TPT1 was associated with poor response to anti-PD-L1 therapy and survival outcomes of patients.

[0156] Then, the present inventors questioned whether TCTP is responsible for anti-PD-L1 therapy refractory properties. It has been reported that multi-gene signatures are associated with the clinical efficacy of ICB therapy. In this regard, the response outcomes to ICB therapy are predictable by evaluating the functionality of infiltrated CD8+ T cells that can be measured most robustly via the expression of CD8+ T cell signature genes. Interestingly, the present inventors found that TPT1 expression was inversely correlated with T cell infiltration in the patients (FIG. 1C).

[0157] That is, the higher the expression level of the TPT1 gene is, the lower are the survival rate of the patients and the index of T cell counts in tumor.

[0158] One of the major obstacles to successful cancer immunotherapy is the intrinsic resistance of tumor cells to CTL-mediated apoptosis. The intrinsic resistance of tumor cells to CTL-mediated apoptosis is characterized by the gene signature responsible for the anti-apoptosis pathway. Indeed, the anti-apoptosis signature was higher in NR compared to R to anti-PD-L1 therapy and positively correlated with TPT1 expression (FIG. 1D).

[0159] Taken together, these results strongly indicate that TPT1 mRNA expression is highly associated with immune-refractory phenotypes including non-T cell inflamed tumors and resistance to CTL-mediated killing. Therefore, TPT1 could be a biomarker in predicting the response to anti-PD-L1 therapy and clinical outcomes.

Example 2: Requirement of TCTP for Immune-Refractoriness to Anti-PD-L1 Therapy and Reversal of Immune-Refractoriness Through TCTP Gene Expression Suppression

[0160] To explore the mechanisms responsible for the refractory phenotypes of tumors to ICB therapy, the present inventors newly developed an ICB-refractory CT26 P3 tumor model generated from an ICB-susceptible parental cell line, CT26 P0, through three rounds of in vivo selection by anti-PD-L1 therapy (FIG. 2A).

[0161] While anti-PD-L1 antibody treatment successfully retarded tumor growth and prolonged mouse survival in CT26 P0 tumor-bearing mice, there was no remarkable therapeutic effects in CT26 P3 tumor-bearing mice (FIGS. 2A A-B). As evidenced by decreased levels of overall CD8+ T cells, the ratio of CD8+ T cells to T regs and tumor-reactive CD8+ T cells making granzyme B P3 tumors exhibited non-T cell inflamed immune phenotypes, relative to CT26 P0 tumors, (FIG. 2A C-F).

[0162] Notably, anti-PD-L1 therapy significantly induced T cell-inflamed immune phenotypes and apoptotic cell death in the CT26 P0 tumors. However, refractoriness appeared in CT26 P3 tumors, and these refractory phenotypes of CT26 P3 tumors were not reversed by PD-L1 blockade (anti-PD-L1). Thus, these data indicate that the refractory properties to anti-PD-L1 therapy shown in patients were conserved in our ICB-refractory tumor model constructed by the present inventors.

[0163] To characterize the role of TCTP in ICB-refractory properties, the present inventors constructed a CT26 P3 tumor model by performing three rounds of in vivo selection through anti-PD-L1 therapy and measured the levels of TCTP mRNA and protein in different rounds of selection by anti-PD-L1 therapy (P0 to P3) or IgG treatment (N1 to N3), and found a stepwise increase in the levels of TCTP from P0 to P3 (FIG. 2B).

[0164] On the basis of the fact that tumor cells could regulate T cell trafficking, the present inventors performed an in vitro Transwell-based chemotaxis assay and found that CT26 P3 cells had a much lower capacity to recruit the T cells compared to CT26 P0 cells. Furthermore, the present inventors tested T cell chemotaxis by using conditioned media (CM) derived from CT26 P0 or P3 cells and observed that CT26 P3-derived CM markedly reduced T cell chemotaxis compared to CM from P0 cells (FIG. 2D). These results suggest that ICB-refractory CT26 P3 cells could inhibit T cell infiltration by decreasing the production of soluble factors responsible for T cell chemotaxis.

[0165] In order to confirm the direct association of TCTP gene with ICB-refractory phenotypes of CT26 P3 tumor cells, siRNA (siTPT1 #1, #2, #3) was used to silence TPT1 in CT26 P3 cells (FIG. 2C).

[0166] Notably, T cell migration was increased when incubated with CM derived from siTPT1-transfected CT26 P3 cells, compared to siGFP-transfected CT26 P3 cells (FIG. 2D).

[0167] TPT1 knockdown also increased the sensitivity of CT26 P3 cells to apoptosis induced by AH-1-specific CTLs (FIG. 2E).

[0168] On the basis of the in vitro observations, the present inventors reasoned that in vivo silencing of TPT1 could reverse the refractory phenotypes of CT26 P3 tumors to anti-PD-L1 therapy. To test this, the present inventors treated CT26 P3-bearing mice with anti-PD-L1 therapy along with intravenously-administered chitosan nanoparticles (CNPs) carrying siTPT1 or siGFP for the in vivo delivery of siRNAs to tumors (FIG. 2F).

[0169] While anti-PD-L1 therapy alone had no effect on tumor growth, the combined therapy with anti-PD-L1 antibody and siTPT1-loaded CNPs profoundly retarded tumor growth (FIG. 2G), and prolonged the survival of the mice (FIG. 2H).

[0170] Notably, it was found that the number of functional CD8+ T cells infiltrating the tumor and apoptotic tumor cells was significantly increased in the combined treatment compared to either treatment alone (FIGS. 2I-2L). From the data, it was understood that targeting TCTP could improve the therapeutic efficacy of anti-PD-L1 via reversing immune-refractory tumor phenotypes.

Example 3: Promotion of Immune-Refractory Phenotypes by Ectopic Expression of TCTP, thereby Contributing Resistance to Anti-PD-L1 Therapy

[0171] Given the crucial role of the TCTP in ICB-refractory tumors, the present inventors examined whether TCTP expression alone could promote the immune-refractory phenotypes. The overexpression of TPT1 in CT26 P0 cells reduced T cell chemotaxis and increased resistance to CTL-mediated apoptosis (FIGS. 3A, 3B, and 3D).

[0172] In an effort to elucidate a key molecule in the TCTP-mediated inhibition of T cell chemotaxis, the present inventors noted that chemokines play integral roles in T cell trafficking. Notably, the level of CXCL10 was significantly decreased upon TPT1 overexpression (FIG. 3A), and restoring CXCL10 expression in TCTP-ectopically-expression CT26 P0 cells (FIG. 3B) and CT26 TCTP cells (FIG. 3C) reversed T cell chemotaxis, indicating an important role of CXCL10 in the property mediated by TCTP.

[0173] For the TCTP-mediated anti-apoptotic response to CTLs, the present inventors noted an increase in anti-apoptotic protein MCL-1 in CT26 TCTP cells, relative to CT26-no cells (FIG. 3A). The knockdown of MCL-1 restored the susceptibility of CT26 TCTP cells to CTL-mediated apoptosis (FIGS. 3D and 3E).

[0174] Thus, the present inventors concluded that CXCL10 and MCL-1 were key mediators of the TCTP-induced immune-refractory phenotypes. Consistent with the in vitro results, TCTP overexpression conferred a poor response to anti-PD-L1 therapy in vivo (FIGS. 3F-3H). This was accompanied by decreased numbers of tumor-infiltrated CD8+ T cells, and the ratio of CD8+ T cells to T regs and tumor-reactive CD8+ T cells (FIGS. 3I-3K), as well as the apoptotic cell death of tumor cell populations (FIG. 3L).

[0175] Given these results, the present inventors concluded that TCTP itself was sufficient to promote the non-T cell inflamed immune-phenotype and resistance of tumor cells to CTL killing, thereby contributing to anti-PD-L1 therapy resistance.

Example 4: Enrichment of TCTP.SUP.+ Immune-Refractory Cancer Cells by CTL-Mediated Immune Selection

[0176] As tumor antigen-specific CTLs are key effectors in anti-PD-L1 therapy, the present inventors reasoned that increased TCTP expression under anti-PD-L1 therapy is due to immune selection imposed by CTLs. To test this possibility, the present inventors chose the A375 human melanoma cells, the most typical cancer for the clinical application of adoptive CD8+ T cell transfer therapy (ACT), and established an ACT-refractory A375 P3 model from parental A375 P0 cells by selection with NY-ESO1-specific CD8+ T cells in vivo (FIG. 4A).

[0177] While the adoptive transfer of NY-ESO1-specific CTLs significantly retarded tumor growth and prolonged mouse survival in A375 P0 tumor-bearing NOD/SCID mice, there was no remarkable therapeutic effects in the A375 P3 tumor-bearing mice. Relevant to the ICB-refractory tumor model, A375 P3 cells had immune-refractory properties, including a lower capacity to induce T cell migration and resistance to CTL-mediated killing. Indeed, the levels of TCTP mRNA and protein were increased in different rounds of CTL-mediated immune selection (FIG. 4B)

[0178] The CTL-mediated immune selection was likely due to the enrichment of TCTP+ cells during the ACT, as evidenced by an increased proportion of TCTP+ cells from around 8.9% in the A375 P0 cells to around 94.9% in the A375 P3 cells (FIG. 4C).

[0179] Changes in MCL-1 and CXCL10 protein levels were also observed in the A375 P3 cells compared to the P0 cells (FIG. 4D).

[0180] Notably, TCTP knockdown in A375 P3 cells increased T cell migration and sensitized tumor cells to CTL-mediated killing, which was accompanied by profound changes in CXCL10 and MCL-1 (FIGS. 4E-4G).

[0181] To demonstrate the therapeutic value of inhibiting TCTP, the present inventors inoculated A375 P3 cells into NOD/SCID mice and intravenously administered siTPT1- or siGFP-CNPs (FIG. 4H). The infiltrated functional T cells and apoptotic tumor cells were increased in the siTPT1-treated A375 P3 tumors compared to the siGFP-treated A375 P3 tumors (FIGS. 4I and 4J).

[0182] As shown in FIG. 4K, relative to adoptive T cell transfer efficacy, the percentage of apoptotic cells was increased in the tumors of siTPT1-treated mice compared to siGFP-treated mice, indicating that the combined therapeutic effects of targeting TCTP and ACT were affected by both induced CTL-trafficking to the tumor and increased CTL-mediated apoptotic tumor cells. Consistently, combined therapy with siTPT1-CNPs and ACT profoundly retarded tumor growth (FIG. 4L) and prolonged the survival of the mice (FIG. 4M).

[0183] Taken together, the data indicate that the enrichment of TCTP+ immune-refractory tumor cells under CTL-mediated immune selection could cause the tumor phenotypes refractory to ACT therapy. Therefore, therapeutic strategies targeting TCTP could reverse immune-refractory phenotypes, thereby improving the efficacy of ACT and ICB therapy.

Example 5: Activation of EGFR-AKT Signaling by TCTP Through Phospho-Dependent Binding with Na, K ATPase, thereby Promoting Immune-Refractory Properties of Tumor Cells

[0184] The present inventors next attempted to elucidate the signaling pathway by which TCTP conferred the immune-refractory phenotypes. The present inventors found that hyperactivation of the EGFR-AKT pathway was closely linked to the immune escape of tumor cells. In addition, it was revealed that TCTP activates the EGFR signaling pathway via binding to the Na, K ATPase al subunit. Notably, TCTP overexpression increased the phosphorylation of both EGFR and AKT, and reduced T cell chemotaxis and CTL susceptibility, which were accompanied by CXCL10 downregulation and MCL-1 upregulation (FIG. 5A).

[0185] Conversely, the knockdown of EGFR in A375 TCTP cells robustly dampened the levels of phosphorylated AKT and MCL-1, but increased CXCL10 levels (FIG. 5B), demonstrating activation of the EGFR-AKT-MCL-1/CXCL10 axis by TCTP. That is, the overexpression of TCTP promoted the activation of EGFR, AKT, and MCL-1 and inhibited the activation of CXCL10.

[0186] Consistently, loss of EGFR markedly increased T cell chemotaxis and susceptibility to CTLs in A375 TCTP cells (FIGS. 5C and 5D).

[0187] Taken together, the present inventors concluded that the hyperactivation of EGFR signaling by TCTP drove the immune-refractory phenotypes

[0188] By using two mutant forms of TCTP, including a phospho-loss mutant TCTP (TCTP 546A) and a phospho-mimic mutant TCTP (TCTP S46D). it was confirmed that the phosphorylation of TCTP was crucial for EGFR-AKT signaling as well as the immune-refractory properties.

[0189] Similar to TCTP WT, TCTP S46D transfection into A375 P0 cells led to the activation of the EGFR-AKT signaling pathway, and promoted the immune-refractory properties of the tumor cells (FIGS. 5E-5G).

[0190] In contrast, TCTP S46A failed to reflect the biochemical and functional properties of TCTP WT, demonstrating the important role of phosphorylation in these properties mediated by TCTP (FIGS. 5E-5G).

[0191] In addition, the binding of TCTP to the Na, K ATPase al subunit was found to contribute to the activation of the EGFR signaling pathway. TCTP WT or S46D co-precipitated with Na, K ATPase al, whereas TCTP S46A did not (FIG. 5H), indicating a phosphorylation-dependent interaction between TCTP and Na, K ATPase α1.

[0192] Therefore, it was understood from the data that the phospho-dependent binding of TCTP to Na, K ATPase leads to the activation of EGFR-AKT signaling, indicating that blocking TCTP phosphorylation could be an additional combination strategy with T cell-mediated therapy.

Example 6: Effect of TCTP-Targeting Drug on i) TCTP Inhibition, ii) Sensitization of TCTP.SUP.high .Cancer Cells to T Cell-Mediated Killing, and iii) Increase in T Cell Chemotaxis Capacity of Tumor Cells

[0193] As a result of having explored that targeting TCTP could be a potential therapeutic strategy to overcome immunotherapy refractoriness, the present inventors aimed to screen clinically-actionable drugs that could target TCTP to reverse the immune-refractory phenotypes of TCTP.sup.high tumor cells. It has been suggested that a number of drugs such as dihydroartemisinin (DHA), rapamycin, sertraline, and thioridazine had an inhibitory effect on TCTP function. Indeed, while CT26 TCTP cells were refractory to cisplatin as reported previously, these cells were more susceptible to TCTP-targeting agents, especially to DHA, a clinically-available drug to treat malaria (CT26 No IC.sub.50=407.6 μM, CT26 TCTP IC.sub.50=22.67 μM, about 20-fold) (FIG. 6A).

[0194] To further investigate the effect of each drug on sensitizing TCTP.sup.high tumor cells to CTL-mediated killing, CT26 TCTP tumor cells were incubated with CTLs at various tumor cell-T cell ratios after treatment with a sublethal dose of each drug. Compared to PBS or cisplatin, TCTP-targeting drugs (DHA, rapamycin, sertraline, and thioridazine) augmented CTL-mediated cytotoxicity in a synergistic fashion (FIG. 6B).

[0195] To quantify the synergistic effects of treatment of each drug with CTLs, a combination score was calculated based on changes in the percentage of apoptosis in drug-treated tumor cells with or without CTLs (see the following formula 1).

Combination score=(% of active-caspase 3+tumor cells by drug and CTLs)/(% of active-caspase 3+tumor cells by drug).  Formula 1

[0196] From this analysis, the present inventors found that the score of the combination with DHA was remarkably higher than other drugs at all ratios (FIG. 6C).

[0197] Given these data, the present inventors concluded that DHA was the most effective drug to reverse the immune-refractory phenotypes of TCTP.sup.high tumor cells.

[0198] To verify the phenotypic effects of DHA in multiple types of TCTP.sup.high tumor cells, the present inventors further employed previously established ACT-refractory MDA-MB-231 P3 cells and human cancer cells 526Mel and HCT116 which expressed TCTP at high level. Consistently, the knockdown of TCTP robustly dampened the EGFR-AKT-MCL-1/CXCL10 pathway across all tested cells (FIG. 6D). Notably, DHA treatment resulted in the identical effects on the level of these molecules compared to treatment with siTPT1 (FIG. 6D). Importantly, both siTPT1- and DHA-treated tumor cells were more susceptible to CTL-mediated apoptosis, and they also had increased T cell chemotaxis capacity compared to siGFP- or PBS-treated control cells, respectively (FIGS. 6, E and F).

[0199] These results demonstrated that the biochemical and functional properties of the TCTP axis were conserved across multiple types of cancer cells and that impeding TCTP signaling with DHA is a widely applicable strategy for controlling immune-refractory TCTP.sup.high cancer cells.

Example 7: Targeting TCTP by Using DHA Reverses Resistance to i) ACT Therapy and ii) Anti-PD-L1 Therapy in Preclinical Mode

[0200] Given the observations in vitro, the present inventors reasoned that the in vivo administration of DHA should reverse resistance of TCTP.sup.high tumor cells to T cell-mediated therapy.

[0201] To test this possibility, ACT-refractory A375 P3 tumor-bearing NOD/SCID mice were treated cognate NY-ESO1-specific CTLs with or without DHA (FIG. 7A). While CTLs alone had no effect on tumor growth, dual therapy with CTLs and DHA retarded tumor growth (FIG. 7B), and prolonged survival of the mice compared to the other groups (FIG. 7C).

[0202] The proportion of NY-ESO1-specific CTLs in the tumors was increased in the tumors of DHA-treated mice compared to those in PBS-treated mice (FIG. 7D), and the overall cytotoxic effects of these CTLs were greater after treatment with DHA relative to the PBS control, as indicated by the percentage of apoptotic cells in the tumor populations (FIGS. 7E and 7F).

[0203] Next, the present inventors expanded the preclinical therapeutic value of DHA in ICB therapy. To do this, ICB-refractory CT26 P3 tumor-bearing mice were administered anti-PD-L1 antibody alone or combined with DHA (FIG. 7G).

[0204] Compared to treatment with anti-PD-L1 or DHA alone, combined therapy with anti-PD-L1 and DHA showed a remarkable therapeutic effect in CT26 P3 tumor-bearing mice (FIG. 7H).

[0205] Importantly, while 90% of the mice that received both the anti-PD-L1 blockade agent and DHA survived, all of the mice in the other groups died (FIG. 7I).

[0206] In addition, the numbers of infiltrated functional CD8+ T cells and the cytotoxic effect of these CTLs were significantly higher in the co-treated mice group than in the other mice groups (FIGS. 7J-7M).

[0207] Taken together, it was concluded that targeting TCTP by using the actionable drug, DHA, is potential combinational strategy enhancing the response to ACT as well as ICB therapy.

[0208] In addition, the present inventors reasoned that a TCTP neutralizing antibody targeting TCTP as well as DHA could reverse anticancer immune resistance.

[0209] First, to examine whether extracellular secretion of TCTP also increases in ACT-refractory A375 P3, TCTP protein levels in intracellular and extracellular supernatants of A375 P0 and A375 P3 were analyzed. As a result, the extracellular secretion of TCTP was found to also increase in A375 P3 resistant cancer (FIG. 8A).

[0210] Furthermore, in order to examine the effect of an anti-TCTP neutralizing antibody targeting TCTP on anticancer immune resistance, investigation was made of the effect of treatment with CTL and anti-TCTP neutralizing antibody on apoptosis percentage of tumor cells and the TCTP-AKT-MCL-1, NANOG pathway. As a result, when a TCTP neutralizing antibody was used to target TCTP, it was found that phenotypes to anticancer immune resistance was decreased (FIG. 8B) and the previously reported immune resistance and the expression of the cross resistance (cisplatin resistance) and multiple malignance (cancer metastasis and cancer stemness) regulator NANOG as well as the AKT signaling pathway were reduced (FIG. 8c).

[0211] From the result, it was understood that the TCTP secreted outside tumor cells play a crucial role in the therapeutic resistance and multiple malignancy phenotypes of resistant cancer and the neutralization of TCTP through anti-TCTP neutralizing antibody could reverse the phenotypes of resistant cancer.

[0212] In addition, the present inventors revealed in samples of patients with various carcinomas that there is a significant correlation between immune resistance and the expression of NANOG, a factor regulating cross resistance (cisplatin resistance) and multiple malignancy (cancer metastasis and cancer stemness) depending on the expression level (TCTP.sup.high/TCTP.sup.low) of TCTP.

[0213] The present inventors also found that in TC-1 LP3 constructed as an immune checkpoint antibody therapy-refractory orthostatic lung cancer model, intracellular and extracellular TCTP secretion remarkably increases (FIG. 10) and when TCTP was neutralized through an anti-TCTP neutralizing antibody, the previously reported immune resistance and cross resistance (cisplatin resistance) was decreased and multiple malignancy (cancer metastasis and cancer stemness) was also reduced.