Combination therapy with PD-1 signaling inhibitors

Abstract

Provided is a novel combination therapy with PD-1 signal inhibition therapy. A pharmaceutical composition of the present invention contains a substance that can enhance a T cell receptor (TCR) signal, and is administered before, after, or simultaneously with administration of a PD-1 signal inhibitor. An enhancer of PD-1 signal inhibitory activity of the present invention contains a CD45 inhibitor and / or cells. A TCR signal enhancer of the present invention contains a CD45 inhibitor and / or cells.

Description

[Technical Field]

[0001] Recent clinical trial results have shown that PD-1 signaling inhibitor therapy is more effective than conventional treatments for many types of cancer (1-4) (Non-Patent Literature 1-4). The response rate to anti-PD-1 antibody therapy in terminally ill cancer patients has dramatically improved to 20-30% compared to conventional anticancer drugs. However, the problem remains that about half of patients show no response (5, 6) (Non-Patent Literature 5, 6). Therefore, there is an urgent need to develop combination therapies with PD-1 signaling inhibitor therapy that are effective for these patients. [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] SL Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012). [Non-Patent Document 2] A. Ribas et al., Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma. JAMA 315, 1600-1609 (2016). [Non-Patent Document 3] M. Reck et al., Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 375, 1823-1833 (2016). [Non-Patent Document 4] PS Chowdhury, K. Chamoto, T. Honjo, Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy. J Intern Med 283, 110-120 (2018). [Non-Patent Document 5] W. Zou, JD Wolchok, L. Chen, PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci Transl Med 8, 328rv324 (2016). [Non-Patent Document 6] AL Shergold, R. Millar, RJB Nibbs, Understanding and overcoming the resistance of cancer to PD-1 / PD-L1 blockade. Pharmacol Res 145, 104258 (2019). [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] The present invention aims to provide a novel combination therapy with PD-1 signaling inhibitor therapy. [Means for solving the problem]

[0004] The inhibitory effect on the PD-1 signaling pathway is primarily induced through T cell activation (PS Chowdhury, K. Chamoto, T. Honjo, Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy. J Intern Med 283, 110-120 (2018).; K. Chamoto et al., Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci USA 114, E761-E770 (2017).). This T cell activation is promoted by the T cell receptor (TCR) signaling cascade. Phosphorylation and dephosphorylation of TCR signaling molecules affect signal complex formation and TCR signal transmission. CD45 is a transmembrane dephosphorylating enzyme that plays a major role in regulating TCR signaling by controlling the phosphorylation levels of lymphocyte protein kinase (Lck) and its downstream factor, tyrosine (J. Rossy, DJ Williamson, K. Gaus, How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism. Front Immunol 3, 167 (2012).) (RJ Brownlie, R. Zamoyska, T cell receptor signaling networks: branched, diversified and bounded. Nat Rev Immunol 13, 257-269 (2013).).In young, naive CD8-positive T cells, high CD45 expression inhibits TCR signaling via dephosphorylation of ZAP-70 and PLCg (JH Cho et al., CD45-mediated control of TCR tuning in naive and memory CD8(+) T cells. Nat Commun 7, 13373 (2016)). Therefore, it is thought that enhancing TCR signaling by suppressing CD45 further reinforces T cell activation enhanced by PD-1 signaling inhibition, thereby enhancing the antitumor effect (Figure 17).

[0005] The inventors have found that combining PD-1 signaling inhibition with an inhibitor of the dephosphorylating enzyme CD45 synergistically enhances the antitumor effect. This tumor-suppressing effect was particularly strong in aging mice and resistant cancers that exhibited resistance to PD-1 signaling inhibition. Based on these results, it is thought that CD45 inhibitors, when used in combination with PD-1 signaling inhibitors, support antitumor immunity, maintain a synergistic effect in suppressing cancer proliferation, and are effective even against cancers that are unresponsive to PD-1 signaling inhibition alone. Similar effects were observed in cell transplantation (xenocellular and allogeneic cells). Enhancement of TCR signaling by cell transplantation was also confirmed.

[0006] The gist of this invention is as follows: (1) A pharmaceutical composition comprising a substance capable of enhancing T cell receptor (TCR) signaling, to be administered before, after, or concurrently with the administration of a PD-1 signaling inhibitor. (2) The pharmaceutical composition according to (1), wherein the substance that can enhance TCR signaling is a CD45 inhibitor and / or cells. (3) The pharmaceutical composition according to (2), wherein the CD45 inhibitor is at least one compound selected from the group consisting of 2-(4-Acetylanilino)-3-chloronaphthoquinone, N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide and analogs thereof. (4) The pharmaceutical composition according to (2), wherein the cells are xenogeneic cells, allogeneic cells or a combination thereof. (5) The pharmaceutical composition according to any one of (1) to (4), wherein the PD-1 signal inhibitor is an antibody. (6) The pharmaceutical composition according to (5), wherein the antibody is at least one antibody selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody and an anti-PD-L2 antibody. (7) The pharmaceutical composition according to any one of (1) to (6), which is used as an anticancer agent, an infectious disease therapeutic agent or a combination thereof. (8) A PD-1 signal inhibitory activity enhancer comprising a CD45 inhibitor and / or cells. (9) A TCR signal enhancer comprising a CD45 inhibitor and / or cells. (10) A method for preventing and / or treating cancer, an infectious disease or a combination thereof, which comprises administering to a subject an effective amount of a substance capable of enhancing a T cell receptor (TCR) signal at any time before, after or simultaneously with the administration of a PD-1 signal inhibitor. (11) A method for enhancing PD-1 signal inhibitory activity, which comprises administering to a subject an effective amount of a substance capable of enhancing a T cell receptor (TCR) signal at any time before, after or simultaneously with the administration of a PD-1 signal inhibitor. (12) A method for enhancing a TCR signal, which comprises administering to a subject an effective amount of a CD45 inhibitor and / or cells. (13) A substance capable of enhancing a T cell receptor (TCR) signal for use in preventing and / or treating cancer, an infectious disease or a combination thereof, wherein the substance is administered at any time before, after or simultaneously with the administration of a PD-1 signal inhibitor.

Advantages of the Invention

[0007] By combining a substance capable of enhancing a TCR signal with a PD-1 signal inhibitor, the antitumor effect is synergistically improved. This specification includes the content described in the specification and / or drawings of the Japanese Patent Application No. 2021-31041, which forms the basis of the priority of this application. [Brief explanation of the drawing]

[0008] [Figure 1] Loss of antitumor activity and P4 cell induction in aged PD-1 KO mice. (A and B) MC38 cells were inoculated intracutaneously into juvenile and aged PD-1 KO mice. (A) MC38 tumor size in juvenile (3-4 months old) and aged (15 months old) PD-1 KO mice. (B) Kaplan-Meier plot of survival rate of MC38 tumor-bearing PD-1 KO mice. (C and D) Analysis of CD8-positive T cell subsets in juvenile (C, 2-3 months old) or aged (D, 15-21 months old) PD-1 KO mice with or without MC38 cell inoculation. Peripheral lymph node and regional lymph node cells stained on Day 9. Representative plots showing CD44 and CD62L expression in CD3-positive and CD8-positive cells, and percentages of CD8-positive cell subsets; CD44low / CD62Lhigh (naive; P1), CD44high / CD62Lhigh (central memory; P2), CD44high / CD62Llow (effector / memory; P3), CD44low / CD62Llow (P4). P-values ​​were calculated using the log-rank test or Student's T-test. *p < 0.05; **p < 0.01; ns, no significant difference. Data show the mean ± standard error of the mice (n = 8-10 for A & B; n = 5-6 for C & D). [Figure 2]Antitumor activity of P4 cells via differentiation into P3 cells. (A and B) To obtain OT-I P3 and P4 subsets, MC38-OVA cells were injected into juvenile OT-1 mice via the tail vein, and each subset was isolated from splenocytes. Five days after intracutaneous transplantation of MC38-OVA cells, P3 or P4 subset cells were adopted into CD8 KO mice. (A) Tumor volume in MC38-OVA tumor-bearing CD8 KO mice with or without P3 or P4 cell introduction. (B) FACS analysis of transplanted CD8-positive T cells in peripheral blood on Day 11. (C) Scheme for isolation of CD8-positive T cell subsets from splenocytes of juvenile PD-1 KO mice. (D to G) FACS analysis of P4(D), P1(E), or P2(F) cells isolated from young (2 to 3 months old) or aged (16 to 18 months old) PD-1 KO mice, stimulated with anti-CD3 / CD28 antibody and IL-2, and cultured for 2 or 3 days. Representative plots showing CD44 and CD62L expression and the percentage of CD8-positive T cells in the cultured cells. Data are shown as the mean ± standard error of the mice (n = 3-5). **p < 0.01 (Tukey's test after evaluation by ANOVA). [Figure 3]Increased expression of 1C metabolic genes in P4 cells. (A to E) Microarray analysis of P1, P2, P3, and P4 cells isolated from young PD-1 KO mice (1 to 3 months old; mixed of 9 mice). (A) Hierarchical clustering heatmap of all genes. (B) Scatter plot showing the normalized log intensity of each probe. The dotted line shows the difference in log 2-fold. Genes that have been shown to be involved in the activation and differentiation of CD8-positive cells are listed. (C) Top 10 gene ontology (GO) involving upregulated genes in P4 cells. GO terms involved in 1C metabolism are shown in red. (D) Schematic diagram of the 1C metabolic pathway. THF, tetrahydrofolate. (E) Heatmap showing the expression of 1C metabolic genes in a CD8-positive T cell subset. (F) mRNA expression of 1C metabolic genes in a CD8-positive T cell subset derived from splenocytes of young PD-1 KO mice. (G) Microarray analysis of wild-type or PD-1 KO CD8-positive T cells in peripheral or regional lymph nodes of juvenile (2 months old, 3 mouse mixture) or aged (17 months old, 6 mouse mixture) mice with or without MC38 tumor (day 9). The heatmap shows the expression of 1C metabolic-related genes in the cells shown in the figure. (H and I) Oxygen consumption rate (OCR) of wild-type or PD-1 KO CD8-positive T cells derived from regional lymph nodes of juvenile (2 to 3 months old) or aged (15 to 19 months old) mice was measured using a Seahorse XFe96 analyzer. OCR trace (H), basal respiration and respiratory reserve capacity were calculated from OCR values ​​(I). *p < 0.05; **p < 0.01 (Student's T-test). Data are shown mean ± standard error (n = 4). [Figure 4]P4 cell induction in aging CD8-positive T cells is attenuated by TCR signaling suppression via high CD45RB expression. (A and B) Analysis of CD8-positive T cell subsets in regional lymph nodes of juvenile (2-3 months old) (A) or aged (15-19 months old) (B) OT-1 mice with or without MC38-OVA cell injection via tail vein. Typical figure showing CD44 and CD62L expression on CD8-positive T cells and the percentage of each CD8-positive T cell subset in mice shown in the figure 5 days after MC38-OVA transplantation. (C) Percentage of pZAP70-positive CD8-positive T cells in peripheral lymph nodes from juvenile or aged OT-1 mice with or without MC38-OVA transplantation. (D) Representative plots of CD45RB expression levels and fluorescence intensity (MFI) in total CD8-positive T cells or their respective subsets from juvenile (2-3 months old) or aged (14-17 months old) OT-1 mice. (E) CD8-positive T cells were isolated from the peripheral lymph nodes of young or aged OT-1 mice and cultured for 1 day with or without MMC-treated MC38-OVA cells. During the last hour of culture, a solvent or CD45 inhibitor (0.05 μM) was added. Data are shown as mean ± standard error (n = 3-6). *p < 0.05; **p < 0.01; ns, no significant difference (Tukey's test or Student's T-test after evaluation by ANOVA). [Figure 5]Restoration of P4 cells and antitumor activity in aging mice by xenocellular transplantation. (A) Schematic diagram of the experimental schedule. (B to D) Young (3 to 4 months old) and aged (16 to 18 months old) PD-1 KO mice were transplanted with Daudi cells via the tail vein. Ten days after transplantation (day 0), peripheral lymph node cells from these mice were analyzed by FACS and real-time PCR. (B) Percentage of P1 to P4 subsets in CD8-positive T cells from peripheral lymph nodes. (C) Expression levels of 1C metabolism-related genes in CD8-positive T cells. (D) CD45RB expression levels in P1 cells derived from peripheral lymph nodes. (E to I) Young (3 to 4 months old) and aged (17 to 18 months old) PD-1 KO mice were transplanted with Daudi cells via the tail vein (day -10). Ten days after transplantation (day 0), these mice were transplanted intracutaneously with MC38 (E to G) or MC38-OVA (H and I) cells and used in the following experiments: MC38 tumor size (E) and survival rate (F); (G) Percentage of pZAP70-positive cells among CD8-positive T cells derived from regional lymph nodes on Day 6; (H and I) Percentage of OVA tetramer-positive CD8-positive T cells in regional lymph nodes or tumor tissue on Day 6. Data are shown mean ± standard error (n = 9-10 for B; n = 4-6 for CI). *p < 0.05; **p < 0.01 (Tukey's test, log-rank test or Student's T-test after evaluation by ANOVA). [Figure 6]Loss of antitumor activity and P4 cell induction in aged wild-type mice. (A and B) MC38 cells were inoculated intracutaneously into young (2-month-old) and aged (14-month-old) C57BL / 6 wild-type mice. These mice were then administered anti-PD-L1 antibodies on days 5, 11, and 17. (A) MC38 tumor size in young and aged wild-type mice. (B) Kaplan-Meier plot of survival rates in MC38 tumor-bearing wild-type mice. P-values ​​were calculated by log-rank test. (C and D) Analysis of CD8-positive T cell subsets in young (C, 2-month-old) or aged (D, 15-month-old) wild-type mice with or without MC38 cell inoculation. Stained peripheral and regional lymph node cells on day 9. Representative plots showing CD44 and CD62L expression in CD3-positive and CD8-positive T cells, and percentages of CD8-positive T cell subsets; CD44low / CD62Lhigh (naive; P1), CD44high / CD62Lhigh (central memory; P2), CD44high / CD62Llow (effector / memory; P3), CD44low / CD62Llow (P4). *p < 0.05; **p < 0.01; ns, no significant difference (Student's T-test). Data show the mean ± standard error of the mice (n = 5 for A, C & D; n = 9-11 for B). [Figure 7] Similar binding affinity of TCRs to OVA antigens between young and aged OT-I CD8-positive cells. Representative plots (A) and graphs (B) of mean fluorescence intensity (MFI) of OVA-specific MHC tetramers in young or aged OT-1 mice. Data show the mean ± standard error of the mice (n = 4-6). ns, no significant difference (Student's T-test). [Figure 8]High CD45RB expression in CD8-positive T cells from aged wild-type or PD-1 KO mice. CD45RB expression levels in CD8-positive T cells derived from peripheral lymph nodes of wild-type (A) or PD-1 KO (B) mice were measured by FACS analysis using an anti-CD45RB antibody. Data show the mean ± standard error of the mice (n = 3-6). *p < 0.05; **p < 0.01; ns, no significant difference (Student's T-test). [Figure 9] Recovery of age-related unresponsiveness to PD-1 inhibitor therapy in wild-type mice by xenocellular transplantation. Young (1 month old) and aged (17 to 19 months old) C57BL / 6 wild-type mice were transplanted intravenously with Daudi cells. A control group (Ctrl) was provided without cell transplantation. Ten days after transplantation, these mice were transplanted intracutaneously with MC38 cells. Administration of PD-L1 antibody was started on day 5 and administered every 6 days for three doses. (A) Schematic diagram of the experimental schedule. (B and C) MC38 tumor size in young (B) and aged (C) wild-type mice. (D and E) Survival rates of young (D) and aged (E) wild-type mice bearing MC38 tumors. Data show the mean ± standard error of the mice (n = 5-6). *p < 0.05; **p < 0.01; ns, no significant difference (Tukey's test or log-rank test after ANOVA evaluation). [Figure 10] Recovery of age-related mitochondrial dysfunction through xenocellular transplantation. MC38-OVA cells were transplanted into the skin of young (3-4 months old) and aged (17-18 months old) PD-1 KO mice that had been transplanted with PBS(Ctrl) or Daudi cells 10 days prior. Six days after transplantation, the OCR of CD8-positive T cells isolated from the regional lymph nodes of the mice shown in the figure was measured using a Seahorse XFe96 analyzer. OCR trace (A), basal respiration (B), and respiratory reserve capacity were calculated from the OCR values ​​(C). Data are shown as mean ± standard error (n = 3). *p < 0.05; **p < 0.01 (Tukey's test after ANOVA evaluation). [Figure 11]Restoration of PD-1 inhibitor resistance associated with aging by allogeneic cell transplantation. MMC-treated splenocytes derived from C57BL / 6 (Ctrl) or Balb / c (Allo) mice were transplanted by tail vein injection into young (1 month old) and aged (14 to 18 months old) C57BL / 6 wild-type mice. Subsequent experimental conditions were the same as in Figure 9A. MC38 tumor size in young (A) and aged (B) C57BL / 6 wild-type mice. Data are shown as mean ± standard error (n = 3). *p < 0.05; ns, no significant difference (Tukey's test after evaluation by ANOVA). (C and D) Survival rates of young (C) and aged (D) C57BL / 6 wild-type mice bearing MC38 tumors. P-values ​​were calculated by log-rank test (n = 5-6). [Figure 12] Names and structural formulas of CD45 inhibitors. A) 2-(4-Acetylanilino)-3-chloronaphthoquinone, also known as "211". B) N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, also known as "PTP". [Figure 13] The method and procedure of the experiment. [Figure 14] Enhanced PD-1 signaling inhibition effect by CD45 inhibition. Pre-administration: PD-1 knockout (KO) mice were administered a solvent (control) or 211, and MC38 cells (1 × 10⁶) were transplanted 3 days later. Post-administration: 211 was administered every 2 days for a total of 3 doses, starting on Day 5, 5 days after MC38 cell transplantation in PD-1 KO mice. A) Tumor volume increase curve, B) Tumor volume 17 days after MC38 cell transplantation. Data show the mean ± standard error of 5 mice. *p < 0.05; **p < 0.01; ns, no significant difference. [Figure 15]Improved efficacy against age-related PD-1 signaling inhibition resistance by CD45 inhibition. Young (1-2 months old) or aged (14-16 months old) PD-1 KO mice were administered 211, and MC38 cells were transplanted 3 days later. A) Tumor volume increase curve, B) Tumor volume 28 days after MC38 cell transplantation. Data show the mean ± standard error for 6 mice. **p < 0.01. [Figure 16] Improved efficacy against PD-1 signaling inhibition-resistant cancers by CD45 inhibition. A) Experimental method and procedure. Young (1-2 months old) wild-type mice were administered 211 or PTP every two days for a total of three doses, starting on Day 7, seven days after transplantation of LLC cells resistant to immunotherapy. Anti-PD-L1 antibody was administered every six days for a total of three doses, starting on Day 7. B and D) Tumor volume increase curves, C and E) Tumor volume 24 days after MC38 cell transplantation. Data show the mean ± standard error of 5-7 mice. **p < 0.01; ns, no significant difference. [Figure 17] Model diagram of TCR signaling. [Figure 18] The effects of immune checkpoint inhibitors are enhanced by the administration of allogeneic cells. Young (6-week-old) C57BL / 6 mice were intradermally injected with MC38 cancer cells (5 x 10^5 cells). At 9, 16, and 23 days, anti-PD-L1 antibody (clone 1-111A.4) (1.5 mg / kg) was administered intraperitoneally. In addition, Balb / c (allogeneic) spleen cells (5 x 10^5 cells / mouse or 2 x 10^6 cells / mouse) treated with mitomycin C were intravenously administered at (A) 6 days, (B) 9 days, or (C) 9, 16, and 23 days, and the change in tumor size over time was measured. The points in each line graph represent the mean value for 5 to 6 mice, and the error bars represent the standard error. * indicates a p-value of less than 0.05, and ns indicates no statistically significant difference (comparison by Tukey's test after one-way ANOVA). [Modes for carrying out the invention]

[0009] The present invention will be described in detail below.

[0010] The present invention provides a pharmaceutical composition comprising a substance capable of enhancing T cell receptor (TCR) signaling, which is administered before, after, or concurrently with the administration of a PD-1 signaling inhibitor.

[0011] T cell receptors (TCRs) are antigen receptor molecules expressed on the cell membrane of T cells. When an antigen binds to a TCR, a signal is transmitted into the cell via phosphorylation of downstream factors such as Lck and ZAP-70, which triggers T cell activation.

[0012] Enhancement of TCR signaling can be confirmed by evaluating the phosphorylation of ZAP-70 in CD8-positive T cells under antigen stimulation. A higher level of ZAP-70 phosphorylation indicates enhanced TCR signaling. ZAP-70 phosphorylation can be detected by flow cytometry analysis using an anti-p-ZAP-70 antibody.

[0013] By enhancing TCR signaling, the antitumor immune effect of CD8-positive T cells can be enhanced. Therefore, enhancing TCR signaling can boost the antitumor effect of PD-1 inhibitor therapy, potentially producing therapeutic effects even in aging animals and humans that are resistant to PD-1 signaling inhibition, as well as against resistant cancers.

[0014] Examples of substances that can enhance TCR signaling include CD45 inhibitors and / or cells. These substances may also induce P4 cells. P4 cells are defined as CD45 inhibitors. low CD62L low This is a subset of CD8-positive T cells. In mice, three major types of CD8-positive T cells are distinguished using surface markers such as CD44 and CD62L (L-selectin): naive (also called P1; CD44) low CD62L high ) and central memory (P2; CD44 high CD62L high ), Effects / Memory (P3; CD44 high CD62L low)。Remaining CD44 low CD62L low (P4) The CD8-positive T cell subset is a very minor population.

[0015] Examples of CD45 inhibitors include 2-(4-Acetylanilino)-3-chloronaphthoquinone, N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, and their analogs.

[0016] As used herein, "analog" is a concept that includes salts of compounds, derivatives of compounds, prodrugs for active metabolites, active metabolites for prodrugs, their solvates, and the like.

[0017] When a compound has an amino group (including a substituted amino group) or an amide group, examples of salts formed with acids such as hydrochloric acid, mesylic acid (methanesulfonic acid), fumaric acid, phosphoric acid, etc. can be given.

[0018] A derivative of a compound is a compound that has been modified to such an extent that it does not significantly change the structure and properties of the parent compound, such as introduction or substitution of functional groups, oxidation, reduction, replacement of atoms, etc. Examples of derivatives of 2-(4-Acetylanilino)-3-chloronaphthoquinone include those in which the chloro (-Cl) group is substituted with another halogen group (e.g., fluoro (-F) group, bromo (-Br) group, etc.), and those in which the acetyl (CH3CO-) group is substituted with another functional group (e.g., hydroxy group (hydroxyl group), amino (-NH2) group, pivaloyl group, benzoyl group, etc.). Examples of derivatives of N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide include those in which the dimethylpropionyl group is substituted with another alkyl group (e.g., methyl group, ethyl group, propyl group, benzyl group, etc.).

[0019] Examples of prodrugs include compounds in which the amino group of the active compound has been acylated, alkylated, or phosphorylated (for example, compounds in which the amino group of the active compound has been eicosanoylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo-1,3-dioxolenn-4-yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated, or t-butylated), and compounds in which the amide group of the active compound has been alkylated (for example, methylated, ethylated, or propylated).

[0020] Examples of solvates include solvates with solvents such as water, methanol, ethanol, and acetonitrile. The solvent may be a single solvent or a mixture of several solvents.

[0021] Cells capable of enhancing TCR signaling may be xenocells (differentiated cells), allocells (allogeneic cells), or a combination thereof, in humans or animals to which the pharmaceutical composition is administered. In the examples described below, differentiated Daudi cells and allogeneic splenic cells were transplanted into mice. In this specification, xenocells are differentiated cells, and allocells are allogeneic cells with different HLA (Human leukocyte antigen) or MHC (major histocompatibility complex).

[0022] Various cells (xenocells, allocells) and tissues are known to be administered to the body for the prevention or treatment of diseases, and these cells may also be used in the present invention. For example, allogeneic cells such as breast cancer cell lines, chronic myeloid leukemia cell lines, lung cancer cells, non-small cell lung cancer cell lines, melanoma cell lines, monocytes, pancreatic cancer cell lines, prostate cancer cell lines, and renal cancer cell lines have been used to treat various cancers (Human Vaccines & Immunotherapeutics 10:1, 52-63; January 2014). These cells may be irradiated to prevent proliferation, and may be genetically modified to secrete immune-activating factors such as GM-CSF. Clinical trials have also been conducted using heterogeneous cells such as mouse melanoma B16 and Lewis lung cancer (LLC) cells, administered to melanoma patients (Eur J Dermatol 2016 Apr 1;26(2):138-43). Other possible uses include cells used in blood transfusion products (red blood cell preparations, platelet preparations, whole blood preparations) and the preparations themselves, as well as hematopoietic stem cells used in bone marrow transplantation. These cells may be either allocells or xenocells.

[0023] In this specification, "PD-1 signaling" refers to the signal transduction mechanism mediated by PD-1, one example being the signal transduction mechanism in which PD-1, in cooperation with its ligands PD-L1 and PD-L2, suppresses T cell activation. PD-1 (Programmed cell death-1) is a membrane protein expressed on activated T cells and B cells, while its ligands, PD-L1 and PD-L2, are expressed on various cells, including antigen-presenting cells such as monocytes and dendritic cells, and cancer cells. PD-1, PD-L1, and PD-L2 act as inhibitors that suppress T cell activation. Certain cancer cells and virus-infected cells express PD-1 ligands to suppress T cell activation and evade host immune surveillance.

[0024] Examples of PD-1 signaling inhibitors include substances that specifically bind to PD-1, PD-L1, or PD-L2. Such substances may include proteins, polypeptides, oligopeptides, nucleic acids (including natural and artificial nucleic acids), low-molecular-weight organic compounds, inorganic compounds, cell extracts, and extracts from plants, animals, or soil. The substances may be natural or synthetic. Preferred PD-1 signaling inhibitors are antibodies, more preferably anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, etc. The antibodies may be any type that can inhibit PD-1 signaling, and may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, or human-type antibodies. Methods for producing such antibodies are well known. The antibodies may be derived from any organism, such as humans, mice, rats, rabbits, goats, or guinea pigs. Furthermore, in this specification, the term "antibody" includes low-molecular-weight compounds such as Fab, F(ab)'2, ScFv, Diabody, VH, VL, Sc(Fv)2, Bispecific sc(Fv)2, Minibody, scFv-Fc monomer, and scFv-Fc dimer. As PD-1 signaling inhibitors, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), cemiplimab (Regeneron / Sanofi), camrelizumab (Jiangsu Hengrui), sintilimab (Innovent Biologics / Lilly), and toripalimab (Shanghai Junshi Bioscience), which target PD-1, and atezolizumab (Genetech / Roche), durvalumab (AstraZeneca), and avelumab (Merck / Pfizer), which target PD-L1, have been approved in various countries worldwide. In addition, tislelizumab (BeiGene), dostarlimab (AnaptysBio / Tesaro), HLX-04 (Shanghai Henlius Biotech), AK-105 (Akeso Biopharma), and spartalizumab, which target PD-1, have also been approved.(Novartis), BCD-100 (Biocad), MGA-012 (Incyte), genolimzumab (Genor Biopharma), HX-008 (Taizhou Hanzhong BioMedical), BAT-1306 (Bio-Thera Solutions / Sun Yat-sen University (SYSU)), AMP-224 (National Cancer Institute (NCI)), BI-754091 (Boehringer envafolimab that targets PD-L1 (Jiangsu Alphamab Biopharmaceuticals, bintrafusp alfa (Merck KGaA), SL-279252 (Shattuck Labs), FS-118 (F-star), and CA-170 (Aurigene / Curis) are in clinical development and these PD-1 signaling inhibitors may be used.

[0025] The pharmaceutical composition of the present invention can be used as an anticancer agent, an infectious disease treatment agent, or a combination thereof.

[0026] When the pharmaceutical composition of the present invention is administered as an anticancer agent, the target cancers or tumors include leukemia, lymphoma (Hodgkin's disease, non-Hodgkin's lymphoma, etc.), multiple myeloma, brain tumor, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendiceal cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer (laryngeal cancer, etc.), and oral cancer (floor of the mouth). Examples of cancers include gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, paranasal sinus cancer (maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, etc.), thyroid cancer, kidney cancer, lung cancer, osteosarcoma, prostate cancer, testicular cancer, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer (basal cell carcinoma, squamous cell carcinoma, malignant melanoma, actinic keratosis, Bowen's disease, Paget's disease, etc.), and anal cancer.

[0027] When the pharmaceutical composition of the present invention is administered as an infectious disease treatment agent, the target infectious diseases include bacterial infections (various infections caused by streptococci (group A β-hemolytic streptococci, pneumococci, etc.), Staphylococcus aureus (MSSA, MRSA), Staphylococcus epidermidis, enterococci, Listeria, meningococci, Neisseria gonorrhoeae, pathogenic Escherichia coli (O157:H7, etc.), Klebsiella (Klebsiella pneumoniae), Proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia marcescens, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, etc.), tuberculosis, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, and syphilis. , tetanus, leprosy, Legionnaires' disease (veterans disease), leptospirosis, Lyme disease, tularemia, Q fever, etc.), rickettsial infections (typhus, scrub typhus, Japanese spotted fever, etc.), chlamydia infections (trachoma, genital chlamydia infection, psittacosis, etc.), fungal infections (aspergillosis, candidiasis, cryptococcosis, ringworm, histoplasmosis, Pneumocystis pneumonia, etc.), parasitic protozoan infections (amebic dysentery, malaria, toxoplasmosis, leishmaniasis, cryptosporidiosis, etc.), parasitic helminth infections (echinococcosis, schistosomiasis japonica, filariasis, roundworm infection, diphyllobothrium latum infection, etc.), viral infections (influenza, viral hepatitis, viral meningitis, acquired immunodeficiency syndrome) Examples include AIDS, adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, common cold, rabies, cytomegalovirus infection, severe acute respiratory syndrome (SARS), progressive multifocal leukoencephalopathy, chickenpox, herpes zoster, hand-foot-and-mouth disease, dengue fever, erythema infectiosum, infectious mononucleosis, smallpox, rubella, polio, measles, pharyngoconjunctival fever (pool fever), Marburg hemorrhagic fever, hantavirus renal hemorrhagic fever, Lassa fever, mumps, West Nile fever, herpangina, chikungunya fever, etc.

[0028] The pharmaceutical composition of the present invention comprises a substance capable of enhancing TCR signaling and is administered before, after, or concurrently with the administration of a PD-1 signaling inhibitor.

[0029] In the pharmaceutical composition of the present invention, the PD-1 signaling inhibitor and the substance that can enhance TCR signaling may be administered separately at different times or at the same time, or they may be administered together at the same time. When the PD-1 signaling inhibitor and the substance that can enhance TCR signaling are administered together at the same time, it is preferable to provide a combination preparation containing the PD-1 signaling inhibitor and the substance that can enhance TCR signaling.

[0030] The pharmaceutical compositions of the present invention are administered to subjects orally or parenterally, either systemically or topically. Subjects include humans and non-human animals.

[0031] PD-1 signaling inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies) should be dissolved in a buffer such as PBS, physiological saline, or sterile water, filtered and sterilized as needed, and then administered to the subject by injection or intravenous infusion. Additives (e.g., colorants, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonic agents, etc.) may also be added to this solution. Possible routes of administration include intravenous, intramuscular, peritoneal, subcutaneous, and intradermal administration.

[0032] The content of PD-1 signaling inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies) in a formulation varies depending on the type of formulation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. The formulation should be prepared into a unit-dose formulation.

[0033] The dosage, number of doses, and frequency of administration of PD-1 signaling inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies) vary depending on the subject's symptoms, age, weight, administration method, and form of administration. For example, typically, for an adult, it is recommended to administer 0.1 to 100 mg / kg body weight of the active ingredient, preferably 1 to 10 mg / kg body weight, at least once, at a frequency that allows the desired effect to be confirmed.

[0034] Substances that can enhance TCR signaling may be included in formulations containing PD-1 signaling inhibitors, or they may be formulated alone or in combination with excipients or carriers into tablets, capsules, powders, granules, liquids, syrups, aerosols, suppositories, injections, etc. The excipients or carriers may be those commonly used in this field and pharmaceutically acceptable, and their type and composition may be changed as appropriate. For example, water and vegetable oil can be used as liquid carriers. As solid carriers, sugars such as lactose, sucrose, and glucose, starches such as potato starch and corn starch, and cellulose derivatives such as crystalline cellulose can be used. Lubricants such as magnesium stearate, binders such as gelatin and hydroxypropylcellulose, and disintegrants such as carboxymethylcellulose may be added. In addition, antioxidants, colorants, flavoring agents, preservatives, etc. may be added.

[0035] Substances that can enhance TCR signaling can be administered via various routes, including oral, nasal, rectal, percutaneous, subcutaneous, intravenous, and intramuscular.

[0036] The content of the substance that can enhance TCR signaling in a formulation varies depending on the type of formulation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. For example, in the case of a liquid formulation, the content of the substance that can enhance TCR signaling in the formulation is preferably 1 to 100% by weight. In the case of capsules, tablets, granules, and powders, the content of the substance that can enhance TCR signaling in the formulation is usually about 10 to 100% by weight, preferably 50 to 100% by weight, with the remainder being a carrier. The formulation is preferably formulated into a unit dose formulation.

[0037] The dosage, number of doses, and frequency of administration of substances that can enhance TCR signaling vary depending on the type of substance, the subject's symptoms, age, weight, method of administration, and form of administration. For example, if the substance that can enhance TCR signaling is a CD45 inhibitor, it is usually appropriate to administer at least once, at a frequency that allows the desired effect to be confirmed, an amount of 0.005 μg (or μl) to 25,000 mg (or ml) / kg body weight, preferably 0.05 μg (or μl) to 12,500 mg (or ml) / kg body weight per adult, converted to the amount of active ingredient. If the substance that can enhance TCR signaling is cells, it is usually 1 x 10⁶ 6 Individual / person~1 x 10 11 Approximately one per person, preferably 5 x 10 6 Individual / person~2 x 10 10 It is recommended to administer the drug at a dose of approximately one dose per person at least once, at a frequency that allows the desired effect to be confirmed.

[0038] The appropriate ratio of a PD-1 signaling inhibitor (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody) to a substance that can enhance TCR signaling is 1:0.1 to 1:100 (mass ratio) when the substance that can enhance TCR signaling is a CD45 inhibitor, preferably 1:1 to 1:50. When the substance that can enhance TCR signaling is cells, the ratio is 1 x 10⁶ cells per 240 mg of PD-1 signaling inhibitor. 6 ~1 x 10 10 A suitable number is 5 x 10. 6 ~2 x 10 9 It is an individual.

[0039] The present invention also provides a method for preventing and / or treating cancer, infection, or a combination thereof, comprising administering to a subject a pharmaceutically effective amount of a substance capable of enhancing TCR signaling at any time before, after, or concurrently with the administration of a PD-1 signaling inhibitor. Furthermore, the present invention provides the use of a substance capable of enhancing TCR signaling for the treatment of cancer, infection, or a combination thereof, wherein the substance capable of enhancing TCR signaling is administered at any time before, after, or concurrently with the administration of a PD-1 signaling inhibitor. Furthermore, the present invention also provides the use of a substance capable of enhancing TCR signaling for use in a method for treating cancer, infection, or a combination thereof, wherein the substance capable of enhancing TCR signaling is administered at any time before, after, or concurrently with the administration of a PD-1 signaling inhibitor. The present invention also provides a substance capable of enhancing T cell receptor (TCR) signaling for use in preventing and / or treating cancer, infection, or a combination thereof, wherein the substance is administered at any time before, after, or concurrently with the administration of a PD-1 signaling inhibitor.

[0040] Furthermore, the present invention provides a drug that enhances PD-1 signaling inhibitory activity, comprising a substance capable of enhancing TCR signaling. The present invention also provides a method for enhancing PD-1 signaling inhibitory activity, comprising administering to a subject an effective amount of a substance capable of enhancing T cell receptor (TCR) signaling at any time before, after, or concurrently with the administration of a PD-1 signaling inhibitor.

[0041] The drug of the present invention can be used as a combination drug or in combination with a PD-1 signaling inhibitor. The combination and combination formulation of a PD-1 signaling inhibitor with a substance that can enhance TCR signaling has been described above. In addition to its use as a pharmaceutical, the drug of the present invention can also be used as an experimental reagent.

[0042] Furthermore, the present invention provides a TCR signaling enhancer comprising a CD45 inhibitor and / or cells. The present invention also provides a method for enhancing TCR signaling, comprising administering a CD45 inhibitor and / or cells to a subject in an effective amount. CD45 inhibitors and cells are described above. The agents of the present invention can be used not only as pharmaceuticals but also as experimental reagents. By enhancing TCR signaling, effects such as improved antitumor effects of PD-1 signaling inhibitors and enhanced therapeutic effects against infectious diseases may be obtained. [Examples]

[0043] The present invention will be described in more detail below with reference to examples. [Example 1] [Summary] CD8-positive cells play a major role in antitumor responses, directly killing cancer cells. Severe suppression of CD8-positive cell immunity in the elderly is associated with cancer and other age-related diseases. This study investigated the role of T cell function in PD-1 inhibitor resistance associated with aging. We investigated CD44 in the regional lymph nodes of aged tumor-bearing mice. low CD62L lowWe found that the formation of CD8-positive T cells was reduced. Initially, P4 cells were induced from naive CD8-positive T cells by in vitro stimulation, and then differentiated into effector / memory cells. In P4 cells, the expression of genes related to one-carbon (1C) metabolism, which is important for antigen-specific T cell activation and mitochondrial function, was specifically elevated. Consistent with this finding, the expression of 1C metabolism-related genes and mitochondrial respiration were reduced in aged CD8-positive T cells compared to juvenile CD8-positive T cells. In aged mice into which the albumin (OVA)-specific T cell receptor (TCR) gene was introduced, ZAP-70 activation was not induced by transplantation of tumor cells expressing OVA. This suppression of TCR signaling was suggested to be due to increased expression of CD45RB phosphatase in aged CD8-positive cells. Surprisingly, strong stimulation by transplanting non-self cells into aged PD-1-deficient mice reduced CD45RB expression to normal levels, restoring P4 cell induction, one-carbon metabolic enzyme expression in CD8-positive T cells, and antitumor activity. These findings suggest that impaired induction of the P4 subset is involved in age-related PD-1 inhibitor resistance, and that it can be restored by strong TCR stimulation.

[0044] [Significant Statement] Although aging is known to suppress antitumor immunity, the detailed mechanisms were largely unknown. Here, we introduce CD44, a pre-effector-like T cell. low CD62L low We found that resistance to PD-1 inhibitory antitumor therapy in aging mice can be explained by the suppression of the formation of a CD8-positive T cell subset (P4). Increased CD45RB expression in aging naive CD8-positive T cells suppresses TCR signaling, resulting in a decrease in P4 cells, a subset that highly expresses 1C metabolic genes. This decrease in P4 cells and the suppression of antitumor activity were restored by strong immune stimulation with non-self cells. These findings provide useful insights into the mechanisms of suppression of antitumor immunity associated with aging and will serve as a basis for developing therapeutic strategies for elderly cancer patients.

[0045] [Introduction] Aging affects many physiological functions and, as a result, contributes to the development of a variety of diseases. The risk of cancer increases with age due to factors such as the accumulation of mutations in the genome and a decline in immune surveillance against cancer cells (1, 2). Immunotherapy, which reactivates suppressed immune function, has emerged as an effective method for treating cancer. Among various cancer immunotherapies, antibodies that block the PD-1 / PD-L1 pathway have shown very high efficacy in patients with various types of cancer (3-6). However, clinical trials have shown that many cancer patients do not respond to PD-1 inhibitory therapy (7, 8). Such therapies have not shown efficacy in aging mouse models, and a similar trend has been shown in several clinical trials (9-12).

[0046] Immunosenosis results from quantitative and qualitative changes in immune cells. One of the major quantitative changes in T cells is a decrease in the diversity of the TCR repertoire due to thymic regression, which reduces naive cells in the periphery (13-15). In addition, functional impairments such as TCR signaling disorders, suppression of differentiation into effector and memory cells, and decreased cytokine production accumulate in peripheral T cells in the elderly (16, 17). In fact, age-related changes significantly affect the proportion of clearly defined CD8-positive T cell subsets (18-20). In mice, three major CD8-positive T cells are distinguished using the surface markers CD44 and CD62L (L-selectin): naive (also called P1; CD44) low CD62L high ) and central memory (P2; CD44 high CD62L high ), Effects / Memory (P3; CD44 high CD62L low ). CD44 remains in naive mouth low CD62L lowThe (P4)CD8-positive T cell subset is a very minor population. Naive T cells differentiate into effector T cells upon antigen stimulation (14, 21). Some effector cells differentiate into memory cells, which can quickly respond to the same antigen stimulation and differentiate back into effector cells. In aged humans and mice, T cell production from the thymus decreases, so self-antigen-dependent proliferation (homeostatic proliferation) is required to maintain T cells (22, 23). In aged individuals, this homeostatic proliferation gradually promotes the differentiation of naive T cells, and as a result, increases the proportion of differentiated T cells, including effector and memory cells.

[0047] T cell proliferation and differentiation are promoted by the T cell receptor (TCR) signaling cascade. Phosphorylation and dephosphorylation of TCR signaling molecules affect signal complex formation and TCR signal propagation. CD45 is a transmembrane dephosphorylating enzyme that plays a major role in regulating TCR signaling by controlling the tyrosine phosphorylation level of lymphocyte protein kinase (Lck). Lck acts as an activator of downstream TCR signaling factors such as TCRz, z-chain-associated protein (ZAP)-70, and the linker of activated T cells (LAT) (24, 25). In young, naive CD8-positive T cells, high CD45 expression inhibits TCR signaling via dephosphorylation of ZAP-70 and PLCg (26). However, despite the known effects of aging on TCR signaling, the molecular mechanisms remain largely unknown (27, 28).

[0048] In activated and proliferating cells, 1C metabolism is generally enhanced, supporting survival and differentiation through purine and thymidine biosynthesis, amino acid production, epigenetic regulation, and redox regulation (29, 30). Recent reports have shown that 1C metabolism is one of the most readily induced metabolic pathways during the initial activation of CD4-positive or CD8-positive T cells (31, 32). After T cell activation, enhanced 1C metabolism promotes serine processing necessary for the biosynthesis of new nucleic acids derived from the 1C unit, which is required for the proliferation, differentiation, and effector function of antigen-specific T cells. This 1C metabolic pathway is also involved in energy production through the regulation of mitochondrial protein synthesis by mitochondrial tRNA methylation (33, 34). Mitochondrial function and 1C metabolism are lower in aging CD4-positive T cells than in younger CD4-positive T cells (35), suggesting that T cell aging may be involved in the inactivation of 1C metabolism.

[0049] To elucidate the mechanisms of age-related attenuation of antitumor effects by PD-1 inhibition, we compared young and aged PD-1 knockout (KO) or wild-type (WT) CD8-positive T cells with and without tumor transplantation. We found that resistance to PD-1 deficiency or inhibitory antitumor therapy in aged mice is due to the inhibition of P4 subset induction, and revealed that P4 cells are intermediate between naive and effector cells and strongly express 1C metabolism-related genes. This suppression in aged mice is due to suppression of TCR signaling via increased CD45RB expression and is reversible with strong immune stimulation. These findings will provide new clues to understanding the mechanisms of age-dependent changes in CD8-positive T cell subsets related to metabolic changes and antitumor activity.

[0050] [result] CD44 in aging mice low CD62L low (P4) Suppression of CD8 positive T cell induction Young PD-1 KO mice strongly suppressed tumor growth, while older PD-1 KO mice did not exhibit tumor rejection (Figure 1A&B). We also observed that older wild-type mice were resistant to PD-1 inhibitory tumor treatment (Figure 6A&B). Comparative analysis of CD8-positive T cell subsets in peripheral lymph nodes and regional lymph nodes from PD-1 KO mice with and without tumors showed that the percentage of P4, a minor CD8-positive T cell subset, increased in young mice after tumor transplantation but not in older mice (Figure 1C&D). Changes in other CD8-positive T cells after tumor transplantation were smaller than the changes in the P4 subset in both young and older PD-1 KO mice (Figure 1C&D). Similar results were observed in wild-type mice (Figure 6C&D). These results suggest that the P4 subset is induced during tumor rejection and may be necessary for the antitumor activity observed with PD-1 inhibition or deficiency.

[0051] P4 cells are an intermediate between P1 and P3 cells and retain the potential for antitumor activity. To investigate whether the suppression of P4 cell induction is involved in PD-1 inhibitor resistance in aged mice, we examined the functional characteristics of CD8-positive P4 subsets. We first examined the antitumor activity of P4 cells by transplanting isolated P4 cells into young, tumor-bearing CD8 KO mice. To prevent bias in the TCR repertoire, we used TCR gene-transformed OT-1 mice, which harbor CD8-positive T cells expressing a TCR specifically responsive to the OVA peptide. CD8-positive T cell P3 and P4 subsets were induced by intravein administration of OVA-expressing MC38 tumor cells (MC38-OVA) into the tail vein of OT-1 mice and isolated from the spleen five days later. The same number of P3 or P4 cells were adopted into young CD8 KO mice transplanted with MC38-OVA. MC38-OVA proliferation was strongly suppressed in mice transduced with P3 or P4 cells (Figure 2A). Notably, all transplanted P4 cells had differentiated into P3 cells in peripheral blood six days after transplantation (Figure 2B). These results suggest that P4 cells retain the ability to reject tumors by differentiating into P3 cells.

[0052] While P1 and P2 cells are known to differentiate into P3 cells during activation, P4 cells are few in number and their characteristics are largely unknown. To investigate which subset is the origin of P4 cells and which subset they differentiate into, P1, P2, and P4 cells were isolated from juvenile PD-1 KO mice and stimulated in vitro with anti-CD3 / CD28 antibody and IL-2 (Figure 2C). Consistent with in vivo results, isolated P4 cells differentiated mainly into P3 cells under these conditions (Figure 2D). Next, we investigated whether P4 cells are formed from P1 or P2 cells. As shown in Figures 2E and 2F, P4 cells were formed from P1 cells 2 days after in vitro stimulation, but not from P2 cells. These results indicate that stimulated naive P1 CD8-positive T cells form P4 cells and further differentiate into P3 cells. In vivo experiments have shown that P4 cells are not induced in aging mice (Figure 1C&D), so we investigated the ability of aging P1 cells to produce P4 cells in vitro. Surprisingly, P4 and P3 cells were induced from aged P1 cells as well as juvenile P1 cells after culturing with anti-CD3 / CD28 antibody and IL-2 (Figure 2G). These findings suggest that P1 cells derived from aged mice can differentiate into P4 cells with strong in vitro TCR stimulation such as anti-CD3 / CD28 antibody, but not with normal antigen stimulation.

[0053] Upregulation of one-carbon metabolism-related genes in P4 cells To understand the role of P4 cells in antitumor activity in aging mice, we compared gene expression profiles among CD8-positive T cell subsets (P1, P2, P3, and P4) in young PD-1 KO mice. Hierarchical clustering of global gene expression showed that each subset of CD8-positive T cells had different gene expression characteristics (Figure 3A). Genes expressed in activated T cells (such as Ctl14, Prdm1, and Il2rb) showed higher expression in P4 cells than in P1 cells (Figure 3B). On the other hand, genes expressed in differentiated T cells (such as Il7r, Ifng, and Tbx21) showed lower expression in P4 cells than in P2 and P3 cells (Figure 3B). These results suggest that P4 cells are activated but in a pre-mature state and are different from more differentiated CD8-positive T cell subsets such as P2 and P3 cells, supporting our hypothesis that P4 cells are pre-effector-like cells derived from P1 cells.

[0054] Next, gene ontology enrichment analysis was performed to investigate which biological processes were upregulated in these CD8-positive T cells. In P4 cells, the most significantly upregulated processes were those related to the 1C metabolic pathway, which is involved in the conversion of serine, glycine, and folate analogs (Figure 3C&D). The expression of 1C metabolic-related genes was highest in P4 cells, as shown by microarray analysis and qPCR (Figure 3E&F). These results indicate that the 1C metabolic pathway is selectively upregulated in P4 cells during the differentiation of CD8-positive T cells.

[0055] In total CD8-positive T cells from young wild-type and PD-1 KO mice, tumor inoculation increased the expression of 1C metabolism-related genes (Figure 3G), which can be explained by P4 cell formation (Figures 1C and 6C). Consistent with the high efficiency of P4 cell induction in PD-1 KO mice (Figures 1C and 6C), CD8-positive T cells from young PD-1 KO mice expressed most 1C metabolism-related genes at higher levels in response to MC38 tumor cell inoculation compared to wild-type mice (Figure 3G). In contrast, the expression of these genes in total CD8-positive T cells from aged mice did not show any significant change upon tumor inoculation, regardless of mouse genotype.

[0056] During immune responses, enhanced mitochondrial function is strongly involved in the activation of CD8-positive T cells (38, 39). Since P4 cells exhibit high expression of Shmt2, a regulator of mitochondrial tRNA modification, we hypothesized that activation of 1C metabolism would promote antitumor activity, partly by enhancing mitochondrial function. To investigate mitochondrial activity in young and aged mice, we compared the oxygen consumption rate (OCR) of total CD8-positive T cells in tumor-bearing young and aged mice. Aged CD8-positive T cells from wild-type or PD-1 KO mice showed clear abnormalities in OCR, accompanied by a significant decrease in basal and reserve respiratory capacity (Figure 3H&I). Taken together, these results suggest that the lack of P4 cell induction in aged mice leads to decreased mitochondrial function in CD8-positive T cells, likely through the suppression of 1C metabolism activation.

[0057] Increased CD45RB expression in aging mice Since it has been shown that P4 cells are formed from P1 cells upon in vitro stimulation, we hypothesized that the decreased P4 cell induction associated with tumor transplantation observed in aged mice could be caused by inefficient transmission of TCR signaling in aged P1 cells. To investigate the relationship between TCR signaling and P4 cell formation, we injected MC38-OVA into OT-1 mice. The percentage of P4 cells in peripheral lymph nodes increased significantly in young mice but not in aged mice (Figure 4A & B). To evaluate TCR signaling, we examined phosphorylation of ZAP-70, which is important for TCR signaling, in CD8-positive T cells overall from young and aged OT-1 mice (40). Injection of MC38-OVA increased the percentage of CD8-positive T cells expressing phosphorylated ZAP-70 in young mice (Figure 4C). This suggests that antigen-stimulated activation of TCR signaling is inhibited in aged OT-1 mice. Notably, no significant differences were observed in the intensity of OVA-specific MHC tetramers (Figure 7A & B), indicating no difference in TCR expression levels or binding affinity between OT-I T cells in young and aged mice. Therefore, it is thought that the inhibition of TCR signaling activation by antigen stimulation observed in aged mice is due to mechanisms other than the interaction between TCR and MHC / antigen.

[0058] Since the response of naive CD8-positive T cells to TCR stimulation is weaker in cells with a high density of CD45 on the cell surface (26), we analyzed the expression levels of CD45RB, an isoform of CD45 mainly expressed in naive and memory T cells of mice. CD45RB expression in CD8-positive T cells as a whole was significantly higher in aged OT-1 mice than in juvenile OT-1 mice (Figure 4D). The effect of aging on CD45RB expression was greater in P1 and P4 cells than in P2 and P3 cells (Figure 4D). Similarly, the highest increase in CD45RB expression was observed in P1 cells of aged wild-type and PD-1 KO mice (Figure 8A&B), suggesting that the inhibition of P4 cell induction may be due to increased CD45RB expression in P1 cells. To investigate whether CD45RB expression suppresses the activation of CD8-positive T cells by TCR signaling, we evaluated the phosphorylation of ZAP-70 in OT-I CD8-positive T cells after treatment with a CD45-specific inhibitor. Similar to in vivo results, the proportion of p-ZAP-70-positive CD8-positive T cells increased in young CD8-positive cells but not in aged CD8-positive T cells (Figure 4E). Under these conditions, the addition of a CD45 inhibitor increased the induction of p-ZAP-70-positive cells in aged CD8-positive cells, similar to that in young cells. These data suggest that age-related suppression of tumor rejection may be due to increased CD45RB expression in aged CD8-positive T cells.

[0059] Xenotherapy promotes P4 cell induction in aging mice and restores the antitumor activity of CD8-positive T cells. As shown above, the suppression of the transition from P1 to P4 in aging mice is restored by strong in vitro stimulation. Therefore, we investigated whether strong stimulation using xenogeneic cells (human Burkitt lymphoma, Daudi cells) could restore the induction of P4 CD8-positive T cells in aging PD-1 KO mice (Figure 5A). Ten days after tail vein injection of Daudi cells (day 0: before MC38 tumor cell transplantation), the proportion of P4 cells increased significantly in the peripheral lymph nodes of young and aging PD-1 KO mice, but the proportion of P1 to P3 cells did not increase significantly (Figure 5B). As expected, the expression levels of 1C metabolism-related genes increased in CD8-positive T cells derived from aging PD-1 KO mice after Daudi cell injection (Figure 5C). Consistent with these findings, Daudi cell injection reduced the expression of CD45RB in P1 cells derived from aging PD-1 KO mice (Figure 5D).

[0060] Daudi cell injection strongly suppressed MC38 tumor growth and extended survival in aging mice (Figure 5E & F). Age-related unresponsiveness to anti-PD-L1 antibodies in wild-type mice was also restored by Daudi cell injection (Figure 9A-E). Six days after tumor transplantation, the proportion of p-ZAP-70 positive cells increased in PD-1 KO mice injected with Daudi cells (Figure 5G), suggesting that heterologous stimulation enhances TCR signaling to tumor antigens. We further investigated whether Daudi cell therapy enhances the proliferation of tumor antigen-reactive CD8-positive T cells. MC38-OVA cells were transplanted into PD-1 KO mice 10 days after Daudi cell injection, and the appearance of OVA tetramer-positive CD8-positive T cells was monitored. The proportion of OVA tetramer-positive CD8-positive T cells increased in the regional lymph nodes and tumor tissue of aged PD-1 KO mice after Daudi cell injection (Figure 5H&I), indicating that this treatment consequently increased the induction of antigen-specific CD8-positive T cells. Assuming that pre-stimulation with Daudi cells enhances P4 cell induction and 1C metabolism in CD8-positive T cells of aged mice, the increase in both basal and reserve respiratory capacity after Daudi cell injection is a reasonable result (Figure 10A-C). Furthermore, injection of Balb / c mouse-derived splenocytes as an alloantigen also enhanced the antitumor effect of PD-1 inhibitory treatment in aged wild-type mice (Figure 11A-D). These data suggest that strong immune stimulation by xenogeneic or allocellular therapy restores the inhibited antitumor response of CD8-positive T cells in aged mice and improves the antitumor effect of PD-1 deficiency or inhibition. In summary, these findings suggest that strong immune stimulation by non-self cells restores inadequate antitumor responses in aging mice through the restoration of TCR signaling and induction of P4 subsets, thereby enhancing the effects of PD-1 deficiency or inhibitory therapies.

[0061] [Consideration] Elderly individuals and aging animals are known to be unresponsive to immunotherapy due to age-related suppression of CD8-positive T cell responses (9-11). The mechanisms by which CD8-positive T cells suppress the response to tumor antigens are largely unknown. We found that aged tumor-bearing mice have fewer P4 cells because TCR signaling is attenuated due to increased CD45RB expression. The characteristics of these P4 cells were previously unknown. The P4 subset differentiates from the naive P1 subset and then matures into the effector / memory subset (P3), which is important for antitumor activity (42, 43). The P4 subset is characterized by high expression of 1C metabolism-related genes, which are involved in development and the survival of proliferating cells (29, 32, 44, 45). These age-related inhibitions of CD8-positive T cell differentiation and PD-1 inhibitory therapy are restored by strong immune stimulation using non-self cells.

[0062] It has been suggested that age-related bias in the naive T cell repertoire is a major cause of suppressed T cell response in aged animals (46, 47). However, we found that OT-1 gene-transformed mice in which all CD8-positive T cells possess an OVA-specific TCR became insensitive to specific antigens expressed on tumor cells with age. Our study further revealed that strong TCR stimulation by xenotherapy in vitro or in vivo restored P4 cell induction. Similarly, impaired antitumor response observed in aged OT-II CD4-positive T cells expressing an OVA-specific TCR is caused by Th1 differentiation inhibition due to age-related IL-6 increase (48). These results suggest that repertoire limitation may not be the main cause of suppressed T cell response in aged animals.

[0063] Previous studies have reported that TCR signaling is suppressed with age, leading to cytoresponsive dysfunction (49, 50). Increased p-ZAP-70 levels due to T cell activation are decreased in elderly and aged mice (51, 52). Consistent with these reports, we also found that ZAP-70 phosphorylation was low in aged mice transplanted with tumor cells. Our data showed that this change is caused by increased CD45RB expression in aged P1 cells. CD45 negatively regulates TCR signaling by dephosphorylating tyrosine 394 of the tyrosine kinase Lck, leading to suppression of ZAP-70 activation (53). Indeed, the induction of p-ZAP-70-positive CD8-positive T cells was restored by treatment with a CD45 inhibitor. These results strongly support the hypothesis that inhibition of the CD8-positive T cell response in aged animals is caused by TCR signaling deficiency in naive T cells via increased CD45RB expression.

[0064] Many T cells are activated directly (e.g., donor peptides presented by donor MHC) or indirectly (e.g., donor peptides presented by recipient MHC) by allo- or heterologous MHC molecules (54-56). We hypothesize that P4 cell recovery in aging mice is due to TCR recognition of non-self antigens. Since TCRs inherently recognize a wide range of different peptides (57, 58), P4 cells or pre-activated P1 cells recovered by non-self antigens cross-react with tumor antigens, leading to an increased antitumor response. Our data also suggest that xenologous cell therapy retains the potential to reduce CD45RB expression in CD8-positive T cells in aging mice. Further research is needed to elucidate the molecular mechanisms by which CD45RB expression is regulated by aging and non-self cells.

[0065] [Materials and Methods] Mice and cells All mice were reared in an SPF environment free from specific pathogens that could interfere with experiments at the Animal Experiment Facility of the Graduate School of Medicine, Kyoto University, or the RIKEN Center for Life Science Technologies, and were used under appropriate experimental guidelines. PD-1- / - (PD-1KO) mice were produced in our laboratory by homologous recombination (59). C57BL / 6 wild-type mice were obtained from Charles River Laboratories Japan. OT-1 TCR gene transfection and CD8 - / - (CD8 KO) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) (deposited by MB Bevan of Washington Medical Center or T. Mak of the University of Toronto). The mouse colorectal cancer (MC38) cell line was donated by JP Allison, Memorial Sloan-Kettering Cancer (New York, NY), and ovalbumin (OVA)-expressing MC38 cells were generated by transforming MC38 cells with pMXs-based OVA-F2A-EGFO retrovirus. Cells were cultured in RPMI 1640 (Gibco; 11875-093) containing 10% inanimate fetal bovine serum and 1% penicillin-streptomycin (Nacalai Tesque; 26253-84) under mycobacterial-free conditions.

[0066] Mouse therapeutic model MC38 cells are (5 × 10 5 or 2 × 10 6The graft was implanted intradermally in the right flank of the mouse (day 0). Five days after MC38 cell transplantation (day 5), these mice were intraperitoneally administered anti-PD-L1 antibody (clone 1-111A.4, produced and stored at the Department of Immunology and Genome Medicine, Graduate School of Medicine, Kyoto University (Immunology Letters 84(2002) 57-62)) (6 mg / kg). This anti-PD-L1 antibody administration was repeated three times every six days (days 5, 11, and 17). The anti-PD-L1 antibody isotype (Rat IgG2a) was used as a control. Tumor growth was monitored by measuring tumor size using a measuring instrument, and the volume was calculated using the formula π (length × width × height) / 6. Non-autologous cell transfer was performed 10 days prior to tumor cell transplantation using Burkitt lymphoma Daudi cells or mitomycin C (MMC)-treated BALB / c or C57BL / 6N-derived splenocytes (2 × 10⁶). 6 This was performed by injection into the tail vein of cells (or mice).

[0067] Cell isolation for analysis and culture Peripheral and regional lymph node cells and tumor-infiltrating lymphocytes were isolated as previously described (39). For splenocyte analysis, the spleen was quickly disintegrated and suspended in potassium ammonium chloride solution to lyse red blood cells. Splenocytes were washed and resuspended in PRMI 1640 intracellular solution (T cell culture medium) containing 10% fetal bovine serum, L-glutamine, 55 μM 2-mercapsules and ethanol, penicillin-streptomycin, 1 mM sodium pyruvate (Gibco), and 1% MEM non-essential amino acid solution (Gibco). We further isolated a subset of CD8-positive T cells using CD8a(Ly-2) microbeads (Miltenyi Biotec; 130-117-044) and FACSAria (BD Biosciences). For peripheral blood cell analysis, the collected peripheral blood cells were treated with potassium ammonium chloride solution and washed with T cell culture medium. The purified cells were stimulated in a T cell culture medium supplemented with anti-CD3 / 28 Dynabeads (Gibco) and recombinant human IL-2 (20 U / mL; PeptoTech). To stimulate CD8-positive T cells using MC38-OVA cells, CD8-positive T cells isolated from the lymph nodes of OT-1 mice were co-cultured with MMC-treated MC38-OVA cells in T cell culture medium. One day after stimulation, the cells were treated with 0.05 μM CD45 inhibitor VI (EDM Millipore) for 1 hour.

[0068] Flow cytometry analysis The following antibodies were used for analysis: CD3 (clone 145-2C11) and CD8 (clone 53-6.7) (Invitrogen); phospho-ZAP70 / Syk (clone n3kobu5) (Invitrogen), CD45.1 (clone A20), CD45.2 (clone 104), CD44 (clone IM7) and CD62L (clone MEL-14) (TONBO biosciences or BioLegend); CD8 (clone KT15, MBL Life Science); CD45RB (clone 16A, BD Pharmingen). H2Kb Negative (SIY) Tetramer-SIRYYGL-APC and H-2Kb OVA Tetramer-SINFEKL-APC (TS-M008-2) were analyzed by MBL Life Science. Flow cytometry analysis was performed using FACSCant II (BD Biosciences) and FlowJo software (FlowJo LLC).

[0069] CD8 KO and OT-1 mouse model To induce P3 and P4 cells, MC38-OVA cells (1-2 × 10⁶) 6 The cells were transferred to OT-1 mice via tail vein injection. Five days later, P3 and P4 cells were isolated from these mice. Each isolated CD8-positive T cell was 2 × 10⁶ five days prior. 5 MC38-OVA cells were injected intradermally into the tail vein of CD8 KO mice. Peripheral blood was then obtained 6 days later to analyze the differentiation of the transplanted cells.

[0070] Microarray analysis Total RNA was extracted from total CD8-positive T cells fused from 3 to 6 mice or CD8-positive T cell subsets fused from 9 mice using Nucleospin RNA (Macherey-Nagel) according to the producer's protocol. Microarray analysis was performed using a mouse 8×60K v2 microarray by Macrogen, Inc. The data were registered in the GEO repository as related numbers GSE161659 and GSE161660 (http: / / www.ncbi.nlm.nih.gov / geo / ). Gene ontology enrichment analysis was performed using the Annotation, Visualization, and Integrated Discovery (DAVID; http: / / david.abcc.ncifcrf.gov / ) database. Gene expression changes were visualized using R3.1.2 or RStudio version 1.1.383.

[0071] Real-time PCR Total RNA was extracted from cells using Nucleospin RNA (Macherey-Nagel) and used for cDNA synthesis with RevatraAce reverse transcriptase (Toyobo) and random primers according to the manufacturer's manual. Real-time PCR was performed to amplify the mRNAs shown using the Applied Biosystems 7500 Fast Real-Time PCT system (ABI) and PowerUp SYBR Green Master Mix (Applied Biosystems). The expression levels of each gene were normalized to the β-actin mRNA level. The primer sequences are as follows: Shmt1 forward (Fw), 5'-CCAGAGTGCTGTGGCAACTC-3' (SEQ ID NO: 1); Shmt1 reverse (Rv), 5'-GCAAACACAGGCTGTTCCTG-3' (SEQ ID NO: 2); Shmt2 Fw, 5'-GACAGTTGAGGACACCTGGC-3' (SEQ ID NO: 3); Shmt2 Rv, 5'-CCAGAGAGGAGTGACATCTC-3' (SEQ ID NO: 4); Phgdh Fw, 5'-TGGCCTCGGCAGAATTGGAAG-3' (SEQ ID NO: 5); Phgdh Rv, 5'-TGTCATTCAGCAAGCCTGTGGT-3' (SEQ ID NO: 6); Psat1 Fw, 5'-GATGAACATCCCATTTCGCATTGG-3' (SEQ ID NO: 7); Psat1 Rv, 5'-GCGTTATACAGAGAGGCACGAATG-3' (SEQ ID NO: 8); β-actin Fw, 5'-TAAGGCCAACCGTGAAAG-3' (SEQ ID NO: 9); and β-actin Rv, 5'-GAGGCATACAGGGACAGCAC-3' (SEQ ID NO: 10).

[0072] Oxygen Consumption Rate (OCR) Analysis The OCR assay was performed with some modifications, following the previous description (39). The oxygen consumption rate of CD8-positive T cells isolated from regional lymph nodes was analyzed using the Seahorse XFe96 Extracellular Flux assay kit and the Seahorse XF Cell Mito Stress test kit (Agilent Technologies) with the XFe96 Extracellular Flux analyzer (Agilent Technologies). CD8-positive T cells were (3 × 10⁻⁶). 5 Seeds were seeded on XFe96 plates coated with Cell-Tak (Corning). Respiratory reserve was calculated from OCR graphs as previously described (60). Basal respiration was calculated by subtracting non-mitochondrial respiration (value before oligomycin addition minus value after rotenone / antimycin A addition) (61).

[0073] statistical analysis Data were analyzed using Prism 7 (Graphpad), and results are expressed as mean ± standard error. When comparing two groups, Student's test (two-tailed test for independent two groups) was performed. When comparing two or more groups, statistical significance was assessed for multiple groups using one-way analysis of variance (ANOVA), followed by multiple comparisons (Tukey's post hoc test) to evaluate the differences. Survival rates were assessed using the Kaplan-Meier method, and statistical significance was determined by the log-rank test.

[0074] Data Availability All sequence data is registered with NCBI GEO as accession IDs GSE161659 and GSE161660.

[0075] References 1. T. Finkel, M. Serrano, MA Blasco, The common biology of cancer and aging. Nature 448, 767-774 (2007). 2. K. Tomihara, T. J. Curiel, B. Zhang, Optimization of immunotherapy in elderly cancer patients. Crit Rev Oncog 18, 573-583 (2013). 3. S. L. Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012). 4. A. Ribas et al., Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma. JAMA 315, 1600-1609 (2016). 5. M. Reck et al., Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 375, 1823-1833 (2016). 6. P. S. Chowdhury, K. Chamoto, T. Honjo, Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy. J Intern Med 283, 110-120 (2018). 7. W. Zou, J. D. Wolchok, L. Chen, PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci Transl Med 8, 328rv324 (2016). 8. A. L. Shergold, R. Millar, R. J. B. Nibbs, Understanding and overcoming the resistance of cancer to PD-1 / PD-L1 blockade. Pharmacol Res 145, 104258 (2019). 9. J. Brahmer et al., Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med 373, 123-135 (2015). 10. C. Helissey, C. Vicier, S. Champiat, The development of immunotherapy in older adults: New treatments, new toxicities? J Geriatr Oncol 7, 325-333 (2016). 11. A. Padron et al., Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 105, 146-154 (2018). 12. J. Sceneay et al., Interferon Signaling Is Diminished with Age and Is Associated with Immune Checkpoint Blockade Efficacy in Triple-Negative Breast Cancer. Cancer Discov 9, 1208-1227 (2019). 13. K. Naylor et al., The influence of age on T cell generation and TCR diversity. J Immunol 174, 7446-7452 (2005). 14. M. Ahmed et al., Clonal expansions and loss of receptor diversity in the naive CD8 T cell repertoire of aged mice. J Immunol 182, 784-792 (2009). 15. Q. Qi et al., Diversity and clonal selection in the human T-cell repertoire. Proc Natl Acad Sci U S A 111, 13139-13144 (2014). 16. J. Nikolich-Zugich, G. Li, J. L. Uhrlaub, K. R. Renkema, M. J. Smithey, Age-related changes in CD8 T cell homeostasis and immunity to infection. Semin Immunol 24, 356-364 (2012). 17. M. T. Ventura, M. Casciaro, S. Gangemi, R. Buquicchio, Immunosenescence in aging: between immune cells depletion and cytokines up-regulation. Clin Mol Allergy 15, 21 (2017). 18. E. J. Wherry, R. Ahmed, Memory CD8 T-cell differentiation during viral infection. J Virol 78, 5535-5545 (2004). 19. J. C. Nolz, G. R. Starbeck-Miller, J. T. Harty, Naive, effector and memory CD8 T-cell trafficking: parallels and distinctions. Immunotherapy 3, 1223-1233 (2011). 20. J. Nikolich-Zugich, The twilight of immunity: emerging concepts in aging of the immune system. Nat Immunol 19, 10-19 (2018). 21. B. Youngblood, J. S. Hale, R. Ahmed, T-cell memory differentiation: insights from transcriptional signatures and epigenetics. Immunology 139, 277-284 (2013). 22. D. Sauce et al., Lymphopenia-driven homeostatic regulation of naive T cells in elderly and thymectomized young adults. J Immunol 189, 5541-5548 (2012). 23. N. Minato, M. Hattori, Y. Hamazaki, Physiology and pathology of T-cell aging. Int Immunol 32, 223-231 (2020). 24. J. Rossy, D. J. Williamson, K. Gaus, How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism. Front Immunol 3, 167 (2012). 25. R. J. Brownlie, R. Zamoyska, T cell receptor signalling networks: branched, diversified and bounded. Nat Rev Immunol 13, 257-269 (2013). 26. J. H. Cho et al., CD45-mediated control of TCR tuning in naive and memory CD8(+) T cells. Nat Commun 7, 13373 (2016). 27. A. C. Maue et al., T-cell immunosenescence: lessons learned from mouse models of aging. Trends Immunol 30, 301-305 (2009). 28. J. Nikolich-Zugich, Aging of the T cell compartment in mice and humans: from no naive expectations to foggy memories. J Immunol 193, 2622-2629 (2014). 29. M. Yang, K. H. Vousden, Serine and one-carbon metabolism in cancer. Nat Rev Cancer 16, 650-662 (2016). 30. G. S. Ducker, J. D. Rabinowitz, One-Carbon Metabolism in Health and Disease. Cell Metab 25, 27-42 (2017). 31. N. Ron-Harel et al., Mitochondrial Biogenesis and Proteome Remodeling Promote One-Carbon Metabolism for T Cell Activation. Cell Metab 24, 104-117 (2016). 32. E. H. Ma et al., Serine Is an Essential Metabolite for Effector T Cell Expansion. Cell Metab 25, 345-357 (2017). 33. R. J. Morscher et al., Mitochondrial translation requires folate-dependent tRNA methylation. Nature 554, 128-132 (2018). 34. D. R. Minton et al., Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs. Mol Cell 69, 610-621 e615 (2018). 35. N. Ron-Harel et al., Defective respiration and one-carbon metabolism contribute to impaired naive T cell activation in aged mice. Proc Natl Acad Sci U S A 115, 13347-13352 (2018). 36. C. H. June, Principles of adoptive T cell cancer therapy. J Clin Invest 117, 1204-1212 (2007). 37. N. S. Joshi, S. M. Kaech, Effector CD8 T cell development: a balancing act between memory cell potential and terminal differentiation. J Immunol 180, 1309-1315 (2008). 38. N. E. Scharping et al., The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity 45, 701-703 (2016). 39. K. Chamoto et al., Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci U S A 114, E761-E770 (2017). 40. H. Wang et al., ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol 2, a002279 (2010). 41. M. Perron, H. U. Saragovi, Inhibition of CD45 Phosphatase Activity Induces Cell Cycle Arrest and Apoptosis of CD45(+) Lymphoid Tumors Ex Vivo and In Vivo. Mol Pharmacol 93, 575-580 (2018). 42. M. Sharpe, N. Mount, Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech 8, 337-350 (2015). 43. R. W. Jenkins, D. A. Barbie, K. T. Flaherty, Mechanisms of resistance to immune checkpoint inhibitors. Br J Cancer 118, 9-16 (2018). 44. R. J. DeBerardinis, N. S. Chandel, Fundamentals of cancer metabolism. Sci Adv 2, e1600200 (2016). 45. N. M. Chapman, M. R. Boothby, H. Chi, Metabolic coordination of T cell quiescence and activation. Nat Rev Immunol 20, 55-70 (2020). 46. E. S. Egorov et al., The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging. Front Immunol 9, 1618 (2018). 47. C. S. Palmer et al., Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure. J Immunol 196, 4437-4444 (2016). 48. H. Tsukamoto, S. Senju, K. Matsumura, S. L. Swain, Y. Nishimura, IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age. Nat Commun 6, 6702 (2015). 49. R. A. Miller, Effect of aging on T lymphocyte activation. Vaccine 18, 1654-1660 (2000). 50. G. Pawelec, K. Hirokawa, T. Fulop, Altered T cell signalling in ageing. Mech Ageing Dev 122, 1613-1637 (2001). 51. R. L. Whisler, M. Chen, B. Liu, Y. G. Newhouse, Age-related impairments in TCR / CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of zeta-chains and p59fyn / p56lck in human T cells. Mech Ageing Dev 111, 49-66 (1999). 52. T. Fulop, Jr., A. Larbi, G. Dupuis, G. Pawelec, Ageing, autoimmunity and arthritis: Perturbations of TCR signal transduction pathways with ageing - a biochemical paradigm for the ageing immune system. Arthritis Res Ther 5, 290-302 (2003). 53. T. Furukawa, M. Itoh, N. X. Krueger, M. Streuli, H. Saito, Specific interaction of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta chain. Proc Natl Acad Sci U S A 91, 10928-10932 (1994). 54. K. F. Lindahl, D. B. Wilson, Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors. J Exp Med 145, 508-522 (1977). 55. M. Cascalho, J. L. Platt, Xenotransplantation and other means of organ replacement. Nat Rev Immunol 1, 154-160 (2001). 56. N. Degauque, S. Brouard, J. P. Soulillou, Cross-Reactivity of TCR Repertoire: Current Concepts, Challenges, and Implication for Allotransplantation. Front Immunol 7, 89 (2016). 57. L. J. D'Orsogna, D. L. Roelen, Doxiadis, II, F. H. Claas, TCR cross-reactivity and allorecognition: new insights into the immunogenetics of allorecognition. Immunogenetics 64, 77-85 (2012). 58. M. Hebeisen et al., Molecular insights for optimizing T cell receptor specificity against cancer. Front Immunol 4, 154 (2013). 59. H. Nishimura, N. Minato, T. Nakano, T. Honjo, Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses. Int Immunol 10, 1563-1572 (1998). 60. PS Chowdhury, K. Chamoto, A. Kumar, T. Honjo, PPAR-Induced Fatty Acid Oxidation in T Cells Increases the Number of Tumor-Reactive CD8(+) T Cells and Facilitates Anti-PD-1 Therapy. Cancer Immunol Res 6, 1375-1387 (2018). 61. MD Brand, DG Nicholls, Assessing mitochondrial dysfunction in cells. Biochem J 435, 297-312 (2011).

[0076] [Example 2] [Materials and Methods] Mice and cells All mice were reared in an SPF environment free from specific pathogens that could interfere with the experiments at the Animal Experiment Facility of the Graduate School of Medicine, Kyoto University, and were used under appropriate experimental guidelines. C57BL / 6 was obtained from Charles River Laboratories Japan. PD-1- / - (PD-1KO) mice were produced in our laboratory by homologous recombination (see Nishimura et al.) (11). MC38 cells derived from mouse colon cancer were obtained from JP Allison, Memorial Sloan-Kettering Cancer (New York, NY). LLC cells derived from Lewis lung cancer were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Gibco; 11875-093) containing 10% denatured fetal bovine serum and 1% penicillin-streptomycin (Nacalai Tesque; 26253-84) under conditions free from mycobacterial infection.

[0077] Mouse therapeutic model CD45 inhibitors (Figure 12): 2-(4-Acetylanilino)-3-chloronaphthoquinone (211) (530197, EDM Millipore) (12) (3 mg / kg) or N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide (PTP) (540215, Sigma-Aldrich) (13) (3 mg / kg) were administered intraperitoneally to mice before or after tumor cell transplantation (5 or 7 days later). MC38 cells (5 × 10) 5 or 1 × 10 6 ) or LLC cells (2 × 10 6The LLC cells were transplanted intradermally into the right flank of the mice (day 0). Seven days after LLC cell transplantation (day 7), these mice were administered anti-PD-L1 antibody (clone 1-111A.4) (2 mg / kg) intraperitoneally. This anti-PD-L1 antibody administration was repeated three times every six days (days 7, 13, and 19). Tumor volume was measured using a measuring instrument and calculated using the formula π (length × width × height) / 6.

[0078] statistical analysis Data were analyzed using Prism 7 (Graphpad). When comparing two or more groups, statistical significance was assessed for multiple groups using one-way analysis of variance (ANOVA), and then differences were evaluated using multiple comparisons (Tukey's post hoc test). When comparing two groups, Student's T-test was performed.

[0079] Investigation of the effect of CD45 inhibitors on enhancing antitumor effects through PD-1 signaling inhibition. In this example, 2-(4-Acetylanilino)-3-chloronaphthoquinone (211) or N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide (PTP) were used as CD45 inhibitors, and the tumor growth inhibitory effect when used in combination with PD-1 signaling inhibition was investigated (Figure 12).

[0080] PD-1 knockout mice (2 months old) were administered the CD45 inhibitor 211 at a rate of 3 mg / kg intraperitoneally either once (pre-administration) 3 days before transplantation of MC38 mouse colorectal cancer cells or three times (post-administration) 5 days after transplantation (Figure 13A & B). MC38 cells were 1 × 10⁶ 6 The tumors were intradermally transplanted into the right ventral side of mice. As a result, compared to the control group administered only the solvent, the groups administered 211 before or after MC38 transplantation showed a significant reduction in tumor volume (Figure 14A & B). These results suggest that CD45 inhibition enhances the antitumor effect mediated by PD-1 signaling inhibition.

[0081] Investigation of the effect of CD45 inhibitors on improving PD-1 signaling inhibition resistance associated with aging. Young (1-2 months old) or older (14-16 months old) PD-1 knockout mice were administered the CD45 inhibitor 211 intraperitoneally at a rate of 3 mg / kg. Three days after administration of 211, MC38 cells were divided into 5 × 10⁶ cells. 5 The tumor was intradermally transplanted into the right ventral side of mice in proportion to the tumor size, and tumor growth was examined. As a result, the suppression of tumor growth observed in young PD-1 KO mice was not seen at all in aged PD-1 KO mice, revealing resistance to PD-1 signaling inhibition (Figure 15A & B). On the other hand, when 211 was administered to aged mice, tumor growth was strongly suppressed (Figure 15A & B). These results indicate that CD45 inhibition suppresses the growth of tumors that exhibit resistance to PD-1 signaling inhibition as a result of aging.

[0082] Investigation of the improvement effect of CD45 inhibitors on cancers exhibiting resistance to PD-1 signaling inhibition. LLC cells were introduced into the skin of young (1-2 months old) wild-type mice, with a sample size of 2 × 10⁶ cells. 6 The tumors were transplanted to the right ventral side of mice according to their proportion (Figure 16A). Starting 7 days later, tumor growth was examined after treatment with either anti-PD-L1 antibody alone or in combination with a CD45 inhibitor (211 or PTP) (Figure 16A). As a result, tumor suppression, which was hardly observed with anti-PD-L1 monotherapy, was significantly suppressed with combination therapy with a CD45 inhibitor (Figures 16B & C). Both of these CD45 inhibitors similarly showed an inhibitory effect on tumor growth when used in combination with an anti-PD-L1 antibody (Figures 16B & C). Furthermore, when the effect of CD45 inhibitor monotherapy was examined under the same conditions, it did not show a significant inhibitory effect on LLC tumor growth (Figures 16D & E). These results suggest that CD45 inhibition may suppress the progression of unresponsive cancer by enhancing the effect of PD-1 signaling inhibition therapy. [Example 3] Young (6-week-old) C57BL / 6 mice were intradermally injected with MC38 cancer cells (5 x 10^5 cells). At 9, 16, and 23 days, anti-PD-L1 antibody (clone 1-111A.4) (1.5 mg / kg) was administered intraperitoneally. In addition, Balb / c (allogeneic) spleen cells (5 x 10^5 cells / mouse or 2 x 10^6 cells / mouse) treated with mitomycin C were intravenously administered at (A) 6 days, (B) 9 days, or (C) 9, 16, and 23 days, and the change in tumor size over time was measured (Figure 18). The points in each line graph represent the mean value of 5 to 6 mice, and the error bars are the standard error. * indicates a p-value of less than 0.05, and ns means there is no significant difference (comparison by Tukey's test after one-way ANOVA). The results showed that administering allogeneic cells three times resulted in a greater combined effect with immune checkpoint inhibitors than administering them once.

[0083] References 1. SL Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012). 2. A. Ribas et al., Association of Pembrolizumab With Tumor Response and Survival Among Patients With Advanced Melanoma. JAMA 315, 1600-1609 (2016). 3. M. Reck et al., Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 375, 1823-1833 (2016). 4. P. S. Chowdhury, K. Chamoto, T. Honjo, Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy. J Intern Med 283, 110-120 (2018). 5. W. Zou, J. D. Wolchok, L. Chen, PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci Transl Med 8, 328rv324 (2016). 6. A. L. Shergold, R. Millar, R. J. B. Nibbs, Understanding and overcoming the resistance of cancer to PD-1 / PD-L1 blockade. Pharmacol Res 145, 104258 (2019). 7. K. Chamoto et al., Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci U S A 114, E761-E770 (2017). 8. J. Rossy, D. J. Williamson, K. Gaus, How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism. Front Immunol 3, 167 (2012). 9. R. J. Brownlie, R. Zamoyska, T cell receptor signalling networks: branched, diversified and bounded. Nat Rev Immunol 13, 257-269 (2013). 10. J. H. Cho et al., CD45-mediated control of TCR tuning in naive and memory CD8(+) T cells. Nat Commun 7, 13373 (2016). 11. H. Nishimura, N. Minato, T. Nakano, T. Honjo, Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses. Int Immunol 10, 1563-1572 (1998). 12. M. D. Perron et al., Allosteric noncompetitive small molecule selective inhibitors of CD45 tyrosine phosphatase suppress T-cell receptor signals and inflammation in vivo. Mol Pharmacol 85, 553-563 (2014). 13. B. Y. Ma et al., The lectin Jacalin induces human B-lymphocyte apoptosis through glycosylation-dependent interaction with CD45. Immunology 127, 477-488 (2009). 14. SM Krummey et al., CD45RB Status of CD8(+) T Cell Memory Defines T Cell Receptor Affinity and Persistence. Cell Rep 30, 1282-1291 e1285 (2020). All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0084] The pharmaceutical composition of the present invention can be used as an anticancer agent, an infectious disease treatment agent, or a combination thereof. [Sequence Listing Free Text]

[0085] <Sequences 1-10> The base sequence of the primer is shown.

Claims

1. A pharmaceutical composition comprising a substance capable of enhancing T cell receptor (TCR) signaling, for administration before, after, or concurrently with the administration of a PD-1 signaling inhibitor, The substance that can enhance the TCR signal is at least one compound selected from the group consisting of 2-(4-Acetylanilino)-3-chloronaphthoquinone, its salts and solvates, and N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, its salts and solvates. The pharmaceutical composition wherein the PD-1 signaling inhibitor is at least one antibody selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies.

2. A pharmaceutical composition comprising a substance capable of enhancing T cell receptor (TCR) signaling, for administration before, after, or concurrently with the administration of a PD-1 signaling inhibitor, The substances that can enhance the TCR signal are (i) and / or (ii) below: The pharmaceutical composition wherein the PD-1 signaling inhibitor is at least one antibody selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. (i) At least one compound selected from the group consisting of 2-(4-Acetylanilino)-3-chloronaphthoquinone, its salts and solvates, and N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, its salts and solvates. (ii) Xenocytes, allocells, or combinations thereof that can enhance the TCR signaling of naive T cells and restore their induction into effector precursor T cells.

3. The pharmaceutical composition according to claim 2, for patients who are unresponsive to monotherapy with PD-1 signaling inhibitors.

4. A pharmaceutical composition according to any one of claims 1 to 3, used as an anticancer agent.

5. A PD-1 signaling inhibitory activity enhancer comprising a CD45 inhibitor and / or cells, The CD45 inhibitor is at least one compound selected from the group consisting of 2-(4-Acetylanilino)-3-chloronaphthoquinone, its salts and solvates, and N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, its salts and solvates. The PD-1 signaling inhibitory activity enhancer is a xenocell, allocell, or a combination thereof, which allows cells to enhance the TCR signaling of naive T cells and restore induction into effector precursor T cells.

6. A TCR signaling enhancer comprising a CD45 inhibitor and / or cells, The CD45 inhibitor is at least one compound selected from the group consisting of 2-(4-Acetylanilino)-3-chloronaphthoquinone, its salts and solvates, and N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide, its salts and solvates. The TCR signaling enhancer is a xenocell, allocell, or a combination thereof, which allows cells to enhance the TCR signaling of naive T cells and restore their induction into effector precursor T cells.

Images