Vectors for cancer treatment

Abstract

The present invention relates to an adenoviral or adeno-associated viral vector comprising a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope, wherein the vector is capable of inducing an expansive memory CD8+ T cell response, and the vector does not contain nucleic acid encoding an additional cancer-specific T cell epitope. It also relates to methods and uses of the vector.

Description

[Technical Field]

[0001] This invention relates to vectors capable of inducing an expansive memory CD8+ T cell response. These vectors that induce an expansive memory CD8+ T cell response are suitable for use in cancer treatment. This invention also relates to methods for producing such vectors and methods for inducing an expansive memory CD8+ T cell response. [Background technology]

[0002] Anti-cancer strategies aimed at activating the immune CD8 T cell arm have shown remarkable efficacy. There is considerable overlap between the requirements for a good CD8 T cell response to chronic infection and those for cancer—they must be durable, functional, persistent, home to the correct site, and resistant to exhaustion from long-term TCR stimulation.

[0003] Epitope-based cancer vaccines are one strategy used to activate T-cell responses to specific tumor-associated antigens. Initially, single-epitope vaccines based on peptides were used, but these showed poor clinical responses because they did not sufficiently activate the innate immune system. To enhance immune activation, multi-peptide vaccines, in which multiple epitopes are administered together, have been developed.

[0004] This approach of administering multiple epitopes has also been implemented using adenovirus vectors. By using adenovirus vectors capable of encoding large transgenes, multiple epitopes can be encoded and delivered as concatemers (Bei and Scardino., J Biomed Biotechnol 2010;2010:102758). Alternatively, full-length antigens can be encoded and delivered. However, the need to enhance immune activation against cancer cells remains. [Overview of the project] [Means for solving the problem]

[0005] This invention stems from the remarkable finding that a vector encoding a single cancer-specific CD8+ and / or CD4+ T cell epitope, referred to herein as a minigene vector, can induce an expansive memory CD8+ T cell response. Memory expansion explains the long-term development of a stable, expanded CD8+ T cell memory pool in which cells possess distinct phenotypes and functions. This expansive memory response results in a long-lived pool of epitope-specific T cells that remain large and functional even beyond the acute phase of infection (Klernerman, Immunol Rev 2018 283(1):99~11). The expansive memory cell feature is thought to potentially lead to an enhancement of the antitumor response.

[0006] The inventors have developed a vaccine platform based on a replication-deficient AdHu5 adenovirus vector backbone into which only the target CD8+ T cell epitope is inserted. In this form, the need for antigen processing is avoided, which allows for the development of an expanding response, or otherwise, a non-expanding epitope. A single priming injection of the vector has been demonstrated to produce a large epitope-specific CD8+ T cell response, in which the T cells exhibit an expanding memory phenotype. Surprisingly, the response produced was long-lived and was able to control tumors even later than 50-90 days after immunization in prophylactic immunization experiments and when administered to mice already with tumors. These responses were detectable for long periods and showed low PD-1 levels, as well as low levels of checkpoint inhibitors, Lag-3 and Tim-3. In comparison, administration of a vector encoding a full-length protein antigen did not produce a CD8+ T cell response of the same magnitude or phenotype.

[0007] Adenovirus vectors generally offer the advantage of large transgene packaging capabilities by removing one or more viral genes. As such, previous approaches to epitope-based vaccines using adenovirus vectors have encoded multiple T cell epitopes as concatemers. However, this approach has found that long-lasting and durable immune responses can be induced by adenovirus vectors (referred to herein as minigene vectors) containing relatively small insertion fragments of approximately 70 bp and minimal enhancer elements. Surprisingly, it has been shown that short nucleic acid sequences can be transcribed in vivo, successfully presented on MHC molecules, and generate peptide-specific CD8+ T cells.

[0008] Additionally, the scale and durability of the CD8+ T cell response induced by the minigene are significantly higher than those previously observed in responses induced using adenovirus vectors containing multiple CD8+ T cell epitopes, even at later post-delivery times (beyond day 50). By providing an adenovirus or adeno-associated virus encoding a short epitope peptide sequence, the encoded peptide is thought to bypass the need for normal antigen processing for presentation on MHC molecules. This, in turn, results in a T cell response that is easier to predict, more reliable, more extensive, and more robust and effective.

[0009] These mini-gene vectors offer several advantages over traditional peptide-based and DNA vaccines. Firstly, adenovirus vector mini-genes can induce a proper priming response (co-stimulation) within infected cells. This results in the development of a potent antigen-specific CD8+ T cell response. DNA and peptide vaccines cannot induce a priming response unless combined with an adjuvant. Secondly, adenovirus vector mini-genes can persistently infect cells. This property allows the vector to act as a long-term source of antigen, thereby maintaining the size of the antigen-specific T cell pool. Thirdly, peptide and DNA vaccines cannot produce a long-lived antigen-specific CD8+ T cell response unless administered in a multi-dose priming-boosting regimen, and usually in combination with an adjuvant. In contrast, a large pool of long-lived antigen-specific CD8+ T cells is generated from a single injection of the mini-gene. These long-lived tumor-specific CD8+ T cell responses are found in the blood and therefore present throughout the body. Therefore, they can play an important role in suppressing micrometastases after primary tumor control. Finally, adenovirus vector minigenes also have the advantage of being easy to design and fabricate due to the simplicity of the vector and coding sequence.

[0010] As such, the present invention relates to a single cancer-specific CD8+ and / or CD4+ This invention relates to an adenovirus vector comprising a nucleotide sequence encoding a T cell epitope, wherein the vector has the ability to induce an expansive memory CD8+ T cell response. This specification encompasses the following disclosures of the invention. [1] An adenovirus vector or adeno-associated virus vector comprising a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope, wherein the vector is capable of inducing an expansive memory CD8+ T cell response, and the vector does not contain nucleic acids encoding further cancer-specific T cell epitopes. [2] The vector described in [1] has the ability to induce the production of CD8+ T cells characterized by a marker selected from the group including CX3CR1+, KLRG-1+, CD44+, and CD62L-. [3] The vector described in [1] has the ability to induce the production of CD8+ T cells characterized by a marker selected from the group including CX3CR1+, KLRG-1+, CD44+, CD62L-, CD27-(low), and CD127-(low). [4] A vector according to any of [1] to [3], comprising a nucleotide sequence encoding a cancer-specific CD8+ T cell epitope, comprising 12 to 45 nucleotide base pairs. [5] A vector according to any of [1] to [4], comprising a nucleotide sequence encoding a cancer-specific CD8+ T cell epitope, comprising 24 to 45 nucleotide base pairs. [6] A vector described in any of [1] to [5], wherein the cancer-specific CD8+ T cell epitope is derived from a tumor-associated antigen. [7] A vector described in any of [1] to [6], wherein the T cell epitope is mutated in cancer cells. [8] A vector described in any of [1] to [7], wherein the T cell epitope is overexpressed in cancer cells. [9] A vector described in any of [1] to [8], wherein the T cell epitope is derived from a tumor-associated antigen selected from the group consisting of TRP-1, CEA, TAG-72, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, Melan-A / MART-1, tyrosinase, CLPP, cyclin-A1, cyclin-B1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2a, CD45, glypican-3, IGF2B3, kallikrein-4, KIF20A, lengusin, meroe, MUC5AC, survivorbin, PRAME, SSX-2, NY-ESO-1 / LAGE1, gp70, MC1R, TRP-1 / -2, β-catenin, BRCA1 / 2, CDK4, and fetal protein SIM1.

[10] The vector according to [9], wherein the T cell epitope contains SEQ ID NO: 2 (SLLMWITQC).

[11] The vector according to any one of [1] to [6], wherein the cancer-specific CD8+ T cell epitope is specific to colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein-Barr virus-driven cancer, human papillomavirus-driven cancer, and soft tissue sarcoma.

[12] The vector according to any one of [1] to

[11] , which is human serotype 5 (AdHu5).

[13] The vector according to any one of [1] to

[12] , which contains a CMV promoter.

[14] The vector according to any one of [1] to

[13] , which contains a TATA box.

[15] The vector according to any one of [1] to

[14] , which lacks E1 and E3 proteins.

[16] An immunogenic composition comprising the vector according to any one of [1] to

[15] .

[17] An immunogenic composition comprising at least two vectors according to any one of [1] to

[15] .

[18] The composition according to

[17] , wherein each vector encodes a different cancer-specific CD8+ T cell epitope.

[19] The composition according to any one of

[16] to

[18] , which contains one or more additional active ingredients, a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[20] A host cell comprising the vector according to any one of [1] to 15 or the immunogenic composition according to any one of

[16] to

[19] .

[21] The vector or composition according to any one of [1] to

[19] for use in therapy.

[22] The vector or composition according to any one of [1] to

[19] for use in the treatment or prevention of cancer. A method of treating or preventing cancer, comprising the step of administering a therapeutically effective amount of the vector according to any one of [1] to

[15] or the composition according to any one of

[16] to

[19] .

[24] A method of inducing an expansive memory CD8+ T cell response, comprising the step of administering a therapeutically effective amount of the vector according to any one of [1] to

[15] or the composition according to any one of

[16] to

[19] to a subject that needs it, wherein the CD8+ T cells are characterized by a marker selected from the group including CX3CR1+, KLRG-1+, CD44+, and CD62L-.

[25] The vector or composition according to

[21] or

[22] , the method according to

[23] or

[24] , wherein the vector or composition is administered intravenously or intramuscularly.

[26] The vector or composition according to

[21] or

[22] , the method according to

[23] or

[24] , wherein the vector or composition is administered as a single dose.

[27] The vector or composition according to

[21] or

[22] , the method according to

[23] or

[24] , wherein the vector or composition is administered as multiple doses.

[28] The vector or immunogenic composition according to

[21] or 22], the method according to

[23] or

[24] , wherein the vector or immunogenic composition is administered prophylactically to a subject.

[29] The vector or immunogenic composition according to

[21] or 22, the method according to

[23] or

[24] , wherein the vector or immunogenic composition is administered therapeutically to a subject.

[30] The vector or composition according to

[21] or

[22] , the method according to

[23] or

[24] , wherein the vector or composition is administered in combination with an immunomodulatory molecule, either separately, sequentially, or simultaneously.

[31] The method, vector, or composition according to

[30] , wherein the immunomodulatory molecule is an immune checkpoint inhibitor.

[32] The method, vector, or composition according to

[31] , wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, LAG-3, B7-H3, B7-H4, B7-H6, A2aR, BTLA, GAL9, and IDO.

[33] i) A step of synthesizing nucleic acid sequences encoding an epitope as sense and antisense primers, ii) The step of cloning the nucleic acid sequence encoding the epitope sequence into the first plasmid, iii) A step of cloning a sequence containing the nucleic acid sequence encoding the epitope into a second vector containing adenovirus DNA, A method for producing a vector, including any of the vectors described in [1] to

[15] . A kit comprising a vector as described in any of

[34] [1] to

[15] or a composition as described in any of

[16] to

[19] , one or more additional active ingredients, a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant, and optionally instructions for use. A method for inducing a T cell immune response in animals against a cancer-specific CD8+ T cell epitope, comprising the step of contacting cells with a vector described in any of

[35] [1] to

[15] or a composition described in any of

[16] to

[19] .

[0011] In one embodiment, the present invention relates to an adenovirus vector or adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope, wherein the vector has the ability to induce an expansive memory CD8+ T cell response. In one embodiment, the vector has the ability to induce the production of CD8+ T cells characterized by a marker selected from the group comprising CX3CR1+, KLRG-1+, CD44+, and CD62L-. In one embodiment, the vector has the ability to induce the production of CD8+ T cells characterized by a marker selected from the group comprising CX3CR1+, KLRG-1+, CD44+, CD62L-, CD27(low), and CD127(low). In one embodiment, the nucleotide sequence encoding the cancer-specific CD8+ or CD4+ T cell epitope comprises 12 to 45 nucleotide base pairs. In one embodiment, the nucleotide sequence encoding the cancer-specific CD8+ and / or CD4+ T cell epitope contains 24 to 45 nucleotide base pairs. In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope is derived from a tumor-associated antigen. In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope is mutated in cancer cells. In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope is overexpressed in cancer cells.In one embodiment, cancer-specific CD8+ and / or CD4+ T cell epitopes are derived from tumor-associated antigens selected from the group consisting of TRP-1, CEA, TAG-72, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, Melan-A / MART-1, tyrosinase, CLPP, cyclin-A1, cyclin-B1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2a, CD45, glypican-3, IGF2B3, kallikrein-4, KIF20A, lengusin, meroe, MUC5AC, survivin, PRAME, SSX-2, NY-ESO-1 / LAGE1, gp70, MC1R, TRP-1 / -2, β-catenin, BRCA1 / 2, CDK4, and fetal protein SIM1. In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope comprises SEQ ID NO: 1 (SPSYVYHQF) or SEQ ID NO: 2 (SLLMWITQC). In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope is specific to colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein-Barr driven cancers, human papillomavirus driven cancers, and soft tissue sarcoma. In one embodiment, the vector is human serotype 5 (AdHu5). In one embodiment, the vector comprises a CMV promoter. In one embodiment, the vector comprises a TATA box. In one embodiment, the vector lacks E1 and E3 proteins. In another embodiment, the vector does not contain any additional nucleotide sequences encoding cancer-specific CD8+ and / or CD4+ T cell epitopes.Therefore, the vector has a single cancer-specific CD8+ T cell epitope and may contain other vector elements necessary for the transcription of its nucleic acid, but does not contain nucleic acid sequences encoding cancer-specific epitopes that are not CD8+ T cell epitopes, such as CD4+ T cell epitopes. Furthermore, it does not contain more than one cancer-specific CD8+ or CD4+ T cell epitope. Thus, the presence of multiple anti-cancer T cell epitopes in the vector is excluded. This excludes multiple copies of the same anti-cancer T cell epitope or copies of different anti-cancer T cell epitopes. The vector does not have concatemers, i.e., long, continuous DNA molecules containing multiple copies of the same cancer-specific T cell epitope linked in series.

[0012] In one embodiment, the present invention relates to an immunogenic composition comprising a vector according to the present invention. In one embodiment, the present invention relates to an immunogenic composition or vaccine composition comprising at least one, two, three, four, five, six, seven, eight, nine, or up to ten, twenty, thirty, forty, or fifty vectors according to the present invention.

[0013] In one embodiment, the present invention relates to a host cell comprising a vector or an immunogenic composition according to the present invention. In one embodiment, the present invention relates to a vector or composition according to the present invention for use in therapeutic applications.

[0014] In one embodiment, the present invention relates to a method for treating or preventing cancer, comprising the step of administering a therapeutically effective amount of a vector or composition according to the present invention. In one embodiment, the present invention relates to a method for inducing an expansive memory CD8+ T cell response, comprising the step of administering a therapeutically effective amount of a vector or composition according to the present invention to a subject in need thereof, wherein the CD8+ T cells are characterized by a marker selected from the group including CX3CR1+, KLRG-1+, CD44+, and CD62L-.

[0015] In one embodiment, the present invention is a method for producing the above-mentioned vector: i) A step of synthesizing nucleic acid sequences encoding epitopes as sense and antisense primers, ii) The step of cloning the nucleic acid sequence encoding the epitope sequence into the first plasmid, iii) A step of cloning a sequence containing the nucleic acid sequence encoding the epitope into a second plasmid containing adenovirus DNA, This includes methods.

[0016] In one embodiment, the present invention relates to a kit comprising a vector according to the present invention, one or more additional active ingredients, a pharmaceutically acceptable carrier, a diluent, an excipient, or an adjuvant, and optionally, instructions for use.

[0017] In one embodiment, the present invention relates to a method for inducing a T cell immune response in animals against a cancer-specific CD8+ and / or CD4+ T cell epitope, comprising the step of contacting cells with a vector or composition according to the present invention. [Brief explanation of the drawing]

[0018] [Figure 1]Immunization of Balb / c mice with an AdHu5 replication-deficient vector encoding the AH1 CD8+ T cell tumor epitope stimulates a persistent CD8+ T cell response in the periphery. Figure 1A is a schematic diagram of the construct used to construct an AdHu5 vector expressing the MHC-1 binding CT26-specific cancer epitope. Figure 1B is a FACS plot showing %CD8+ AH1 tetramer+(tet+) cells in the blood of mice vaccinated with AH1 (left) and Ad-I8V (right). Figure 1C shows the AH1 tetramer-specific CD8+ T cell response in the blood at day 7 (left) and day 50 (right) from two independent experiments. Figure 1D is a FACS plot showing the presence of indicated markers in the CD8+ AH1-tet+(left) and AH1-tet-(right) populations in the blood from the same sample. Figure 1E shows the phenotype of AH1-tet-CD8+ T cells compared to AH1-tet-CD8+ T cells from the same group at day 7 (left) and day 50 (right) from two independent experiments. Geo M FI = Geometric mean fluorescence intensity. [Figure 2-1] Expansive memory AH1-specific T cells inhibit CT26 tumor growth in Balb / c mice, both after prophylactic and therapeutic vaccination with Ad-AH1. (A) Figure shows the experimental setup for prophylactic vaccination (two doses, P1 and P2, performed independently) and therapeutic vaccination (T1). Stars indicate the presence of palpable tumors. (B-D) Figures show tumor growth curves for different groups (N=5 per group) in prophylactic (P1 and P2) (B and C) and therapeutic vaccination setups (T1) (D). In T1, arrows indicate the time of vaccination. Mice vaccinated with Ad-AH1 (1 × 10⁸ IU) are shown in green, Ad-AH1 Low (1 × 10⁷ IU) in orange, Ad-AH1 (1 × 10⁸ IU) + Ad-GSW11 (1 × 10⁸ IU) in red, Ad-GSW11 (1 × 10⁸ IU) in light purple, Ad-I8V (1 × 10⁸ IU) in gray, and naive mice in black. TF = no tumor. [Figure 2-2](E, G, L) Figures showing statistically significant differences in tumor size between groups at 18 days post-exposure. Points represent individual mice. (F, H, J) Graphs show the slope of the tumor growth curve determined by linear regression from the day when the tumor showed clear tumor growth (7 days post-exposure for controls, and 18 days post-exposure for Ad-AH1 vaccinated mice). (K) Tumor growth rate was recalculated to determine the specific growth rate. The tumor growth rate between transplantation and the humane endpoint was quantified using the parameter of specific growth rate (SGR, % / day), calculated using the following equation:

[15] SGR=ln(V2 / V1) / (t2-t1), where V1 and V2 are the tumor volumes at 1 day post-transplantation (V1 fixed at 0.01 mm) (t1=day0) and the endpoint (t2), respectively. [Figure 3] AH1-specific CD8+ T cells differ in both abundance and phenotype between tumors and spleens. (A) Representative FACS plots showing %CD8+ AH1-tet+ cells in tumors (upper panel) and spleens (lower panel) from Ad-AH1-vaccinated mice. For negative controls, tumor and spleen samples were stained with a full range of fluorescent dye conjugate antibodies and an unrelated H2-Ld-conjugated tetramer (pp89) for tumor samples or no tetramer (no tet) for spleen samples. (B) Graphs showing %CD8+ AH1-tet+ cells in tumors (upper panel) and spleens (lower panel) from prophylactic (left panel) and therapeutic (right panel) vaccinated mice. (C) Heatmaps showing the phenotype of AH1-specific CD8+ T cells in tumors and spleens from prophylactic-vaccinated mice (Ad-AH1) and control mice (Ad-I8V and naive). Values ​​in cells represent the mean of two independent experiments (N=5-10). The markers quantified by geometric MFI are standardized to a 0-100% scale. [Figure 4](A) In tumors derived from Ad-AH1-vaccinated mice, the presence of regulatory T cells (CD4+ FoxP3+ cells) appears to be lower compared to control mice, both after prophylactic vaccination (left) and after therapeutic vaccination (right). Data from Ad-AH1 and Ad-AH1+Ad-GSW11-vaccinated mice were grouped, as were Ad-I8V-vaccinated mice and naive mice (shown as vaccinated and control, respectively). (B) AdHu5-AH1-MG immunization increases the percentage of Trm tet+ cells in tumors. Mice immunized with AdHu5-AH1-MG alone or in combination show an increased percentage of AH-1-specific CD8 T cells (TILs) in tumors with a resident memory phenotype compared to control mice (naive or immunized with an unrelated AdHu5 construct (AdHu5-I8V-MG or AdHu5-GSW11)). [Figure 5] AdHu5-AH1-MG immunization induces AH-1+ CD8 T cells in the spleen that remain functional during tumor growth. Splenocytes and TILs were stimulated with the AH-1 peptide, and their cytotoxic potential was measured based on IFN-gamma secretion. AH-1 peptide-specific splenocytes from immunized animals were able to respond to peptide stimulation (A and B). In contrast, CD8 T cells in TILs did not respond to peptide stimulation (C and D); however, the levels of IFN-g secreted in response to PMA-ionomycin were also low, indicating an overall state of CD8 T cell downregulation in tumors. [Figure 6](A) Figure shows the correlation between the slope of the tumor growth curve for each animal (shown as dots) and the percentage of CD8+ AH1-specific T cells in the blood (left) and spleen (center), and the absolute number of CD8+ AH1 tet+ cells in the tumor (right). Data are shown for two independent prophylactic experiments (P1 and P2) and a single therapeutic experiment (T1). Lower tumor growth rates correlate with increased levels of AH1-specific CD8+ T cells in the spleen and blood after tumor exposure, but only weakly with the absolute number of AH1-specific CD8 T cells in the tumor. (B) shows a comparison of antigen-specific cells from various compartments regarding specific growth rates. [Figure 7A] (A) Therapeutic immunization with the AdHu5 vector encoding full-length gp90 (AdHu5-gp90FL) did not provide a similar level of tumor control. The relative tumor growth rate in each group was compared using the Mann-Whitney U test. *p<0.05, **p<0.005. [Figure 7B] (B) Mice in which tumors were eliminated by therapeutic and prophylactic immunization continued to have circulating AH-1 specific cells. Blood samples were taken from mice in which tumors had been completely eliminated 6 months prior and stained for AH1+ CD8 T cells by tetramer staining. [Figure 8-1]HHD mice immunized with the AdHu5-NY-ESO-1157-165 minigene construct developed a long-lived, circulating population of NY-ESO-1157-165 Tet+ CD8 T cells with an expansive memory phenotype. (A) Levels of NY-ESO-1157-165 Tet+ cells were measured by tetramer staining in the blood of groups of HHD mice (N=4-5 per group) after immunization with 1 × 10⁸ IU AdHu5-NY-ESO-1 mini or 1 × 10⁹ IU AdHu5-NY-ESO-1-FL. A schematic diagram of the constructs used is shown. These cells were phenotyped by surface staining for (B) CD44 and CD62L to determine the memory subset, the markers for swollen cells (C) CX3CR1 and (D) KLRG-1, and the markers for exhaustion (E) PD-1, (F) Tim3, and (G) Lag-3. The results shown are from 4-5 mice per group from one of two independent experiments. [Figure 8-2] HHD mice immunized with the AdHu5-NY-ESO-1157-165 minigene construct developed a long-lived, circulating population of NY-ESO-1157-165 Tet+ CD8 T cells with an expansive memory phenotype. (A) Levels of NY-ESO-1157-165 Tet+ cells were measured by tetramer staining in the blood of groups of HHD mice (N=4-5 per group) after immunization with 1 × 10⁸ IU AdHu5-NY-ESO-1 mini or 1 × 10⁹ IU AdHu5-NY-ESO-1-FL. A schematic diagram of the constructs used is shown. These cells were phenotyped by surface staining for (B) CD44 and CD62L to determine the memory subset, the markers for swollen cells (C) CX3CR1 and (D) KLRG-1, and the markers for exhaustion (E) PD-1, (F) Tim3, and (G) Lag-3. The results shown are from 4-5 mice per group from one of two independent experiments. [Figure 9]Mice primed with a single dose of the AdHu5-NY-ESO-1157-165 minigene generated a higher percentage of circulating NY-ESO-1157-165 Tet+ CD8 T cells after tumor exposure, demonstrating better control of tumor growth. (A) Animals were subcutaneously injected with either 1 × 10⁸ IU AdHu5-NY-ESO-1 mini or 1 × 10⁹ IU AdHu5-NY-ESO-1 FL at day 53 (solid line) or day 99 (dashed line) with either 1 × 10⁶ (solid line) or 5 × 10⁵ (dashed line) HHD-NY-ESO-1 sarcoma cells. As a negative control, groups of mice were either immunized with 1 × 10⁸ IU of an unrelated AdHu5-minigene construct (N=5) or left naive (N=10). Tumors were measured every 1-2 days using a digital calyx. (B) Circulating levels of NY-ESO-1157-165 Tet+ cells were measured by tetramer staining in blood collected 14 days after tumor exposure. (C-D) Tumor size measured at (C) early (day 14) and (D) late, day 27 / 28, relative to the level of NY-ESO-1157-165 Tet+ detected in blood before tumor exposure is shown. Statistical measurements were performed by t-tests. The data shown are from two separate, independent experiments. [Figure 10] Tumor-derived NY-ESO-1157-165 Tet+ CD8 T cells (TILs) showed elevated levels of exhaustion and activation markers. Mice were sacrificed when a humane endpoint was reached, either due to unhealed ulcers or when their size approached 1300 mm3, and the spleen and tumors were removed and analyzed. Lymphocytes were isolated from both compartments, and (A) the percentage of CD8 T cells was measured. (B) The percentage of NY-ESO-1157-165 Tet+ cells was also determined, along with the levels of exhaustion markers (C) PD-1, (D) Tim-3, and (E) Lag-3, as well as the apoptosis marker (F) FasL. [Figure 11]Following AdHu5-NY-ESO-1157-165 minigene immunization, CX3CR1 was preferentially upregulated on NY-ESO-1157-165 Tet+ CD8 T cells in the spleen and TILs. Lymphocytes isolated from TILs or spleen upon reaching a humanitarian endpoint were stained with tetramers, and the levels of the following molecules on Tet+ cells were determined: (A) the swelling marker CX3CR1 in the spleen and (B) TILs; (C) the effector memory phenotype markers CD44 and CD62L, and (D) the resident memory markers CD103 and CD69; and (E) the levels of CD4+ regulatory T cells (Tregs) in both compartments were also determined by intracellular staining. [Figure 12-1] CX3CR1+ CD8 T cells are more resistant to oxidative stress and contain higher levels of healthy, polarized mitochondria. (A) Intracellular reactive oxygen species (ROS) levels in CX3CR1+ / - GFP splenocytes from Ad-lacZ or MCMV-infected mice were detected by CellROX Red assay after 51 days post-infection (N=2 independent experiments). (B) Peripheral blood lymphocytes from C57BL / 6 mice infected with MCMV or AdHu5 recombinant adenovector (Ad-I8V) more than 100 days prior were stained with MitoTracker Green (detects all mitochondria) and MitoTracker DeepRed (detects only healthy, polarized mitochondria), then surface-stained with anti-mouse CD8, anti-mouse CX3CR1, and LiveDead nearIR Fixable Marker, and subsequently analyzed by LSRII, with data calculated using FlowJo. Antigen-specific CX3CR1+ swollen cells contain healthier mitochondria and exhibit enhanced redox resilience. [Figure 12-2](D) and (E) show that when incubated in serum-free medium (i.e., stress), the CX3CR1+ population survived significantly better than CX3CR1-negative T cells in the bulk (Figure D) and antigen-specific populations (Figure E). (F) shows the levels of reactive oxygen species (ROS) under serum starvation, indicating that CX3CR1+ T cells (bulk and antigen-specific) intrinsically have lower levels of ROS and are more resistant to oxidative stress. [Figure 13] Prophylactic immunization with the HPV16 E749-57 minigene vector provides protection from tumor exposure. E749-57 specific cells can migrate to the tumor transplantation site, providing protection from tumor exposure. [Figure 14] Synergistic effects after immunization at suboptimal doses of panel minigenes encoding CD8 T cell epitopes against MCMV. A panel of three minigenes against known MCMV-specific CD8 T cell epitopes, namely M45 (985HGIRNASFI993), M38 (316SSPPMFRV325), and m139 (419TWYGFCLL426), was constructed. These were intravenously injected into C57BL / 6 mice either as individual minigenes or as a cocktail. The minigenes encoding M38 and m139 were injected at a suboptimal dose of 1 × 10⁷ infection units (IU), while the minigene encoding M45 was injected at an optimal dose of 1 × 10⁸ IU. Levels of M38-specific cells in the blood were measured at 6 days post-immunization. Surprisingly, mice that received a mixed minigene vaccine containing suboptimal doses of M38 and m139 minigene vectors, plus an optimal dose of M45 minigene, generated higher levels of M38-specific T cells compared to the group injected with suboptimal doses of M38 minigene vector alone. This unexpected result suggests that delivery of a cocktail of minigene vectors at suboptimal doses may produce an additive effect, enhancing the scale of antigen-specific T cells more than that observed with immunization using a single vector alone. [Figure 15]CD8 T cells derived from tumors of AdHu5-AH1-MG immunized mice express higher levels of granzyme B. 15A shows the levels of granzyme B in all CD8 T cells of the tumor 23 days post-transplantation and 16 days post-immunization, as well as the tumor size at the time of analysis. 15B shows the levels of the transcription factors T-bet and Eomes in AH1-specific CD8 T cells of the tumor 23 days post-transplantation and 16 days post-immunization. [Figure 16] Testing of the GP70423-431(AH1) minigene as a therapeutic vaccine in combination with anti-PD-L1. 16A shows that groups of mice immunized with the indicated adenovector 7 days after tumor exposure were subsequently treated with anti-PD-L1 or isotype control. Individual mouse tumor sizes are shown. 16B shows the survival curves for all mouse groups. 16C shows the percentage of circulating GP70423-431(AH1)Tet+ cells 15 days after immunization (22 days after tumor exposure). 16D shows that the relative tumor growth rates in each group were compared using the Mann-Whitney U test. *p<0.05, **p<0.005 [Figure 17-1] Single cells derived from spleen and tumors from prophylactic (A, C) or therapeutic (B, D) vaccinations were stimulated ex vivo with AH1 peptide (4 μg / ml) or PMA-ionomycin (IO) for 7 hours, and then stained for intracellular cytokine production of IFNγ. Low-level background activation (culture medium only) was subtracted for each sample. [Figure 17-2] Single cells derived from spleen and tumors from therapeutic vaccination with 17E-H anti-PD-L1 were stimulated ex vivo with AH1 peptide (4 μg / ml) or PMA-ionomycin (IO) for 7 hours, and then stained for intracellular cytokine production of IFNγ. Low-level background activation (culture medium only) was subtracted for each sample. CD8 T cell responses in spleen (17E) and tumor (17G), and CD4 T cell responses in spleen (17F) and tumor (17H) are shown. [Figure 18]A pilot experiment to determine whether two minigenes encoding two tumor antigens improve tumor control. A shows the protocol used: tumor transplantation on day 0, vaccination with one of the following at day 7: AdHu5-AH1 minigene (MG), AdHu5-e2F8-27mer MG, Combo (both MGs - AdHu5-AH1 and AdHu5-e2F8-27mer), unrelated AdHu5-MG, no vaccination, N=6 per treatment group. On days 12, 16, and 19 post-transplant, half of each group was treated with the checkpoint inhibitor anti-PD-1 and the other half with an isotype control. Blood samples were taken on days 13 and 20. Figures 18B, C, D, E, and F show tumor growth over time. [Figure 19] This paper compares mixed minigene treatment with anti-PD-1 to vaccination with negative controls and single minigenes. [Figure 20] Tumor growth rates calculated by linear regression for mixed minigene treatment, single minigene treatment, and negative controls. [Figure 21] %CD8+ AH1-tet+ cells and %CD8+ ef28-tet+ cells produced from vaccination with mixed minigene treatment and vaccination with single minigenes AdHu5-AH1 and AdHu5-e2F8-27mer, and measured 6 days after vaccination. [Figure 22-1] Simultaneous intravenous immunization with two mini-gene constructs / vaccines (combo-AdHu5-AH1 and AdHu5-e2F8) induces antigen-specific populations of similar size and phenotype to those of a single vaccine, as measured 11 days after vaccination. [Figure 22-2] Simultaneous intravenous immunization with two mini-gene constructs / vaccines (combo-AdHu5-AH1 and AdHu5-e2F8) induces antigen-specific populations of similar size and phenotype to those of a single vaccine, as measured 11 days after vaccination. [Figure 23]Immunization with two minigenes (AdHu5-AH1 and AdHu5-e2F8) that target CD8 T cell epitopes in cancer cells controls tumor growth. Linear regression data in Figure 20 are recalculated as relative growth rates. [Figure 24] Atypical Subset of Memory T Cells: Transcriptional Profiling of Expanding Memory T Cells. (A) PCA of Expanding / Non-Expanding CD8 T Cells. 3D PCA showing the distribution of transcriptional profiles of two independent models: an expanding sample (M38, D8V (later period), i.e., expanding memory, circled in blue) and a non-expanding sample (M45, I8V (later period), i.e., central memory, circled in brown), as well as a naive sample, at the acute phase (day 7 or 21) and later periods (day 50 or 100). (B) PCA of Exhausted / Non-Exhausted CD8 T Cells. 3D PCA showing the distribution of transcriptional profiles of an exhausted model (Cl13 at day 30, tetrahedrons circled in gray), along with a non-exhausted sample (Arm at day 30, spheres circled in blue), and a naive sample, at different time points. Timing: Day 6 (yellow), Day 8 (brown), Day 15 (pink), Day 30 (black), Naive (green). [Figure 25]The inflatable memory subset expresses different gene modules compared to other T cell memory subsets. (A) Weighted co-expression network analysis of inflatable samples. Co-expression network analysis detected six gene modules (merge distance = 0.25, soft-thresholding power β = 9); blue module (highlighted) genes are enriched with respect to immune-related GO categories and include relevant genes such as Tbx21, Eomes, Zeb2, and E2f2. (B) PCA of inflatable / exhausted samples based on blue module genes. PCA plot based on the gene set of 588 genes detected as blue modules in the co-expression network analysis of inflatable samples only, using the first three principal components. The plot shows the distribution of naive (green), non-inflatable and non-exhausted (blue), as well as inflatable and exhausted (red) samples (sphere: exhausted studies; tetrahedron: inflatable studies) (the inflatable memory population is the red tetrahedron surrounded by a blue circle). (C) Hierarchical clustering of expandable / exhausted samples based on blue module genes. Dendrogram plot showing sample clustering analysis (Euclidean distance) for expandable-exhausted merged sets based on the gene set of 469 genes detected as blue modules in the repeat gene co-expression network analysis of expandable samples after removing outliers (Soft-thresholding power β=20). Memory expandable clusters are contained within rectangles. [Figure 26A] (A) A schematic diagram of the AdHu5 adenovirus with a mini-gene immunogen cassette and a close-up view of the mini-gene immunogen cassette are shown. [Figure 26B] (B) A schematic diagram of the AAV ITR with the mini-gene immunogen cassette, and a close-up view of the mini-gene immunogen cassette are shown. [Modes for carrying out the invention]

[0019] Herein, the present invention is further described. In the following paragraphs, various aspects of the present invention are defined in more detail. Each of the aspects thus defined may be combined with any other aspect unless otherwise clearly indicated. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous. The practice of the present invention employs the usual techniques of immunology, molecular biology, chemistry, biochemistry, and recombinant DNA technology, which are within the scope of the skills of those skilled in the art, unless otherwise specified. Such techniques are well described in the literature, see, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0020] This invention is based on the surprising finding that an adenovirus vector encoding a single cancer-specific epitope elicits an expansive memory CD8+ T cell response. The term expansive memory response refers to a sustained, functional, and durable CD8+ T cell response. The resulting pool of CD8+ T cells can resist exhaustion that may occur due to prolonged TCR stimulation. T cell exhaustion can be characterized by the upregulation of markers such as PD-1, Tim-3, and Lag-3.

[0021] Expansion memory CD8+ T cells are characterized by a unique phenotype compared to other CD8+ memory subsets, exemplified by the expression of markers CX3CR1 and KLRG-1. These cells also exhibit a transcriptional profile distinct from both the central memory T cell subset and the exhausted memory T cell subset. Furthermore, they exhibit features such as enhanced redox resilience and resilience to oxidative stress, possibly due to lower levels of reactive oxygen species intrinsically. In particular, the transcriptional profile is driven by the transcription factor Tbx21, with minimal contribution from Eomes. This results in a CD8+ T cell phenotype that is long-lived and present in large quantities in peripheral organs while retaining effector function. Antigen-specific expansion memory CD8+ T cells arise through a unique set of processing, presentation, and co-stimulatory conditions. Epitope processing occurs independently of immunoproteasomes, and later presentation by non-hematopoietic atypical APCs may help preserve this phenotype. While not bound by theory, it is believed that using the vector of the present invention, which encodes a single epitope of interest, avoids the need for antigen processing, thereby generating an expansive memory response.

[0022] In one embodiment, the present invention relates to an adenovirus vector comprising a nucleotide sequence encoding a single cancer-specific CD8+ and / or CD4+ T cell epitope, wherein the vector has the ability to induce an expansive memory CD8+ T cell response. In one embodiment, the adenovirus vector comprises a nucleotide sequence encoding a single cancer-specific CD8+ and / or CD4+ T cell epitope, e.g., a single cancer-specific CD8+ T cell epitope, and the vector does not contain any additional cancer-specific CD8+ and / or CD4+ T cell epitopes. As such, the vector of the present invention encodes a single cancer-specific CD8+ and / or CD4+ T cell epitope, e.g., a single cancer-specific CD8+ T cell epitope. The present invention does not extend to adenovirus vectors encoding more than one or more cancer-specific CD8+ and / or CD4+ T cell epitopes.

[0023] In one embodiment, the present invention relates to an adenovirus vector comprising a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope, wherein the vector has the ability to induce an expansive CD8+ T cell response. In one embodiment, the adenovirus vector comprises a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope, and the vector does not contain any additional cancer-specific CD8+ T cell epitopes. As such, the adenovirus vector of the present invention encodes a single cancer-specific CD8+ T cell epitope. The present invention does not extend to adenovirus vectors encoding more than one or more cancer-specific CD8+ T cell epitopes.

[0024] The vector of the present invention, encoding a single cancer-specific CD8+ T cell epitope, can generate a sustained, functional, and durable CD8+ T cell response from a single dose. The resulting CD8+ T cell pool can resist exhaustion that may occur due to prolonged TCR stimulation. The resulting CD8+ T cell pool may also exhibit enhanced redox resilience and low levels of reactive oxygen species.

[0025] As used herein, the term “vector” refers to a nucleic acid sequence capable of transporting another nucleic acid to which the vector sequence is ligated into a cell. The vector of the present invention is an adenovirus and comprises a nucleotide sequence encoding a single cancer-specific CD8+ or CD4+ T cell epitope, which contains a gene construct in a form suitable for expression by cells (e.g., ligated to a transcriptional regulatory element).

[0026] As used herein, the term “epitope” refers to a portion of an antigen recognized by the immune system, which may be a short protein sequence. “Cancer-specific CD8+ and / or CD4+ T cell epitopes” refer to epitopes that may be presented by antigen-presenting cells bound to MHC molecules and subsequently recognized by T cell receptors (TCRs). CD4+ T cells express a CD4 coreceptor that binds to MHC II and recognize peptides presented by MHC II molecules. CD8+ T cells express a CD8 coreceptor that binds to MHC I and recognize peptides presented by MHC I molecules.

[0027] Expansion memory T cells can be characterized by the presence of specific markers and cell surface markers. Methods for identifying and quantifying these markers are well known in the art. Examples of appropriate methods include, but are not limited to, affinity-based separation methods, magnetic cell sorting techniques, and fluorescence-based cell sorting techniques such as FACS (fluorescence-activated cell sorting). Expansion memory CD8+ T cells can be characterized by the presence of several markers, examples of which include, but are not limited to, CX3CR1, KLRG-1, and CD44. Expansion memory CD8+ T cells can also be characterized by the low expression of several markers, examples of which include, but are not limited to, CD62L, CD27, and CD127. The term "low expression" may refer to cells that do not express the marker, or it may refer to cells that express the marker at a lower level compared to other cells in a sample.

[0028] In one embodiment, expanding memory CD8+ T cells are characterized by markers selected from the group including CX3CR1+, KLRG-1+, CD44+, and CD62L-, where the symbol (+) indicates the presence of the marker and the symbol (-) indicates low or absent expression of the marker. If the symbol (-) means low expression, this may be further indicated by "(low)". Expanding memory CD8+ T cells may be characterized by markers selected from the group including CX3CR1+, KLRG-1+, CD44+, CD62L-, CD27-(low), and CD127-(low).

[0029] Expansion memory CD8+ T cells can be characterized by the phenotype CX3CR1+, KLRG-1+, CD44+, CD62L-. Expansion memory CD8+ T cells can also be characterized by the phenotype CX3CR1+, KLRG-1+, CD44+, CD62L-, CD27-(low), CD127-(low).

[0030] CD8+ T cells produced in the expansion memory response may possess several other characteristics. For example, the cells may have a transcriptional profile driven by Tbx21 (also known as T-bet). These cells exhibit sustained expression of Tbx21. These cells may also exhibit sustained expression of E2f2, a transcription factor generally involved in cell growth and proliferation. These cells may also lack or have low expression of the transcription factor Eomes.

[0031] Expanding memory CD8+ T cells may not exhibit classical contraction after exposure to an antigen. During classical memory evolution after antigen exposure, the cells form a contracted central memory pool, comprising less than 1% of the total circulating CD8+ T cells. However, expanding memory cells are maintained as a large pool of cells circulating in the blood. As such, in one embodiment, the resulting expanding memory CD8+ T cells constitute approximately 2% to approximately 20% of the total CD8+ T cells, preferably approximately 8% to approximately 20%, and more preferably approximately 12% to approximately 20%.

[0032] In one embodiment, a large pool of expandable memory CD8+ T cells retains their effector memory phenotype. The resulting expandable memory CD8+ T cells can retain their memory effector phenotype for an extended period, characterized by CD44+, CD62L-. Expandable memory CD8+ T cells can retain their memory effector phenotype for up to 60 days, up to 55 days, up to 50 days, up to 40 days, or up to 30 days after exposure to the vector of the present invention.

[0033] Swelling memory CD8+ T cells may also lack markers of exhaustion. T cell exhaustion can result from excessive TCR (T cell receptor) stimulation. Markers of T cell exhaustion may include upregulation of markers such as PD-1, Tim-3, and Lag-3. As such, in one embodiment, swelling memory CD8+ T cells may lack or show low expression of markers selected from the group consisting of PD-1, Tim-3, and Lag-3.

[0034] The nucleotide sequence encoding a single cancer-specific CD8+ and / or CD4+ T cell epitope may comprise approximately 12 to 45 base pairs; in another embodiment, the nucleotide sequence may comprise approximately 15 to 45 base pairs; in another embodiment, the nucleotide sequence may comprise approximately 18 to 45 base pairs; in another embodiment, the nucleotide sequence may comprise approximately 21 to 45 base pairs; and in a preferred embodiment, the nucleotide sequence may comprise approximately 24 to 45 base pairs. As such, the vector encodes a single cancer-specific CD8+ and / or CD4+ T cell epitope comprising approximately 5 to 15 amino acids; in another embodiment, the vector encodes an epitope comprising approximately 6 to 15 amino acids; in another embodiment, the vector encodes an epitope comprising approximately 7 to 15 amino acids; and in a preferred embodiment, the vector encodes an epitope comprising approximately 8 to 15 amino acids.

[0035] A single cancer-specific CD8+ and / or CD4+ T cell epitope is an immunogenic epitope in that it triggers an immune response. T cell epitopes bind to major histocompatibility complexes to elicit a subsequent immune response. In some embodiments, such an epitope has the ability to bind to and be presented to MHC molecules. There are numerous methods known in the art for identifying epitopes that bind to MHC and thus produce an immune response. These methods include peptide-MHC binding predictive models, and there are numerous publicly available programs of such models.

[0036] In one embodiment, a single cancer-specific CD8+ and / or CD4+ T cell epitope is derived from a tumor-associated antigen (TAA). TAAs are antigenic products produced by cancer, and they provide biomarkers for tumor target identification. TAAs can be broadly classified into abnormally expressed autoantigens, mutated autoantigens, and tumor-specific antigens. As such, TAAs can be upregulated or overexpressed in cancer cells. TAAs can mutate within cancer cells. TAAs may be specific to cancer cells and expressed only within cancer cells; these may also be called tumor-specific antigens.

[0037] In some embodiments, cancer-specific CD8+ and / or CD4+ T cell epitopes are mutated in cancer cells. In some embodiments, cancer-specific CD8+ and / or CD4+ T cell epitopes are overexpressed in cancer cells. In some embodiments, cancer-specific CD8+ and / or CD4+ T cell epitopes are non-coding tumor-specific epitopes. As used herein, the term “non-coding tumor-specific epitope” refers to a peptide found in cancer cells that derives from a nucleotide sequence that is epigenetically repressed in healthy cells. These peptide sequences are abnormally expressed in tumor cells.

[0038] In one embodiment, cancer-specific CD8+ and / or CD4+ T cell epitopes are not latent epitopes. As used herein, “latent epitope” means an epitope that is not immunogenic in an immunocompetent individual.

[0039] In one embodiment, cancer-specific CD8+ and / or CD4+ T cell epitopes may be viral epitopes associated with virally driven cancers. Virally driven cancers may be HPV (human papillomavirus), HTLV (human T-lymphotropic virus), or EBV (Epstein-Barr virus).

[0040] In one embodiment, cancer-specific CD8+ and / or CD4+ T cell epitopes are derived from tumor-associated antigens selected from the group consisting of TRP-1, CEA, TAG-72, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, Melan-A / MART-1, tyrosinase, CLPP, cyclin-A1, cyclin-B1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2a, CD45, glypican-3, IGF2B3, kallikrein-4, KIF20A, lengusin, meroe, MUC5AC, survivin, PRAME, SSX-2, NY-ESO-1 / LAGE1, gp70, MC1R, TRP-1 / -2, β-catenin, BRCA1 / 2, and CDK4.

[0041] Cancer-specific CD8+ and / or CD4+ T cell epitopes may be private epitopes. As used herein, the term “private epitope” refers exclusively to an epitope found in only one antigen in only one cancer. Cancer-specific CD8+ and / or CD4+ T cell epitopes may be public epitopes. As used herein, the term “public epitope” refers to an epitope found in cancers of two or more people.

[0042] In some embodiments, cancer-specific CD8+ and / or CD4+ T cell epitopes may be neoepitopes. As used herein, the term “neoepitope” refers to an epitope arising through mutation within tumor cells, in particular somatic mutations or passenger mutations which may result in the generation of a neoepitope. In some embodiments, cancer-specific CD8+ and / or CD4+ T cell epitopes are not neoepitopes.

[0043] In one embodiment, cancer-specific CD8+ and / or CD4+ T cell epitopes are specific to colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein-Barr-driven cancer, human papillomavirus-driven cancer, and soft tissue sarcoma. As used herein, the term “cancer” refers to a disease having abnormal cell growth, and as used herein, the term refers to both primary tumors and metastases of primary tumors.

[0044] In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope includes SEQ ID NO: 1 (SPSYVYHQF), SEQ ID NO: 2 (SLLMWITQC), or SEQ ID NO: 37 (SLLMWITQV). If the cancer-specific CD8+ and / or CD4+ T cell epitope is a viral epitope associated with a virally driven cancer, the epitope may include SEQ ID NO: 7 (RAHYNIVTF). The virally driven cancer may be selected from EBV-driven cancer, HTL-driven cancer, and HPV-driven cancer. EBV-driven cancers may be selected from Hodgkin lymphoma (HL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), as well as two rarer tumors associated with severe immune deficiencies, plasmablastic lymphoma (PBL) and primary coeloplasmic lymphoma (PEL), T-cell or NK-cell LPD and malignant lymphomas, epithelial nasopharyngeal carcinoma (NPC) and gastric carcinoma, and leiomyosarcoma. HPV-driven cancers may include anogenital cancers, oropharyngeal cancers, oral cancers, head and neck squamous cell carcinomas, and pharyngeal cancers.

[0045] In one embodiment, the cancer-specific CD8+ and / or CD4+ T cell epitope includes one or more of the epitopes in Table 1.

[0046] [Table 1]

[0047] Furthermore, cancer-specific CD8+ and / or CD4+ T cell epitopes can be determined using techniques known in the art, such as proteomics approaches, mass spectrometry approaches, genomic approaches, transcriptome analysis, bioinformatics approaches, and in silico methods. Those skilled in the art can select appropriate epitopes to be encoded in the vectors of the present invention.

[0048] Nucleic acids encoding cancer-specific CD8+ and / or CD4+ T cells may be codons optimized for mammalian codon use frequency. Appropriately, their nucleic acid sequences may be codons optimized for human codon use frequency.

[0049] The vector may contain adeno-associated virus (AAV). The vector may contain adenovirus. Adenovirus vectors or AAV vectors may also have additional features such as enhancer and promoter regions. In some embodiments, the vector may include a strong promoter, examples of which include, but are not limited to, the CMV promoter, the RSV promoter, and the EF1α promoter. In a preferred embodiment, the vector includes a CMV promoter, and a suitable sequence for the CMV promoter is provided in SEQ ID NO: 18. In some embodiments, the vector may include a TATA box. In some embodiments, the vector includes a translation initiation sequence, such as a Kozak sequence. The Kozak sequence has a consensus sequence (gcc)gccRccAUGG, and a suitable Kozak sequence is provided in SEQ ID NO: 19. In some embodiments, the vector includes a termination sequence and / or a polyadenylation sequence. A suitable polyadenylation sequence is provided in SEQ ID NO: 34. AAV vectors may include a reverse-terminal repeat (ITR) sequence. A suitable ITR sequence is provided in SEQ ID NO: 42.

[0050] In one embodiment, the vector does not contain additional cancer-specific CD8+ and / or CD4+ T cell epitopes. The vector encodes only a single cancer-specific CD8+ and / or CD4+ T cell epitope. In one embodiment, the adenovirus vector consists of a vector backbone, a promoter region, and a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope. The adenovirus backbone may include additional features such as an enhancer region, promoter region, TATA box, and translation initiation sequence.

[0051] The AAV vector may be serotype 1, 2, 3, 4, 5, 6, 7, 8, or 9. In a preferred embodiment, the AAV vector may be serotype 2 or 5. The AAV vector may contain an ITR sequence, which in a preferred embodiment is adjacent to an encoded cancer-specific CD8+ and / or CD4+ T cell epitope. There may be an ITR sequence located 5' to the cancer-specific epitope and an ITR sequence located 3' to the cancer-specific epitope. The 5' ITR sequence may include SEQ ID NO: 39. The 3' ITR sequence may include SEQ ID NO: 42. The AAV vector may contain a sequence on the 5' side of the cancer-specific epitope, e.g., SEQ ID NO: 38. The AAV vector may contain a sequence on the 3' side of the cancer-specific epitope, e.g., SEQ ID NO: 41. A helper plasmid may be used to produce an AAV vector containing a cancer-specific CD8+ and / or CD4+ T cell epitope. The helper plasmid may be used to provide genes required for AAV replication or packaging. In one embodiment, the helper plasmid encodes E2A, E4, and VA adenovirus proteins, as well as / or the rep and cap genes of AAV.

[0052] The adenovirus vector may be a serotype of species C. Species C includes serotypes Ad1, 2, 5, and 6. In a preferred embodiment, the adenovirus vector is human serotype 5 (AdHu5). The adenovirus vector may preferably be modified, for example, to reduce immunogenicity and improve the biosafety of the vector. As such, the adenovirus vector may be non-replicable. The adenovirus vector may lack E1 and E3 proteins. The adenovirus vector may contain a sequence on the 5' side of a cancer-specific epitope, e.g., sequence number 13. The adenovirus vector may contain a sequence on the 3' side of a cancer-specific epitope, e.g., sequence number 14.

[0053] Other adenovirus vectors may also be suitable as vectors for the present invention. In some embodiments, the vector may be an adenovirus vector derived from an animal, such as a dog, a monkey, and especially rhesus monkeys and chimpanzees. In some embodiments, the adenovirus vector may be a rare serotype vector derived from a non-human primate. Vectors derived from chimpanzees may be suitable as vectors for the present invention, including, but are not limited to, ChAd63, ChAd3, and ChAdY25.

[0054] In one embodiment, an immunogenic composition comprising a vector as defined above is provided. The immunogenic composition may further comprise one or more additional active ingredients, a pharmaceutically acceptable carrier, a diluent, an excipient, or an adjuvant.

[0055] An immunogenic composition containing the vector according to the present invention may be used in combination with at least one other immunogenic composition containing the vector according to the present invention, each vector encoding a different cancer-specific CD8+ and / or CD4+ T cell epitope. An immunogenic composition containing the first vector according to the present invention may be administered separately, sequentially, or simultaneously with an immunogenic composition containing the second vector according to the present invention.

[0056] In one embodiment, the immunogenic composition may comprise at least two vectors according to the present invention. It is preferable that at least two vectors encode different cancer-specific CD8+ and / or CD4+ T cell epitopes. If further additional vectors are present in the composition, they may encode different cancer-specific CD8+ and / or CD4+ T cell epitopes. The immunogenic composition may further comprise one or more additional active ingredients, pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. While not bound by theory, the use of a cocktail of vectors encoding different epitopes may result in a stronger immune response and, furthermore, a synergistic effect that enhances the immune response.

[0057] If the composition of the present invention comprises at least two vectors as described herein, the vectors may be provided as separate drugs for administration at the same or different time points.

[0058] In some embodiments, if the system includes at least two vectors as described herein, the vectors may be provided as separate drugs for administration at different time points. When administered separately and at different time points, one vector may be administered first. In some embodiments, both may be administered on the same day or on different days, and they may be administered using the same or different schedules throughout the treatment cycle.

[0059] Alternatively, if the composition contains at least two vectors as described herein, the administration of the vectors may be carried out simultaneously. When simultaneous administration is used, the vectors may be formulated as separate pharmaceutical compositions. In a preferred embodiment, at least two vectors may be formulated as a single pharmaceutical composition.

[0060] The compositions of the present invention may take the form of liquids, such as solutions, emulsions, or suspensions. Whether they are solutions, suspensions, or other similar forms, the liquid compositions of the present invention may also include one or more of the following: water, saline solution, preferably physiological saline, Ringer's solution, isotonic saline, synthetic mono or diglycerides, fixing oils such as polyethylene glycol or glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and tonic agents such as sodium chloride or dextrose. The compositions may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials.

[0061] Intravenous formulations of the vector or composition of the present invention may take the form of sterile aqueous or non-aqueous (e.g., oily) solutions or suspensions for injection. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable media and solvents that may be used include water, phosphate buffer solution, Ringer's solution, and isotonic saline. In addition, sterile fixatives may be used as solvents or suspending agents. For this purpose, any non-irritating fixative may be used, including synthetic mono or diglycerides. Furthermore, fatty acids such as oleic acid may be used in the preparation of the intravenous formulation of the present invention.

[0062] Immunogenic compositions can be prepared using methodologies well known in the pharmaceutical field. For example, a composition intended for administration by injection can be prepared by combining the vector of the present invention with water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension.

[0063] In one embodiment, the present invention relates to a host cell comprising a vector or immunogenic composition as described herein. The host cell may be a mammal, such as a human or mouse. The host cell can be transduced with the vector. The host cell may be used to produce an adenovirus stock.

[0064] In one embodiment, the vector or immunogenic composition is for therapeutic purposes. In a preferred embodiment, the vector or immunogenic composition is for the treatment or prevention of cancer. The term “treatment” refers to the medical management of a patient with the intention of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward improvement of the disease, pathological condition, or disorder, and also causal treatment, i.e., treatment directed toward the removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes symptomatic treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, i.e., treatment directed toward minimizing, partially or completely inhibiting, the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement other specific treatments directed toward improvement of the associated disease, pathological condition, or disorder.

[0065] The present invention further relates to a method for treating or preventing cancer, comprising the step of administering a therapeutically effective amount of the vector or composition according to the present invention to a subject in need thereof.

[0066] In one embodiment, the present invention relates to the use of a vector or composition described herein in the manufacture of a drug for the treatment or prevention of cancer. In one embodiment, the present invention relates to the use of a vector or composition described herein in the treatment or prevention of cancer.

[0067] As used herein, the term “therapeutically effective” means that the amount of the composition used is sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement requires only a reduction or change, and not necessarily elimination.

[0068] The present invention also provides a method for inducing an expansive memory CD8+ T cell response, the method comprising the step of administering a therapeutically effective amount of the vector or composition according to the present invention to a subject in need thereof, wherein the CD8+ T cells are characterized by a marker selected from the group including CX3CR1+, KLRG-1+, CD44+, and CD62L-.

[0069] Preferably, CD8+ T cells are characterized by the phenotypes CX3CR1+, KLRG-1+, CD44+, and CD62L-. More preferably, they are characterized by the phenotypes CX3CR1+, KLRG-1+, CD44+, CD62L-, CD27(low), and CD127(low).

[0070] Vectors or immunogenic compositions may be used to treat or prevent colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, and soft tissue sarcoma.

[0071] Vectors or compositions as described herein may be administered by any conventional route. These routes include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, nasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, and inhalation routes. Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. In some embodiments, the vector or composition is administered intravenously or intramuscularly. The composition may take the form of one or more dosage units.

[0072] In certain embodiments, it may be desirable to administer the vector or composition of the present invention topically to an area requiring treatment, such as at the site of a tumor. In other embodiments, it may be desirable to administer the vector or composition by intravenous injection or infusion. The amount of the vector of the present invention that is effective / active in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, optionally, in vitro or in vivo assays may be used to help identify the optimal dosage range. The exact dose used in the composition also depends on the route of administration and the severity of the disease or disorder and should be determined according to the physician's judgment and the circumstances of each patient.

[0073] The composition contains an effective amount of the vector according to the present invention to obtain an appropriate dosage. The correct dosage of the compound varies depending on the specific formulation, mode of administration, and the specific site, host, and disease to be treated. Other factors such as age, weight, sex, diet, time of administration, rate of elimination, host condition, mixed drugs, response sensitivity, and disease severity should be taken into consideration. Administration may be continuous or periodic.

[0074] In the treatment of cancer, the vectors or immunogenic compositions of the present invention may be used in combination with existing therapies. In one embodiment, the vectors or compositions are used in combination with existing therapies or therapeutic agents, such as anti-cancer therapies. Accordingly, in another embodiment, the present invention also relates to combination therapies comprising the administration of the vectors or compositions of the present invention and anti-cancer therapies. Anti-cancer therapies may include therapeutic agents or radiotherapy, and may include gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anti-cancer therapy, or tumor regression agents. Other examples of therapeutic agents include checkpoint inhibitors, anti-cancer agents, immunogenic agents, attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), target small molecules and biological molecules (e.g., components of signaling pathways, e.g., tyrosine kinase modulators and receptor tyrosine kinase inhibitors, as well as agents that bind to tumor-specific antigens, e.g., EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or immunosuppressants, and cells transfected with genes encoding immunostimulatory cytokines (e.g., GM-CSF), and chemotherapy. In one embodiment, the vector or composition is used in combination with surgery. The vector or composition of the present invention may be administered at the same time as or at different time points as other treatments, for example, simultaneously, separately, or sequentially.

[0075] In some embodiments, the vector or composition is used in combination with an immunomodulator. The immunomodulator may be administered simultaneously, sequentially, or separately. In certain embodiments, the immunomodulator may be an immune checkpoint inhibitor, and examples of immune checkpoint inhibitors include, but are not limited to, inhibitors of immune checkpoint proteins selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, LAG-3, B7-H3, B7-H4, B7-H6, A2aR, BTLA, GAL9, and IDO.

[0076] Certain tumor types have been previously reported to be unresponsive to anti-PD-1 and anti-PD-L1 monotherapy. Surprisingly, it is shown herein that immunization with a minigene vector, when administered in combination with a checkpoint inhibitor such as anti-PD-L1 therapy, can result in enhanced tumor control. This has been shown to be effective in tumor models known to be unresponsive to standard checkpoint inhibitor therapy. As such, in some embodiments, the vector or composition may be used in combination with a checkpoint inhibitor for the treatment of checkpoint inhibitor-unresponsive tumors.

[0077] The vector or composition and immunomodulator of the present invention may be provided as separate drugs for administration at the same or different time points. In some embodiments, the vector or composition and immunomodulator of the present invention are provided as separate drugs for administration at different time points. When administered separately and at different time points, either the vector or the immunomodulator may be administered first. In some embodiments, both may be administered on the same day or on different days, and they may be administered using the same or different schedules throughout the treatment cycle.

[0078] Alternatively, the administration of the immunomodulator may be carried out simultaneously with the administration of the vector or immunogenic composition. When simultaneous administration is used, the vector or immunogenic composition and the immunomodulator may be formulated as separate pharmaceutical compositions. The vector or immunogenic composition and the immunomodulator may be formulated as a single pharmaceutical composition.

[0079] The vectors or compositions of the present invention may be administered prophylactically or therapeutically. The term “prophylactically” refers to an administration intended to produce a protective effect against disease. The term “therapeutically” refers to an administration intended to produce a curative effect.

[0080] The vectors or compositions of the present invention may be administered as a single dose. The administration may be provided in a prophylactic or therapeutic setting. In some embodiments, the single dose may be provided as a single-dose unit further comprising one or more active ingredients, a pharmaceutically acceptable carrier, a diluent, an excipient, or an adjuvant.

[0081] The vector or composition of the present invention may be administered in multiple doses. When multiple doses are administered, one or more doses may be administered prophylactically, or one or more doses may be administered therapeutically. When multiple doses are administered, one or more doses may be administered prophylactically and one or more doses may be administered therapeutically. In one embodiment, the vector may be administered as a "prime-boost" regimen, with an initial dose of the adenovirus vector (priming dose), followed by a second dose (boost dose).

[0082] Medication delays and / or dose reductions, as well as schedule adjustments, will be implemented if necessary, depending on the individual patient's tolerance to the treatment. If the immunogenic composition comprises at least two vectors, and the vectors encode different epitopes as described above, a synergistic effect may exist between the vectors. As such, each vector may be administered in a suboptimal dose. The term “suboptimal” dose refers to a dose level that is not intended to completely eliminate or eradicate the tumor, but nevertheless causes some tumor cells or tissue to become necrotic. Those skilled in the art can determine the appropriate dose required to achieve this based on factors such as the patient’s age, the stage of the disease, and the size and location of the tumor or metastasis.

[0083] In one embodiment, a method for producing the vector described above: i) A step of synthesizing nucleic acid sequences encoding epitopes as sense and antisense primers, ii) A step of cloning the nucleic acid sequence encoding the epitope sequence into a first plasmid, iii) A step of cloning a sequence containing the nucleic acid sequence encoding the epitope into a second plasmid containing adenovirus DNA, A method is provided that includes this.

[0084] Appropriate cloning methods are known within the art, and examples of such methods include, but are not limited to, restriction ligation methods, gateway cloning, Gibson assembly, and ligation-independent cloning. Those skilled in the art can determine an appropriate method for cloning the sequence into a plasmid. The cloning method for introducing the nucleic acid sequence encoding the epitope sequence into a first plasmid may be the same as or different from the previously described cloning methods. In one embodiment, the cloning method for introducing the nucleic acid sequence encoding the epitope sequence into a first plasmid is selected from restriction ligation methods, gateway cloning, Gibson assembly, and ligation-independent cloning. In one embodiment, the cloning method for introducing the nucleic acid sequence encoding the epitope into a second plasmid containing adenovirus DNA is selected from restriction ligation methods, gateway cloning, Gibson assembly, and ligation-independent cloning.

[0085] In one embodiment, step iii) comprises cloning a sequence containing an epitope-encoding nucleic acid sequence into a second plasmid containing adenovirus DNA, wherein the sequence containing the epitope-encoding nucleic acid sequence also includes additional features selected from the group including a translation initiation sequence, a promoter, a termination sequence, and a polyadenylation sequence.

[0086] In one embodiment, a method for producing a vector is: i) A step of synthesizing nucleic acid sequences encoding epitopes as sense and antisense primers, ii) A step of annealing the sense and antisense primers, iii) The annealed primers are digested with an appropriate restriction enzyme to enable insertion into the donor plasmid, and iv) The step of transferring the donor plasmid into a second plasmid containing adenovirus DNA, Includes.

[0087] Appropriate restriction enzymes and sites are known to those skilled in the art. Designing appropriate restriction sites within sense and antisense primers to enable insertion into donor plasmids is within the capabilities of those skilled in the art.

[0088] The encoded epitopes are cancer-specific CD8+ and / or CD4+ T cell epitopes. Several cancer-specific epitopes have been identified and are known in the art. Selecting the appropriate epitope to encode in the vector is possible for those skilled in the art. Further methods for identifying cancer-specific epitopes are known in the art and include bioinformatics approaches, transcriptome analysis, and in silico methods.

[0089] A second plasmid encoding an adenovirus vector may include any of the following features. The adenovirus vector may include enhancer and promoter regions, such as strong promoters including the CMV promoter, RSV promoter, and EF1α promoter. In a preferred embodiment, the vector includes the CMV promoter. The vector may include a TATA box. In one embodiment, the vector includes a translation initiation sequence, such as a Kozak sequence. The Kozak sequence has a consensus sequence (gcc)gccRccAUGG. In one embodiment, the vector includes a termination sequence and / or a polyadenylation sequence. The adenovirus vector may be a serotype of species C, such as Ad1, 2, 5, and 6 serotypes. In a preferred embodiment, the adenovirus vector is human serotype 5 (AdHu5). It may be preferable for the adenovirus vector to be modified, for example, to reduce immunogenicity and improve the biosafety of the vector. As such, the adenovirus vector may be non-replicable. The adenovirus vector may lack E1 and E3 proteins.

[0090] The step of transferring the donor plasmid into the second plasmid can be carried out by any method, such as a ligation method. In one embodiment of the present invention, a kit is provided comprising a vector or immunogenic composition as described herein, one or more additional active ingredients, a pharmaceutically acceptable carrier, a diluent, an excipient, or an adjuvant, and optionally, instructions for use.

[0091] Additional active substances may include checkpoint inhibitors, anti-cancer agents, immunogenic agents, attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), target small molecules and biological molecules (e.g., components of signaling pathways, e.g., tyrosine kinase modulators and receptor tyrosine kinase inhibitors, as well as agents that bind to tumor-specific antigens, e.g., EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or immunosuppressants, and cells transfected with genes encoding immunostimulatory cytokines (e.g., GM-CSF).

[0092] Pharmaceutically acceptable carriers, diluents, excipients, or adjuvants may include sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic saline, synthetic mono or diglycerides, fixing oils such as polyethylene glycol or glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and tonic modifiers such as sodium chloride or dextrose.

[0093] In one embodiment, the present invention relates to a method for inducing a T cell immune response in animals against a cancer-specific CD8+ and / or CD4+ T cell epitope, comprising the step of contacting cells with a vector or immunogenic composition as described herein.

[0094] Cells may be brought into contact with a vector or composition in vitro, ex vivo, or in vivo. If cells are brought into contact with a vector or composition either in vitro or ex vivo, the cells may then be administered to the target.

[0095] The T cell immune response may include an expansive memory CD8+ T cell response. In another embodiment, the present invention provides vectors as shown in the examples and / or accompanying drawings.

[0096] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings generally understood by those skilled in the art. The foregoing disclosure provides a general description of the subject matter encompassed within the scope of the invention, including methods for making and using the invention, as well as the best form thereof. The following examples are provided to enable those skilled in the art to practice the invention and to provide a complete written description thereof. However, those skilled in the art will recognize that the details of these examples should not be read as limitations to the invention, and that the scope of the invention should be taken from the claims and their equivalents attached to this disclosure. Various further aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure.

[0097] All documents referenced in this specification are incorporated herein by reference in their entirety. The present invention is further described in non-limiting examples. [Examples]

[0098] Example 1 A single AdHu5 construct encoding the dominant AH1 epitope, a CD8 epitope identified in CT26 colorectal carcinoma, is immunogenic. A series of experiments in a mouse cancer model possessing endogenous neoantigens were conducted to investigate whether minigene vaccination could induce a T cell response to endogenous T cell epitopes. Using the CT26 mouse colorectal carcinoma model, its peptide sequence SPSYVYHQF (named AH-1, SEQ ID NO: 1) was identified as a protein of mouse leukemia virus (MuLV env gp70), an endogenous retrovirus recognized by Balbc mice in an H2-DL-restricted manner. 423-432 ) originated from the "latent" CD8 T cell epitope, GGPESFYCASW (MuLV env gp90 147-158 The mini-gene encoding sequence number 3, named GSW11, was also tested.d The restrictive epitope does not induce a CD8 T cell response in healthy, immunocompetent BALB / c mice—it also originates from MuLV, but it is encoded in a different open reading frame than AH-1. Furthermore, it does not follow a canonical peptide motif, therefore D d It does not stably bind to MHC molecules and therefore has a very rapid half-life of 20 minutes before it is lost from the cell surface. In contrast, AH-1 has a half-life of 60 minutes; consequently, the CD8 T cell-specific response to GSW11 occurs only when regulatory CD4 T (Treg) cells are systemically depleted (James et al., 2010 J.Immunol.185:5048~5055), resulting in a very high level of activation of antigen-presenting cells (Shevach., 2009 Immunity 30(5):636~45). This was included to investigate whether a response to such an unstable “latent” epitope could be induced by minigene immunization in immunocompetent animals without requiring systemic Treg depletion. The two epitopes were constructed as separate minigenes on the AdHu5 backbone as previously described (Figure 1A).

[0099] Ad-AH1 as 1 × 10 8 or 1 × 10 7Injection of BALB / c mice with the minigene vector AdHu5-AH1-MG at an infectious unit (IU) dose induced AH1-specific CD8+ T cells in approximately 25% of the total CD8+ T cells in the blood at 7 days post-vaccination (Figure 1C, left), as detected by AH1 tetramer staining (Figure 1B). This level gradually decreased over time to approximately 5% at 50 days post-vaccination (Figure 1C, right) and approximately 2.5% at 80 days post-vaccination (data not shown). Similar percentages were observed in C57BL / 6 mice after vaccination with the Ad minigene, although at higher levels it plateaued. The AH1-specific response could not be detected in naive mice or in mice vaccinated with the AdHu5 minigene encoding an unrelated epitope (I8V, an epitope derived from β-galactosidase (a bacterial enzyme)) (Figure 1C, left and right).

[0100] The GSW11-specific response could not be detected by either GSW11 tetramer staining or GSW11 peptide stimulation, indicating that such a "latent" unstable epitope cannot generate a CD8 T cell response in immunocompetent animals. Nevertheless, it is interesting to note that there was no difference in the scale of the AH-1+ tetramer response between the group immunized with AdHu5-AH-1 alone and the group immunized with both minigenes, AdHu5-AH-1+AdHu5-GSW11 (Figure 1C, left), suggesting that co-delivery of the AdHu5 minigene construct encoding a non-immunogenic epitope did not interfere with the induction of the AH-1-specific response.

[0101] Although the size of AH1-specific CD8+ T cells decreased over time, their phenotype remained stable – similar to that observed in 15 C57BL / 6 mice, they exhibited an effector memory phenotype (CD44+ CD62L-, Figures 1D and 1E, top row), lower levels of CD27 (Figures 1D and 1E, middle row), and higher levels of PD-1 (Figures 1D and 1E, bottom row) compared to a tetramer-negative (tet-) population in the blood from the same mice (Figures 1D and 1E). In addition, high levels of CX3CR1 and low levels of CD127 were also detected within this population. By day 50, a slight loss of its effector memory phenotype was observed, along with some upregulation of the activation markers CD27 and PD-1 (Figure 1E, left panel, top, middle, and bottom, respectively).

[0102] Example 2 AdHu-5 minigene immunization slows CT26 tumor growth in prophylactic and therapeutic immunization models. To measure the protective efficiency of this prophylactic immunization regimen, immunized animals were subcutaneously injected with CT26 tumor cells 5 days after Ad-AH1 vaccination (Figure 2A, B-C). All animals immunized with Ad-AH1 showed significant suppression of tumor growth, and one of the exposed animals (1 / 15) achieved complete remission (Figure 2B and C). As expected from immunogenicity data, no protection was observed in the group immunized only with the unrelated minigene Ad-GSW11 (Figure 2B-P1) or in the unimmunized group (Figure 2B-P1). Interestingly, low doses of Ad-AH1 (1 × 10⁻⁶) showed no protection. 7 IU) 17Slightly better control was observed in the group vaccinated with CT26 (Figure 2C-P2) (though not statistically significant). A significant difference in tumor size was observed between AD-AH1-vaccinated mice and control mice at 18 days post-tumor exposure (Figures 2E and 2G). In addition, the rate of tumor growth (determined by the slope of the linear regression line fitted to the curve) was lower in Ad-AH1-vaccinated mice compared to control mice from the day tumor growth began (Figures 2F, 2H, and 2K). In conclusion, Ad-AH1 vaccination before and after CT26 tumor exposure slows tumor growth.

[0103] Next, the minigene constructs were tested in a therapeutic exposure model (Figure 2D). A group of mice were subcutaneously injected with tumor cells, and six days later, they were intravenously injected with Ad-AH1 (Figures 2A and 2D). As previously described, immunization with Ad-AH-1 slowed tumor growth - Ad-AH1-immunized mice had significantly smaller tumors at 18 days post-immunization compared to naive and unaffected AdHu5-immunized animals (Figures 2I and 2J), and some animals remained tumor-free (1 / 10).

[0104] Example 3 AdHu5-AH1 minigene immunization alters the phenotype of specific CD8+ T cells. Tumor-bearing mice were selected and sacrificed when they reached a humane endpoint. At that point, the scale of AH-1-specific CD8 T cells in the tumor (TIL) and spleen was determined. AH1 tetramer staining (Figure 3A, top panel) showed high levels of AH1-specific CD8+ T cells in tumors from vaccinated mice, as well as in control mice (Figure 3B, top row left and right). Staining with a complete panel of fluorescent dye-conjugated antibodies (recognizing the MCMV pp89 epitope) along with different / unrelated H2-Ld-binding tetramers showed no positive cells at all (Figure 3A), confirming that the high levels of AH-1 tetramer cells were neither a result of autofluorescence nor nonspecific binding of the H2-Ld tetramer. However, it is noteworthy that a loss of tumor control had occurred at this point.

[0105] In the later stages of prophylactic immunization, populations of AH-1-specific CD8 T cells were detected in the spleen (Figure 3B, bottom row, left) in all groups, with little difference between groups. In contrast, only animals immunized with Ad-Hu5 AH-1 after tumor exposure showed elevated levels of AH-1 tetramer-positive cells in the spleen (Figure 3B, bottom row, right), suggesting that immunization can boost levels of tetramer-positive cells in other compartments.

[0106] Phenotypic analysis showed that in the spleen, these AH1-specific CD8+ T cells from Ad-AH1-vaccinated mice were effector memory cells (CD44+ CD62L-) that largely upregulated CX3CR1, CD127, Fas, and LFA-1, and downregulated CD27 and Trm cell markers (CD69+ CD103+) (Figure 3C, right panel). In contrast, AH1-specific CD8+ T cells in tumors expressed a different phenotype and, although highly upregulated, were exhausted, as evidenced by high levels of PD-1 (Figure 3C, left panel). The PD-1 upregulation is likely due to massive TCR stimulation, given that PD-1 levels on other CD8 T cells in TILs were not as high (Figure 3C, center panel). All indicated markers are elevated except for CD127, which is downregulated, and CD27 and CD69+ and CD103+, which remain the same in both tetramer-positive and non-tetramer-positive TILs (Figure 3C, left and center panels). Therefore, immunization appears to alter the levels of tetramer-positive cells in the lymphoid compartment and distort the phenotype in TILs into one of the effector memory.

[0107] Example 4 The percentage of regulatory T cells appeared lower in tumors derived from Ad-AH1-vaccinated mice compared to control mice, while Trm was increased in TILs after Ad-AH1 immunization. To determine whether immunization induces other changes in the tumor microenvironment, levels of Treg and AH-1-specific resident memory T cells (Trm) were measured. Within the CD4 T cell compartment, we found that the population of Treg (CD4+ FoxP3+) cells was lower in tumors derived from the Ad-AH1 vaccinated group compared to the control group (naive and unrelated Ad-immunized group) (Figure 4A).

[0108] Recently, antigen-specific CD8 T cells expressing the Trm phenotype have been reported to exhibit superior tumor control. Consistent with this, we found that adenovirus vector minigene immunization increased the percentage of AH1+ CD103+ CD69+ Trm cells in TILs. This was statistically significant when the immunized groups were combined and compared to negative control (IrrAd (irrelevant antigen) and naive) groups. In the naive group, despite the presence of a large population of AH1+ tetramers (Figure 8A), there was little Trm phenotype with low CD103+ CD69+ CD62L and high CD44 (Figure 4B). This increase was evident in both prophylactic and therapeutic immunization settings (Figure 4B). In summary, in this cancer model, minigene immunization appears to alter the tumor microenvironment to favorably recognize and kill tumor cells.

[0109] Example 5 Antigen-specific CD8 T cells in TILs are not responsive to congenital peptides, but spleen cells derived from congeners retain their functionality. The high percentage of AH1-specific CD8+ T cells detected in tumors by AH1 tetramer staining (Figure 3A) indicates the number of cells expressing the AH1-specific TCR. However, this does not establish whether TCR signaling and T cell activation occur through interaction with the AH1 peptide. Therefore, single cells derived from spleen and tumors were stimulated with (1) the AH1 peptide to measure the effect of TCR signaling and (2) PMA / IO to measure nonspecific activation. Production of the pro-inflammatory cytokine interferon-gamma (IFNγ) was used as a readout. Corresponding mouse-derived spleen cells were also stimulated with the homologous peptide and PMA / IO.

[0110] As shown in Figures 5A and 5B, CD8+ spleen cells from the minigene immunization group were able to respond (i.e., produce IFNγ) ex vivo when stimulated with the AH1 peptide from both prophylactically vaccinated and therapeutically vaccinated mice, while only a very small response was recorded from spleen cells not immunized with AH1. In contrast, cytokine production observed in TILs in the corresponding immunized animals was very low or absent. PMA / IO stimulation also induced very low IFN-gamma production from immunized TILs, with even lower levels observed in animals not immunized with AH-1. In summary, the results suggest that, at a late time point, antigen-specific CD8 T cells in tumors are dysfunctional, while similar antigen-specific CD8 T cells in other compartments maintain their function. Furthermore, the dysfunction in TILs is likely endogenous, as the cells were unable to respond to PMA / IO stimulation, which does not require intact antigen-presenting cells. Finally, these results also indicate that minigene immunization increases the population of IFN-gamma-producing antigen-specific cells in other compartments, such as the spleen, but this does not occur when antigen-specific cells are elevated in response to tumor cells.

[0111] Example 6 Minigene immunization induces a population of peripheral AH1-specific CD8 T cells that slow tumor growth. The additional effect of immunization was revealed when tumor growth rates were calculated. Growth rates were determined by calculating the slope of a linear regression line fitted to curves taken from the day when tumors showed clear tumor growth (7 days post-transplant for negative controls and 18 days post-transplant for AH1-immunized animals) (Figure 6A). Alternatively, relative growth rates were calculated using the same raw data (Figure 6B). We found that even when tumor escape occurred, tumors grew at a slower rate compared to controls, suggesting that the selective pressure exerted by expansive cells may have resulted in the growth of a less compatible tumor population. Alternatively, tumors may actually be dividing at the same rate as controls, but a certain proportion of them are consistently eliminated by expansive anti-AH1 CD8 T cells. When tumor growth rate (from Figure 2D) was plotted against the percentage of tetramer-positive cells in TILs or spleens after tumor exposure, a strong inverse correlation was observed between tumor growth rate and AH1 tetramer-positive splenic cells from both prophylactic and therapeutic exposure studies (Figure 3B), indicating that higher levels of these cells result in better control of tumor growth.

[0112] Example 7 Immunization with the AdHu5-AH1 minigene construct may provide better tumor control compared to immunization with AdHu5-gp90FL. The protection provided by an adenovirus construct encoding a dominant CD8 T cell epitope and a similar construct encoding the full-length protein gp90 (from which the epitope is derived) was compared in therapeutic immunization experiments. Here, the minigene construct was found to exert better control compared to AdHu5-gp90-FL, as evidenced by statistically significantly lower tumor growth rates (Figure 7A). Blood from tumor-free mice (from Figure 2B) was sampled approximately 6 months after exposure, and a population of AH1 Tet+ cells continued to be detected in circulation, indicating the long-term presence of a functional CD8 T cell response (Figure 7B).

[0113] Example 8 Immunization with the HLA:A2-restricted CD8 T cell epitope AdHu5-NY-ESO-1(157~165) (SEQ ID NO: 2) leads to the development of an HLA:A2-restricted expansive memory response. Minigene constructs expressing a dominant HLA-A2-restrictive epitope derived from the cancer-testicular antigen NY-ESO-1 were constructed by inserting the epitope under the control of a CMV promoter on a replication-deficient AdHu5 backbone lacking E1 and E3 genes (Figure 8A). A control adenovector containing full-length NY-ESO-1 was also constructed. These were intravenously injected into transgenic HHD mice expressing HLA-A2 antigen in a C57BL / 6 background. In HHD mice, knockout of H-2Db and mouse beta-2-microglobulin (b2m) was also performed (in addition to the HLA-A2 HHDb2m hybrid molecule). This resulted in only HLA-A2 as MHC class 1. The CD8 T cell response to the epitope NY-ESO-1 was tracked in the blood by tetramer staining. As shown in Figure 8A, immunized mice responded to both constructs, and tetramer-specific cell populations were measurable on day 7. In the majority of mice immunized with the full-length NY-ESO-1 protein, the response decreased by day 21 and persisted at low but detectable levels (approximately 2–5%) throughout the experimental period, although some mice showed high levels of this response (up to 20%) even at later time points. In contrast, the majority of mice immunized with the minigene constructs showed consistently elevated levels of tetramer-positive CD8 T cells at subsequent time points. This was consistent with previously reported dynamics after immunization with minigene vectors.

[0114] The phenotype of the tetramer - positive cells was determined and found to exhibit an expanded cell phenotype, mainly effector memory (CD44+ CD62L-, Figure 8C), highly differentiated, expressing KLRG1 - hi (Figure 8D) and CX3CR1+ (Figure 8E). These cells were also PD - 1 low at a later time point and, interestingly, appeared to express lower levels of PD - 1 compared to tetramer - positive cells generated by immunization with the full - length construct (Figure 8F). The levels of other exhaustion markers such as Tim - 3 and Lag - 3 were also lower in the mini - gene - induced tetramer + CD8 T cells compared to their full - length - induced counterparts (Figure 8G and H). Taken together, the data indicate that CD8 T cell peptide epitopes on the mini - gene construct can be processed, loaded onto the human HLA - A2 antigen, and then prime and generate an expanded CD8 T cell response. Furthermore, these responses are large and durable, with very low or no expression of checkpoint inhibitors even at late time points after immunization.

[0115] Example 9 Immunization with AdHu5 - NY - ESO - 1(157 - 165) controls tumor exposure To determine whether these responses could control tumors, multiple sarcoma cells (0.5 - 1×10 6The cells were subcutaneously injected into mice. Tumor growth was tracked. The results showed that mice immunized with the AdHu5-NY-ESO-1 minigene were able to delay tumor growth at both early and late time points (Figure 9A), and 2 out of 10 animals showed complete tumor elimination. In addition, this control was observed at both high dose (solid line) and lower dose (dashed line) exposures. In contrast, mice immunized with the FL vector were able to control tumor growth at lower exposure doses but not at higher cell concentrations (solid line). It is worth noting that since the mice are transgenic only for HLA-A2, the human protein NY-ESO-1 was likely immunogenic and recognized by naive mouse CD4 and CD8 T cells upon tumor exposure. Blood samples taken two weeks after tumor exposure were analyzed for the presence of Tet+ cells. All groups developed a detectable circulating tet+ response 14 days after tumor exposure, with the MG-immunized group showing the largest scale (Figure 9B). Animals with more than 2.5% circulating tet+ cells before tumor exposure exhibited better control of tumor growth at both early and late time points (Figures 9C and D). This correlation was not observed in animals immunized with the FL vector. Data from this part of the experiment suggest that single-priming immunization with a minigene vector may provide longevity protection against tumor exposure.

[0116] Example 10 Immunization with AdHu5-NY-ESO-1(157~165) results in a population of antigen-specific T cells in the spleen. The animal had a tumor size of 1300 mm. 3When they reached the endpoint, which was either approaching or when a non-improving ulcer occurred after 48 hours, they were selected and sacrificed. Tumors were removed between days 17–29 for the naive group, between days 28–29 for MG, between days 26–29 for FL, and between days 22–29 for the unrelated Ad group. At that time, lymphocytes were isolated from the tumors and spleens to examine whether immunization altered the composition of the tumor immune microenvironment and the functionality of tumor-specific cells. Splenocytes and tumor-infiltrating lymphocytes (TILs) were isolated, and TILs from all groups were found to contain similar levels of CD8 T cells (Figure 10A). Splenic CD8 T cell levels were slightly elevated in the MG immunized group, but this was not statistically significant. NY-ESO-1 tet+ cells were detected in TILs from all groups, and there was no statistically significant difference in the percentage of Tet+ TILs between the immunized and non-immunized groups (Figure 10B). However, differences were observed in the percentage of Tet+ splenocytes, with higher levels in MG and FL immunized animals compared to non-immunized animals. Tumors were removed because they had reached their endpoints, and at this point, elevated expression of the checkpoint inhibitor PD-1 was found in tet+ TILs in all groups (Figure 10C). Fas was upregulated in splenocytes and also in TILs in all groups (Figure 10D), indicating activation of Tet+ cells. Interestingly, CD8 Tet+ splenocytes from MG and FL immunized animals expressed higher levels of PD-1, with FL immunized animals showing the highest expression of PD-1 on splenocytes. FL immunization also resulted in higher levels of Lag-3 on splenocytes and TILs. Lag-3 and Tim-3 were not upregulated on Tet+ splenocytes under other conditions; however, they were detected at similar levels in Tet+ TILs in all groups (Figures 10E and F).

[0117] Example 11 CX3CR1 is upregulated in antigen-specific splenocytes after immunization with AdHu5-NY-ESO-1(-). TILs and splenocytes were further characterized with respect to markers of expansive memory. In the spleen, as hypothesized, only antigen-specific CD8 T cells from minigene-immunized mice showed a larger population of upregulated CX3CR1 expression, while in the tumor, antigen-specific cells from all groups showed a large percentage of CX3CR1hi cells (Figure 11B). The majority of antigen-specific cells in the spleen and tumors of all groups were effector memory (Figure 11C). To investigate whether adenovirus immunization alters the tumor microenvironment, Treg levels were measured in the tumor and spleen - Treg levels in the spleen were slightly elevated in the full-length immunization group, but Treg levels in the tumor did not differ between groups (Figure 11D). Similarly, unlike what was observed in the CT26 tumor model, there was no difference in the levels of resident memory antigen-specific CD8 T cells in the tumor (Figure 11E).

[0118] Example 12 CX3CR1hi CD8 T cells are more resistant to oxidative stress. Swelling memory cells upregulate several molecules involved in the anti-apoptotic pathway, including Bcl-XL. CX3CR1 expression on human monocytes has been reported to aid cell survival by reducing antioxidant stress. Therefore, we investigated whether CX3CR1 expression has a pro-survival effect on swelling memory cells. Intracellular reactive oxygen species (ROS) levels in CX3CR1+ / -gfp splenocytes from Ad-lacZ or MCMV-infected mice were detected by the CellROX Red assay after 51 days post-infection. In steady state, CX3CR1hi CD8 T cells contained lower levels of ROS compared to CX3CR1-negative (neg) and intermediate (int) CD8 T cell populations (Figures 12A and 12C), suggesting that CX3CR1hi cells intrinsically possess lower levels of ROS. Interestingly, CX3CR1hi cells derived from CX3CR1 gfp / gfp mice also exhibited lower levels of ROS compared to the CX3CR1neg subset, indicating that this effect is not solely dependent on CX3CR1 signaling. Furthermore, ROS levels in bulk CX3CR1+ CD8 T cell subsets (Figure 12F, center) and antigen-specific CX3CR1+ T cells (Figure 12F, right) remained lower during serum starvation compared to their CX3CR1neg counterparts, thus demonstrating enhanced redox resilience. Additionally, when incubated in serum-free medium (i.e., under stress), the CX3CR1+ population showed significantly greater survival compared to CX3CR1neg T cells in bulk (Figure 12D) and antigen-specific populations (Figure 12E).

[0119] Next, peripheral blood lymphocytes from mice persistently infected with MCMV or adenovectors were stained with MitoTracker Green, a marker of mitochondrial mass, and MitoTracker DeepRed, which stains only polarized, healthy mitochondria, to determine the percentage of depolarized mitochondria in these subsets. Depolarized mitochondria are positive for MitoTracker Green but not for MitoTracker DeepRed and can be isolated from polarized mitochondria by flow cytometry. CX3CR1hi CD8 T cells from wild-type C57BL / 6 mice contain a lower percentage of depolarized mitochondria compared to CX3CR1neg CD8 cells.

[0120] T cells (Figure 12B). In summary, these results indicate that mouse CX3CR1hi CD8 T cells have a survival-promoting advantage over their CX3CR1neg CD8 T cell counterparts, which can enhance their long-term persistence and accumulation in the host.

[0121] Importantly, cancer is linked to oxidative stress primarily mediated through reactive oxygen species (ROS) generated by malignant cells, granulocytes, TAMs, and MSDCs in the tumor microenvironment. Therefore, these properties can also protect and preserve the cytotoxic capacity of cells once they are within a tumor.

[0122] Example 13: Immunization with AdHu5-R9F, which encodes the dominant E7 epitope in HPV, protects levels against TC1-HPV E6 / E7 cervical cancer exposure. The protection provided by a minigene construct encoding a dominant CD8 T cell epitope in the E7 protein was compared to that provided by a similar construct encoding the full-length E7 protein in a prophylactic immunization model. Mice immunized with either construct exhibited a large epitope-specific response (Figure 13A) that provided complete protection upon tumor exposure (Figure 13B), and no difference was observed in the level of protection provided by the whole protein versus the epitope alone.

[0123] Example 14 Synergistic effects after immunization with a panel of minigenes encoding CD8 T cell epitopes against MCMV at suboptimal doses. A panel of three minigenes against known MCMV-specific CD8 T cell epitopes, namely M45( 985 HGIRNASFI 993 Sequence ID 10), M38( 316 SSPPMFRV 325 Sequence ID 11), and m139( 419 TWYGFCLL 426 Sequence ID 12) was constructed. These were intravenously injected into C57BL / 6 mice, either as individual minigenes or mixed together as a cocktail. The minigenes encoding M38 and m139 were 1 × 10⁻¹⁶. 7 The drug was injected at a dose below the optimal dose of infectious units (IU), while the minigene encoding M45 was 1 × 10⁶ 8 The mice were injected at the optimal dose of IU. Levels of M38-specific cells in the blood were measured 6 days post-immunization. Surprisingly, mice that received a mixed minigene vaccine containing suboptimal doses of M38 minigenes and m139 minigene vectors, plus the optimal dose of M45 minigene, developed higher levels of M38-specific T cells compared to the group injected with suboptimal doses of the M38 minigene vector alone. This unexpected result suggests that delivery of a mixture of minigene vectors at suboptimal doses may produce an additive effect that enhances the scale of antigen-specific T cells compared to immunization with a single vector at a suboptimal dose.

[0124] Example 15 Minigene immunization alters the tumor environment and generates higher levels of granzyme B. Mini-gene immunization was performed, followed by analysis of granzyme B levels. Granzyme B levels in all CD8+ T cells of the tumor were assessed 23 days after tumor transplantation and 16 days after mini-gene immunization by intracellular cytokine staining, followed by flow cytometry of single-cell suspensions prepared from the tumor. As shown in Figure 15, granzyme B levels were significantly higher in CD8+ T cells immunized with the mini-gene vector compared to those immunized with a full-length epitope vector. Tumor size was also assessed 23 days after tumor transplantation, and Figure 15 shows that mini-gene immunization significantly reduced tumor size compared to controls.

[0125] Tetramer+ CD8 T cells were also evaluated for levels of the transcription factors Eomes and Tbet. Tetramer+ CD8 T cells isolated from animals immunized with minigene vectors expressed higher levels of Tbet and lower levels of Eomes compared to tetramer+ cells isolated from other groups.

[0126] Example 16 Combination therapy with minigene immunization and anti-PD-L1 therapy enhances tumor control. CT26 tumors have been reported to be unresponsive to anti-PD-1 or PD-L1 monotherapy (Selby et al., Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In Vitro Functional Studies, and Cynomolgus Macaque Toxicology. PLoS ONE. Public Library of Science; September 9, 2016; 11(9):e0161779-19). However, these data demonstrate that combination therapy with a minigene and anti-PD-L1 results in enhanced tumor control and survival.

[0127] Groups of mice were immunized with an adenovirus vector, as shown in Figure 16. Seven days after tumor exposure, mice were administered either anti-PD-L1 or an isotype control. Figure 16A shows that enhanced tumor control (i.e., reduction in tumor size) was observed when the minigene was administered in combination with anti-PD-L1 therapy. This combination therapy also resulted in an approximately 33% increase in time to the humane endpoint in all treated animals compared to untreated subjects immunized with unrelated (irr)AdHu5. Survival curves for all groups of mice are shown in Figure 16B. Circulating GP70 15 days after immunization (22 days after tumor exposure) 423-431 The percentage of Tet+ cells was evaluated. Combination therapy increased the level of circulating tetramer+ cells compared to minigene monotherapy (Figure 16C) and significantly reduced the tumor growth rate (Figure 16D).

[0128] Example 17 Analysis of IFNγ production in tumor-derived and spleen-derived cells Spleen-derived and tumor-derived single cells were obtained from mice immunized prophylactically and therapeutically, stimulated ex vivo with AH1 peptide (4 μg / ml) or PMA-ionomycin (IO) for 7 hours, and then stained for intracellular cytokine production of IFNγ. IFNγ-secreting cells were detected and elevated only in the spleens of the prophylactically (Figure 17A) or therapeutically (Figure 17B) immunized groups, while few or no IFNγ-secreting cells were detected in the tumors (Figures 17C and 17D). Experiments with therapeutic vaccination in combination with anti-PD-L1 involved ex vivo stimulation with AH1 peptide (4 μg / ml) or PMA-ionomycin (IO) for 7 hours, followed by staining for intracellular cytokine production of IFNγ. IFNγ-secreting CD8 T cells in both the spleen and tumors were increased in samples treated with combination therapy compared to minigene monotherapy (Figures 17E and 17G). In the CD4 T cell compartment, IFNγ-secreting CD4 T cells could be detected in tumors of the group vaccinated in combination with anti-PD-L1 (Figure 17H), but could not be detected in the spleen (Figure 17F).

[0129] Example 18 Immunization with a combination of two AdHu5 minigenes (MG) encoding two different tumor antigens enhances survival in therapeutic immunization models compared to immunization with a single gene. CT26 tumor cells (5 × 10) subcutaneously in mice 5 Cells (in mice) were transplanted. Six days later, the mice were each given 1 × 10¹⁶ doses of a single minigene vaccine encoding a different CT26 tumor, either AdHu5-AH1-MG or AdHu5-e2F8-27merMG. 8 IU, or both mini-gene vaccines together (Combo, both 1x10) 8 Vaccination was administered (at IU). 12, 16, and 19 days after transplantation, half of each group was treated with the checkpoint inhibitor anti-PD-1, and the other half was treated with an isotype control. Tumor growth was reduced to 1.3 cm. 3 I monitored it until it got close.

[0130] Figures 18B–F show that vaccination with a combination vaccine slows tumor growth compared to negative controls (unvaccinated or vaccinated with AdHu5-MG encoding an unrelated antigen). Figure 19A shows that combination vaccine treatment with anti-PD-1 enhanced survival compared to negative controls, while, as shown in Figure 19B, combination vaccine treatment generally increased median survival compared to negative controls or groups vaccinated with only a single minigene vaccine.

[0131] Tumor growth rate was determined by simple linear regression analysis of tumor size against time to calculate the slope of the curve (steeper slope = faster growth rate) (Figure 20). Alternatively, growth rate was calculated as relative growth rate using the same data (Figure 23). Individual mouse values ​​by vaccination type are shown. Combination vaccination significantly slowed tumor growth compared to the negative control group (Figures 20 and 23). Blood was sampled 6 days after vaccination and stained for surface markers for CD8 and tetramers specific to AH-1 or e2f8 antigen (Figure 21). %Tet+ in living CD8 T cell compartments is shown. Vaccination with the combination vaccine increased the size of the AH-1 tet+ population compared to the group vaccinated with AdHu5-AH-1 MG alone (Figure 21). Figures 22 and 23 show that simultaneous intravenous immunization (combo) with two minigene constructs / vaccines induces both antigen-specific populations at a scale and phenotype similar to that of a single vaccine, and acts to control tumor growth.

[0132] method animal The mouse experiments were conducted in accordance with UK Home Office regulations (project license numbers PBA43A2E4 and PPL 30 / 3293) and approved by the Regional Ethics Review Board at the University of Oxford. Male and female mice were maintained in individually ventilated cages under specific pathogen-free (SPF) conditions and fed a standard solid diet. Transgenic adult HHD mice for HLA-A2 were housed at the aforementioned university's BSL2 facility and provided courtesy of Vincenzo Cerundolo (HIU, University of Oxford, Oxford). Balbc mice aged 6–8 weeks were obtained from Charles River (Margate, UK).

[0133] Adenovirus vector For the NY-ESO-1 study, the full-length NY-ESO-1 gene or the dominant CD8 T cell epitope SLWTQC was cloned into the AdHu5 vector backbone. For the CT-26 study, the full-length mouse leukemia virus gene gp90 or the dominant CD8 T cell epitope SPSYVYHQF (SEQ ID NO: 1) was inserted as described above to create the constructs AdHu5-FL and AdHu5-AH1-MG. These constructs were scaled up, purified, and quantified using cesium chloride centrifugation in 293A cells at the Viral Vector Core Facility (Oxford, UK), and the stocks were stored in PBS at -80°C. A second construct, AdHu5-e2f8-27MG, encoding an immunogenic mutation derived from a CT26 tumor containing the presumed CD8 T cell epitope VILPQAPSGPSYATYLQPAQAQMLTPP (Sequence ID 4), was prepared, scaled up in 293A cells, and purified by membrane purification (Sartorious).

[0134] For the HPV16 E7 study, the full-length HPV16 E7 gene or the dominant CD8 T cell epitope RAHYNIVTF (SEQ ID NO: 7) was cloned into the AdHu5 vector backbone. The control vector consisted of the bacterial enzyme β-galactosidase-derived CD8 T cell epitope ICPMYARV (SEQ ID NO: 8) inserted into the AdHu5 vector backbone.

[0135] Mouse immunization, tumor exposure, and treatment with anti-PD-L1 antibody As shown, the mouse was subjected to 1 x 10⁶ virus 7~9 Intravenous immunization was performed by tail vein injection of infectious units (IU). Transgenic HHD sarcoma cell lines, CT26 colorectal cancer cells, or TC-1 (HPV16 E7 expressing) cell lines for NY-ESO-1 were administered in doses of 0.1–1 × 10⁻⁶. 6 The cells were subcutaneously injected into the flank at a rate of 200 μl per cell. Mycoplasma testing was performed on the cell line before injection, and only mycoplasma-negative cells were used.

[0136] After tumor exposure, mice were monitored, and when a tumor was palpable, its diameter was measured every 1-2 days using a digital caliper. The volume was then expressed as a deformed ellipse, with volume = (width). 2 The tumor growth rate was determined by calculating it using ×length / 2.

[0137] In therapeutic exposure studies, tumor cells were first transplanted subcutaneously into the flanks of mice. After 6-7 days, the animals were subjected to 1 × 10⁻¹⁶ exposure. 7~9 Intravenous immunization was performed via the tail vein with the relevant adenovirus vector in the IU, and the tumors were measured as described above. In several experiments, mice were treated by intravenous injection of either 0.2 mg of anti-mouse PD-L1 (clone 10F, 9G2, Biolegend) or an isotype control on days 14, 17, 20, and 22 after tumor transplantation.

[0138] Isolation of lymphocytes from blood and tissues Blood, spleen, and tumor samples were processed using enzymatic and mechanical digestion to obtain highly viable lymphocyte populations. Tumors were excised and subsequently digested with collagenase and DNase at 37°C for 45 minutes. The digested tumor cells were passed through a 100 μm cell sieve, washed with complete RPMI, and pelletized by centrifugation at 1500 rpm for 5 minutes. The cell pellets were resuspended and then passed through a 40 μm cell sieve before washing and pelletizing as described above. The isolated tumor cells were then resuspended and counted.

[0139] Detection and analysis of tumor and vaccine-specific T cells Details of the tetramers and pentamers used to detect virus and vaccine-specific T cells are shown in Table 2.

[0140] [Table 2]

[0141] The reagents listed in Table 2 were synthesized as monomers and tetramerized by the addition of streptavidin-PE (BD Bioscience) or streptavidin-APC (Invitrogen, Paisley, UK). Peptides for monomer construction were obtained from Proimmune (Oxford, UK). Approximately 50 μl aliquots of whole blood were stained with 50 μl of a solution containing the tetramer class I peptide complex at 37°C for 20 minutes, followed by staining with mAbs and fixable NIR LIVE / DEAD staining.

[0142] Antibody staining Single-cell suspensions were blocked with FcR blocking reagent (CD16 / CD32, eBiosciences) to prevent nonspecific antibody binding (20 minutes at 4°C). Subsequently, cells were immunostained with tetramer (as described above) and various fluorescent dye conjugate antibodies (20 minutes at 4°C). Dead cells were excluded from analysis by adding a fixable viability / dead colorant (LIVE / DEAD® near-infrared dye (Invitrogen)) to all antibody panels. The following antibodies were used in flow cytometry at a concentration of 1:100, except for those marked in the list: CD4-AF700 (RMA4-4, Biolegend), CD8 (53-6.7 eBiosciences or Biolegend), CD11a / CD18 / LFA-1 (H155-78, Biolegend), CD25 (PC61.5, eBiosciences), CD27 (LF.3A10, Biolegend), CD44 (IM7, eBiosciences), CD62L (MEL-14, Biolegend), CD69 (H1.2F3, Biolegend, 1 / 200), CD95 / Fas (Jo2 The following stains were used: BD), CD103 (2E7, Biolegend, 1 / 200), CD127 (SB / 199, Biolegend), CD279 / PD-1 (RMP1-30, Biolegend), CX3CR1 (SA011F11, Biolegend), FoxP3 (FJK-16s, eBiosciences), IFN-γ (XMG1.2, eBiosciences), IL-2 (JES6-5H4, eBiosciences), KLRG1 (2F1, abcam), and TNF-α (MP6-XT22, eBiosciences). Extracellular staining was performed before immobilization and permeabilization of single-cell samples required for intracellular staining (using FoxP3 / transcription factor staining buffer set, Invitrogen). For intracellular cytokine staining, single cells derived from tumors or spleens were stimulated ex vivo with peptides (4 μg / ml) for 2.5 hours along with positive controls (PMA at 2 μg / ml and IO at 4.4 μg / ml) and negative controls (culture medium only). Subsequently, the cells were incubated with GolgiPlug (BD, 1 μl / ml) at 37°C for 4-5 hours.The antibodies used are listed in the table below. Unless otherwise indicated, they were used at a 1:100 dilution.

[0143] [Table 3-1]

[0144] [Table 3-2]

[0145] Flow cytometry All immunohistochemical samples were analyzed by flow cytometry using a BD LSR II flow cytometer. Data analysis was performed using FlowJo v10 software. Cells were gated for lymphocytes, single cells, live cells, and subsequently for relevant markers for analysis.

[0146] CellROX red assay Single-cell splenocytes were prepared from CX3CR1gfp / + or gfp / gfp mice infected with MCMV or Ad-lacZ prior to 50 days of age. These splenocytes were seeded into 96-well plates and cultured in complete medium (RPMI + 10% FCS) for 48 hours. The cells were centrifuged and washed with 200 μl sterile DPBS (Life Technologies). Subsequently, the cells were treated with either serum-free RPMI or RPM + 10% FCS (40 μl added per well). These were incubated at 37°C for 1–1.5 hours. CellROX red reagent (Life Technologies) was diluted 1:50 with serum-free medium, and then 4 μl of the diluted reagent was added to each well and incubated at 37°C for 40 minutes. Subsequently, the cells were stained with appropriate surface antibodies (appropriate tetramer-PE, CD8-eFluor 450, CD62L-AlexaFluor 700, CD44-PerCP-Cy5.5, and a fixable LiveDead marker) at 37°C for 20 minutes. The cells were washed with PBS, then resuspended in PBS and analyzed with LSRII, and the geometric mean of CellROX red for living CD8 T cells was calculated using FlowJo software.

[0147] MitoTracker Assay PBLs derived from C57BL / 6 infected with MCMV or AdHu5 recombinant adenovector (Ad-I8V) more than 100 days prior to the disease date were stained with anti-mouse CD8, anti-mouse CX3CR1, and LiveDead near-infrared fixable markers. Staining with 12.5nm MitoTracker Green and 12.5nm MitoTracker DeepRed (Fisher Scientific) at 37°C for 30 minutes was performed prior to surface staining, followed by analysis using LSRII, and data were calculated using FlowJo.

[0148] statistical analysis Descriptive statistics (percentage mean, standard deviation, count) were calculated using GraphPad PRISM (Graphpad software, Inc., La Jolla, CA). P-values ​​for comparing means were determined using t-tests, one-way ANOVA, and two-way ANOVA, and Holm-Sidak was used for correction of multiple comparisons. Statistical significance was defined as p < 0.05.

[0149] Method for generating recombinant AAV minigenes Recombinant AAV encoding the target minigene is produced by transfecting HEK 293 cells with three plasmids: (1) the AAV-ITR plasmid containing the target minigene [AAV-ITR-minigene], (2) an adenovirus helper plasmid encoding the E2A, E4, and VA adenovirus proteins required for AAV replication, and (3) a helper plasmid encoding the AAV rep and cap genes required to package the AAV-ITR-minigene within the AAV virus particle.

[0150] Vector array: The following provides exemplary sequences that may be used in the vectors of the present invention. Sequence ID 13: AdHu5 adenovirus nucleotide sequence at the 5' end of the mini-gene immunogen cassette: Sequence ID No. 14: AdHu5 adenovirus nucleotide sequence at the 3' end of the mini-gene immunogen cassette: Mini Gene Immunogen Cassette: Sequence ID No. 15: 5' end minigene immunogen cassette nucleotide sequence of a T cell epitope 5' minigene immunogen cassette nucleotide sequence of T cell epitope sequence Sequence ID 16: attR1 sequence Sequence ID 17: attL1 array Sequence ID 18 CMV promoter sequence Sequence ID 19: Kossack Sequence Sequence ID 20 Start codon T cell epitope: Sequence ID 2: NY-ESO-1 epitope Sequence ID 21: Human (Homo sapiens) codon-optimized NY-ESO-1 epitope nucleotide sequence Sequence ID 1: AH1 epitope Sequence ID 22: Mouse (Mus Musculus) codon-optimized AH1 epitope nucleotide sequence Sequence ID 3: GSW11 epitope Sequence ID 23: Mouse (Mus Musculus) codon-optimized GSW11 epitope nucleotide sequence Sequence ID 4 e2f8 epitope Sequence ID 24: Mouse (Mus Musculus) codon-optimized e2f8 epitope nucleotide sequence Sequence ID 5: Mtch1-10mer epitope Sequence ID 25: Mouse (Mus Musculus) codon-optimized Mtch1-10mer epitope nucleotide sequence Sequence ID 6: Mtch1-9mer epitope Sequence ID 26: Mouse (Mus Musculus) codon-optimized Mtch1-9mer epitope nucleotide sequence Sequence ID 8: I8V epitope Sequence ID 27: Mouse (Mus Musculus) codon-optimized I8V epitope nucleotide sequence Sequence ID 9, pp89 epitope Sequence ID 28: Mouse (Mus Musculus) codon-optimized pp89 epitope nucleotide sequence Sequence ID 10: M45 epitope Sequence ID 29: Mouse (Mus Musculus) codon-optimized M45 epitope nucleotide sequence Sequence ID 11: M38 Epitope Sequence ID 30: Mouse (Mus Musculus) codon-optimized M38 epitope nucleotide sequence Sequence ID 12: m139 epitope Sequence ID 31: Mouse (Mus Musculus) codon-optimized m139 epitope nucleotide sequence Sequence ID 7 HPV16 E7 49-57 Epitope Sequence ID 32: Mouse (Mus Musculus) codon-optimized HPV16 E7 49-57 Epitope nucleotide sequence Sequence ID 33: 3' end mini-gene immunogen cassette nucleotide sequence of the T cell epitope sequence 3' side minigene immunogen cassette nucleotide sequence of T cell epitope sequence: Stop codon Sequence ID 34: BGH PolyA sequence Sequence ID 35, attL2 sequence Sequence ID 36, attR2 sequence The above minigene immunogen cassette can be used with an AAV vector. For example, an AAV vector containing a reverse terminal repeat sequence can be used. An example sequence is provided below. Sequence ID 38: 5' AAV nucleotide sequence of the mini-gene immunogen cassette Description of the 5' AAV nucleotide sequence of the mini-gene immunogen cassette: Sequence ID 39 5'ITR nucleotide sequence Sequence ID 40: Extra sequence at the 5' end of the mini-gene immunogen cassette. Sequence ID 41: 3' AAV adenovirus nucleotide sequence of the mini-gene immunogen cassette Description of the AAV adenovirus nucleotide sequence at the 3' end of the mini-gene immunogen cassette: Sequence ID 42 3'ITR nucleotide sequence Sequence ID 43: Extra sequence at the 3' end of a mini-gene immunogen cassette.

Claims

1. A replication-deficient adenovirus vector comprising a nucleotide sequence encoding a polypeptide containing a single cancer-specific CD8+ T cell epitope, wherein the viral vector does not contain nucleic acids encoding other cancer-specific T cell epitopes other than the single cancer-specific CD8+ T cell epitope, the single cancer-specific CD8+ T cell epitope is not a viral immunosource, the nucleotide sequence encoding the polypeptide containing the single cancer-specific CD8+ T cell epitope is 24 to 45 nucleotide base pairs in length, the polypeptide encoded by the nucleotide sequence consists of 8 to 15 amino acids, the single cancer-specific CD8+ T cell epitope encoded by the viral vector avoids the need for normal antigen processing for presentation on MHC molecules, and administration of the viral vector to a target induces an expansive memory CD8+ T cell response to the single cancer-specific CD8+ T cell epitope in the target, thereby inhibiting tumor growth.

2. The viral vector according to claim 1, which can persistently infect cells.

3. Expansive memory CD8+ T cell response, (i) CD8+ / CX3CR1+ / KLRG-1+ / CD44+ / CD62L- / CD27- / CD127- T cells; (ii) CD8+ / CX3CR1+ / KLRG-1+ / CD44+ / CD62L- / CD27(low) / CD127- T cells; (iii) CD8+ / CX3CR1+ / KLRG-1+ / CD44+ / CD62L- / CD27- / CD127(low) T cell; or (iv) CD8+ / CX3CR1+ / KLRG-1+ / CD44+ / CD62L- / CD27(low) / CD127(low) T cells A viral vector according to claim 1 or 2, comprising the production of a viral vector.

4. The viral vector according to any one of claims 1 to 3, wherein a single cancer-specific CD8+ T cell epitope is a tumor-associated antigen overexpressed in cancer cells or an antigen mutated in cancer cells.

5. A viral vector according to any one of claims 1 to 4, wherein a nucleotide sequence encoding a cancer-specific CD8+ T cell epitope encodes a polypeptide containing the cancer-specific CD8+ T cell epitope, and the polypeptide containing the cancer-specific CD8+ T cell epitope is not processed by antigen-presenting cells.

6. A single cancer-specific CD8+ T cell epitope is identified as TRP-1, CEA, TAG-72, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, Melan-A / MART-1, tyrosinase, CLPP, cyclin-A1, cyclin-B1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2a, CD45, glypican-3, IGF2B.

3. A viral vector according to any one of claims 1 to 5, derived from a tumor-associated antigen selected from the group consisting of kallikrein-4, KIF20A, lengusin, meloe, MUC5AC, survivin, PRAME, SSX-2, NY-ESO-1 / LAGE1, gp70, MC1R, TRP-1 / -2, β-catenin, BRCA1 / 2, CDK4, and fetal protein SIM1.

7. A single cancer-specific CD8+ T cell epitope is associated with colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, stomach cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein-Barr driven cancers, and human cancer. A viral vector according to any one of claims 1 to 6, which is specific to human papillomavirus-driven cancers or soft tissue sarcomas.

8. A viral vector according to any one of claims 1 to 7, wherein the viral vector is human serotype 5 (AdHu5).

9. A viral vector according to any one of claims 1 to 8, comprising a CMV promoter and / or a TATA box.

10. The viral vector according to any one of claims 1 to 9, which is a replication-deficient AdHu5 adenovirus vector lacking sequences encoding functional E1 and E3 proteins.

11. It includes a promoter, a translation initiation sequence, a start codon, a T cell epitope codon, a stop codon, and a polyadenylation sequence. (i) The promoter is selected from the CMV promoter, the RSV promoter, and the EF1α promoter and includes the sequence described in SEQ ID NO: 18, (ii) The translation initiation sequence includes the sequence described in sequence number 19, and (iii) The polyadenylated sequence includes the sequence described in Sequence ID No. 34, A viral vector according to any one of claims 1 to 10.

12. A viral vector according to any one of claims 1 to 11, comprising a sequence having at least 90% sequence identity in sequence number 13 and a sequence having at least 90% sequence identity in sequence number 14.

13. The viral vector according to claim 12, further comprising a sequence having at least 90% sequence identity with sequence number 15, and a sequence having at least 90% sequence identity with sequence number 33.

14. A composition comprising at least two viral vectors, wherein each of the at least two viral vectors is a viral vector according to any one of claims 1 to 13, and each of the at least two viral vectors encodes a different single cancer-specific CD8+ T cell epitope.

15. A host cell comprising a viral vector according to any one of claims 1 to 13, or the composition according to claim 14.

16. A pharmaceutical product comprising a viral vector according to any one of claims 1 to 13, the composition according to claim 14, or the host cell according to claim 15, and comprising a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

17. A viral vector according to any one of claims 1 to 13, a composition according to claim 14, or a host cell according to claim 15 for use in the treatment of cancer in a subject requiring treatment for cancer, wherein the pharmaceutical composition comprising (a) a viral vector, composition, or host cell and (b) a pharmaceutically acceptable carrier is administered to the subject.

18. The viral vector, composition, or host cell according to claim 17, wherein the cancer is colorectal cancer, prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, stomach cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein-Barr-driven cancer, human papillomavirus-driven cancer, or soft tissue sarcoma.

19. The viral vector, composition, or host cell according to claim 17 or 18, wherein the pharmaceutical composition is administered as a single dose or as multiple doses.

20. A viral vector, composition, or host cell according to any one of claims 17 to 19, administered in combination with a treatment or therapeutic agent.

21. The viral vector, composition, or host cell according to claim 20, wherein the therapeutic agent comprises an immune checkpoint inhibitor.

22. The viral vector, composition, or host cell according to claim 21, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, LAG-3, B7-H3, B7-H4, B7-H6, A2aR, BTLA, GAL9, and IDO.

23. A method for producing a viral vector according to any one of claims 1 to 13, (i) Synthesize a nucleotide sequence encoding the single cancer-specific CD8+ T cell epitope as sense and antisense primers. (ii) Cloning the nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope synthesized in (i) into the first plasmid, and Cloning a sequence containing a nucleotide sequence encoding a single cancer-specific CD8+ T cell epitope from the first plasmid of (iii)(iii) into a second vector containing adenovirus DNA, Methods that include...

24. The viral vector according to claim 1, wherein the expandable memory CD8+ T cell response comprises the production of CD8+ / CX3CR1+ / KLRG-1+ T cells having sustained expression of Tbx21 and E2f2.

25. The viral vector according to claim 1, wherein the expandable memory CD8+ T cell response includes the production of CD8+ / CX3CR1+ / KLRG-1+ T cells with low Eomes expression.

26. CD8+ / CX3CR1+ / KLRG-1+ T cells in the target population are associated with total circulating CD8+ A viral vector according to claim 24 or 25, which forms 2-20% of T cells.

27. The viral vector according to claim 26, wherein CD8+ / CX3CR1+ / KLRG-1+ T cells retain a memory effector phenotype for at least 30 days.

28. The viral vector according to claim 1, wherein the expandable memory CD8+ T cell response comprises the production of CD8+ / CX3CR1+ / KLRG-1+ T cells with low expression of PD-1, Tim-3, and / or Lag-3.

29. A viral vector according to claim 1, or a composition according to claim 14, wherein the expansive memory CD8+ T cell response can control tumor growth in the target for more than 50 days after administration of the viral vector, composition, or host cells.

30. A nucleotide sequence encoding a cancer-specific CD8+ T cell epitope encodes a polypeptide containing the cancer-specific CD8+ T cell epitope, and upon administration, the polypeptide containing the cancer-specific CD8+ T cell epitope is not processed by the target antigen-presenting cells, inducing an expansive memory CD8+ T cell response in the target, and The viral vector according to claim 1, wherein when a pharmaceutical composition comprising a corresponding viral vector comprising a nucleotide sequence encoding a cancer-specific CD8+ T cell epitope is administered, the nucleotide sequence encodes a polypeptide comprising the cancer-specific CD8+ T cell epitope and, when processed by target antigen-presenting cells at the time of administration, does not induce an expansive memory CD8+ T cell response in the target.

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