Method for inducing antitumor immunity using Sindbis virus vector and tumor-associated antigen

Abstract

To develop new methods of enhancing SV anti-cancer therapy.SOLUTION: A method for treating a mammal harboring a tumor comprises identifying a tumor associated antigen (TAA) expressed by the tumor, and parenterally administering to the mammal a therapeutically effective amount of a Sindbis viral vector carrying a gene encoding the TAA to the mammal sufficient to elicit an immune response directed against the tumor, thereby treating the tumor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The United States Government owns certain rights in this invention pursuant to funding received from US Public Health Grant CA100687 from the National Cancer Institute, National Institutes of Health, and the Department of Health and Human Services. [Background technology]

[0002] Oncolytic viruses (OVs) are viruses that specifically target and replicate in tumor cells [1]. Due to their selectivity and oncolytic properties, OVs have attracted considerable interest as an alternative or adjunct to conventional cancer therapy [2]. However, a major limitation of OV therapy is their poor replication and proliferation at tumor sites [3, 4]. Moreover, for safety reasons, many OVs have been engineered to be replication-deficient to prevent their spread to healthy tissues and further limit their oncolytic potential [5].

[0003] One possible solution to this problem is to supplement direct viral oncolysis with a bystander effect, resulting in the destruction of tumor cells that are not directly infected by OVs. This can be achieved, for example, by inserting therapeutic or cytotoxic genes into the OV genome for delivery to the tumor site [6, 7]. Being naturally immunogenic, some OVs can effectively stimulate the immune system, which opens the possibility of using OVs to induce immunological anticancer bystander effects [8]. This idea has gained further momentum with the identification [9, 10] and recent prioritization

[11] of various clinically relevant tumor-associated antigens (TAA) (OV / TAA)

[12] that can be delivered to the tumor site by OVs. In their natural state, TAAs are often not sufficiently immunogenic

[13] . However, immunogenic OV / TAA may break this immune tolerance by redirecting the antiviral immune response to the TAA. Therefore, the main goal of OV research should be the development of safe and effective OV / TAA agents. Sindbis virus (SV), an alphavirus with a positive-sense, single-stranded RNA genome

[14] , represents one of the select viruses that has shown special potential as an OV [15, 16] and as a viral vaccine

[17] . Replication-deficient SV vectors have previously been shown to target and inhibit the growth of xenograft, syngeneic, and spontaneous tumors in mice [16, 18]. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Application Serial No. 13 / 133,680 [Patent Document 2] U.S. Patent No. 8,282,916 [Patent Document 3] U.S. Patent No. 7,303,898 [Patent Document 4] U.S. Patent No. 7,306,792 [Patent Document 5] U.S. Patent No. 8,093,021

Non-Patent Literature

[0005]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

[0006] Recently, SV has also been found to induce the activation of natural killer (NK) cells and macrophages in tumor-bearing mice.

[19] Also, SV vectors expressing immune-modulating genes, such as interleukin-12 (IL-12), have enhanced antitumor

[16] and immune-stimulating

[19] effects. Nevertheless, these approaches have largely failed to lead to complete tumor remission.

[19] Moreover, some tumor cells cannot be efficiently targeted by SV.

[20] Highlighting the need to develop new methods to enhance SV anticancer therapy.

[0007] It was previously hypothesized that the inherent characteristics of SV vectors that make them effective oncolytic and gene delivery systems (e.g., the ability to spread through the bloodstream

[15] and deliver high levels of heterologous proteins

[21] ) may also be useful for efficient TAA delivery. Moreover, the SV life cycle, characterized by the absence of a DNA phase, which makes the vector safer, also involves the production of high levels of double-stranded RNA (dsRNA), a potent immunological "danger signal"

[22] and subsequent activation of the type I interferon pathway

[23] . The combination of safety, immunogenicity, efficient spread, and high TAA expression makes SV / TAA an attractive OV / TAA candidate. Therefore, what is needed in the art is a method to take advantage of all the above benefits by treating tumor-bearing mammals with SV / TAA. [Means for solving the problem]

[0008] As disclosed herein, the use of SV as an OV / TAA agent was examined using a BALB / c CT26 colon cancer tumor model. Unlike other tumor models tested, CT26 cells were found not to be targeted in vivo by SV. Nevertheless, an SV vector carrying β-galactosidase (SV / LacZ) had a notable therapeutic effect in mice bearing LacZ-expressing CT26 tumors. By using an in vivo imaging system (IVIS)

[24] to sensitively detect luciferase activity in vivo, mediastinal lymph nodes (MLNs) were identified as sites of early and transient heterologous protein expression following intraperitoneal (ip) injection of an SV vector carrying the firefly luciferase gene (SV / Fluc). TAA delivery into MLNs inhibited effector and memory CD8 T cells that exhibited robust cytotoxicity against LacZ positive and negative tumor cells. + This latter phenomenon, known as epitope spreading, has recently been suggested to be a key component of effective cancer immunotherapy in patients

[25] .

[0009] In one aspect, the invention provides a method of treating a mammal bearing a tumor, comprising identifying a tumor-associated antigen (TAA) expressed by the tumor, and treating the tumor by parenterally administering to the mammal a Sindbis virus vector carrying a gene encoding the TAA in a therapeutically effective amount sufficient to elicit an immune response against the tumor.

[0010] In another embodiment, the present invention provides a method for the detection of CD8 markers against tumors in a mammal. + A method for inducing a T cell-mediated immune response comprising the steps of identifying at least one tumor-associated antigen (TAA) expressed by a tumor and administering to a mammal in need of such treatment an immune response against the tumor. CD8 + and parenterally administering a Sindbis virus vector carrying a gene encoding a TAA in an amount effective to elicit a T cell-mediated immune response.

[0011] These and other aspects of the invention will become apparent to those of ordinary skill in the art in light of the specification, claims, and drawings. [Brief description of the drawings]

[0012] [Figure 1a] Figure 1: SV / LacZ inhibits the growth of LacZ-expressing CT26.CL25 tumors. 0.5x106 LacZ-expressing CT26.CL25 (left panel) or LacZ-negative CT26.WT (right panel) cells were injected sc into the right flank of BALB / c mice. Nine days after tumor inoculation, mice were treated ip with SV / LacZ, control SV / Fluc vector, or medium (mock). Tumor volumes (mm3) were measured and plotted (N=3-4). Data are representative of at least two independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 1b] Figure 1: SV / LacZ inhibits the growth of LacZ-expressing CT26.CL25 tumors. Kaplan-Meier survival plots of mice bearing peritoneal CT26.CL25 tumors. 2.5x104 CT26.CL25 cells were injected ip and treatment began on day 4 (N=5). Data for SV / LacZ and mock groups are representative of two independent experiments. Data are presented as mean±SEM. *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 1c] SV / LacZ inhibits the growth of LacZ-expressing CT26.CL25 tumors. Representative IVIS images of SV / LacZ and control treated mice bearing pulmonary CT26.CL25.Fluc tumors are shown (left panel). Relative tumor growth (top right panel) was determined by normalizing luminescence to the first image (day 2) for each mouse, and survival was plotted (bottom right panel) (N=5-8). Data are representative of two independent experiments. Data are expressed as mean ± SEM. *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 2a] Figure 1: TAA expression and T cell activation occur in mediastinal lymph nodes. scCT26.CL25 tumor-bearing mice (left panel) or tumor-free mice (right panel) were treated ip with SV / Fluc. Bioluminescence images were taken to monitor vector-derived Fluc expression 3 hours after the fifth (left panel) or first (right panel) treatment. To determine the source of the upper body signal, MLNs were extracted and imaged separately (right panel). The red circle in the left panel indicates the location of the sc tumor in each mouse. [Figure 2b] Figure 1: TAA expression and T cell activation occurs in mediastinal lymph nodes. Mice bearing pulmonary CT26.CL25.Fluc tumors were treated with SV / LacZ. After 24 hours, mediastinal and inguinal lymph nodes were extracted and stained to determine the percentage of T cells (CD3 positive, MHC class II negative) in the lymph nodes. Representative plots (left panel) and their quantification (right panel; n=3) are shown. Data are representative of two independent experiments (the second experiment was done in mice bearing ip tumors) and are expressed as mean ± SEM. *p<0.05, **p<0.01. Fluc, firefly luciferase; MLN, mediastinal lymph node; ILN, inguinal lymph node; S / L, SV / LacZ (SV, Sindbis virus vector); TAA, tumor-associated antigen. [Figure 2c]Figure 1: TAA expression and T cell activation occur in mediastinal lymph nodes. Expression of CD69 on CD8+ T cells extracted from mediastinal and inguinal lymph nodes of lung tumor-bearing mice 24 hours after ipSV / LacZ injection was analyzed. Representative flow cytometry plots (left panel) and bar graphs showing the percentage of high CD69 cells (right panel; n=3) are shown. Data are representative of two independent experiments (second experiment was done in ip tumor-bearing mice) and are expressed as mean ± SEM. *p<0.05, **p<0.01. Fluc, firefly luciferase; MLN, mediastinal lymph node; ILN, inguinal lymph node; S / L, SV / LacZ (SV, Sindbis virus vector); TAA, tumor-associated antigen. [Figure 3a] Figure 1: SV / LacZ induces strong CD8+ T cell responses. Mice bearing lung CT26.CL25.Fluc tumors were treated with SV / LacZ or medium (mock). Seven days later, peritoneal cells were analyzed. Representative flow cytometry plots (left panel) and calculated numbers of CD8+ T cells (right panel) are shown (mean ± SEM, N=3). *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 3b] Figure 1 shows that SV / LacZ induces strong CD8+ T cell responses. CD8+ T cells from peritoneum and lung were further analyzed using NKG2D and L-selectin as activation markers to determine their activation status. Representative flow cytometry plots (left panel) and calculated percentages of activated (high NKG2D, low L-selectin) cells (right panel) are shown (mean ± SEM, N=3). *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 4a] Figure 1 shows that SV / LacZ induces LacZ-specific CD8+ T cell responses. Two weeks after the initiation of SV / LacZ or mock treatment, splenocytes from CT26.CL25 sc tumor-bearing mice were harvested and analyzed. Representative tetramer plots are shown (N=5). [Figure 4b]Figure 1: SV / LacZ induces LacZ-specific CD8+ T cell responses. Splenocytes from CT26.CL25 sc tumor-bearing mice were harvested and analyzed 2 weeks after the initiation of SV / LacZ or mock treatment. The percentage of tetramer positive cells is shown (N=5). Data are presented as mean±SEM. *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 4c] Figure 1: SV / LacZ induces LacZ-specific CD8+ T cell responses. Cells from ip peritoneal and lung tumor-bearing mice were collected, stained, and analyzed 7 days after the start of treatment (N=4-5). Data are presented as mean±SEM. *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 5a] FIG. 1 shows that lymphocytes acquire LacZ-specific cytotoxicity during SV / LacZ treatment. Lung lymphocytes were extracted from CT26.CL25.Fluc lung tumor-bearing mice 7 days after the initiation of mock (-), SV / GFP (G), or SV / LacZ (L) treatment. Extracted lung lymphocytes were co-cultured with CT26.CL25.Fluc cells (CT26.CL25) or CT26.WT.Fluc cells (CT26.WT) for 2 days as described in Materials and Methods, and cytotoxicity of lung lymphocytes against each tumor cell population was determined in response to co-culture with each tumor cell population (data are presented as mean±SD, N=3). **p<0.01 (significantly different from mock). ND, not detected. SV, Sindbis virus vector. [Figure 5b]FIG. 1 shows that lymphocytes acquire LacZ-specific cytotoxicity during SV / LacZ treatment. Lung lymphocytes were extracted from CT26.CL25.Fluc lung tumor-bearing mice 7 days after the initiation of mock (-), SV / GFP (G), or SV / LacZ (L) treatment. Extracted lung lymphocytes were co-cultured with CT26.CL25.Fluc cells (CT26.CL25) or CT26.WT.Fluc cells (CT26.WT) for 2 days as described in Materials and Methods, and IFN-γ secretion from lung lymphocytes in response to co-culture with each tumor cell population was determined (data are presented as mean ± SD, N=3). **p<0.01 (significantly different from mock). ND, not detected. SV, Sindbis virus vector. [Figure 6a] Figure 1 shows that CD8+ T cells are required for the enhanced therapeutic effect of SV / LacZ. The therapeutic effect of SV / LacZ was compared between intact mice and CD8+ T cell-deficient mice [CD8+ T cell (-)] in a sc tumor model. The size of CT26.CL25 sc tumors at the indicated time points was measured and plotted for each group (N=5). Data are presented as mean ± SEM. *P<0.05, **P<0.01. NS, not significant; SV, Sindbis virus vector. [Figure 6b] Figure 1 shows that CD8+ T cells are required for enhanced therapeutic efficacy of SV / LacZ. The therapeutic efficacy of SV / LacZ was compared between intact mice and CD8+ T cell deficient mice [CD8+ T cell (-)] in an ip tumor model. Survival rates of CT26.CL25 ip tumor-bearing mice were monitored and plotted as Kaplan-Meier survival plots. N=8-9. *P<0.05, **P<0.01. NS, not significant; SV, Sindbis virus vector. [Figure 6c]Figure 1 shows that CD8+ T cells are required for enhanced therapeutic efficacy of SV / LacZ. The therapeutic efficacy of SV / LacZ was compared between intact and CD8+ T cell deficient mice [CD8+ T cell (-)] in a lung tumor model. Tumor growth (left panel) and survival (right panel) were analyzed in CT26.CL25.Fluc lung tumor-bearing mice. Relative tumor growth was quantified as in Figure 1c, and survival is shown as Kaplan-Meier survival plots (N=5). Data are presented as mean ± SEM. *P<0.05, **P<0.01. NS, not significant; SV, Sindbis virus vector. [Figure 7a] FIG. 10: Immunity to endogenous CT26 TAAs develops during SV / LacZ treatment. Splenocytes were extracted from CT26.CL25.Fluc lung tumor-bearing mice 7 days after the initiation of mock (-), SV / GFP (G), or SV / LacZ (L) treatment. Extracted splenocytes were co-cultured with CT26.CL25.Fluc (CT26.CL25) or CT26.WT.Fluc (CT26.WT) cells for 2 days as described in Materials and Methods, and splenocyte cytotoxicity against each tumor cell population was determined in response to co-culture with each tumor cell population (mean±SD, N=3). *p<0.05, **p<0.01 (significantly different from mock or naive). ND, not detected; SV, Sindbis virus vector. [Figure 7b] FIG. 10: Immunity to endogenous CT26 TAAs develops during SV / LacZ treatment. Splenocytes were extracted from CT26.CL25.Fluc lung tumor-bearing mice 7 days after the initiation of mock (-), SV / GFP (G), or SV / LacZ (L) treatment. Extracted splenocytes were co-cultured with CT26.CL25.Fluc (CT26.CL25) or CT26.WT.Fluc (CT26.WT) cells for 2 days as described in Materials and Methods, and splenocyte-derived IFN-γ secretion in response to co-culture with each tumor cell population was determined (mean ± SD, N=3). *p<0.05, **p<0.01 (significantly different from mock or naive). ND, not detected; SV, Sindbis virus vector. [Figure 7c]FIG. 1 shows that immunity to endogenous CT26 TAA develops during SV / LacZ treatment. Naive and CT26.CL25 SV / LacZ-treated tumor-cured mice were inoculated iv more than 60 days after the last SV / LacZ treatment, and tumor growth in the lungs was analyzed by bioluminescence imaging at the indicated time points. The left panel shows representative IVIS images of two independent experiments. The right panel shows quantification of tumor bioluminescence at the indicated time points (mean±SEM, N=8). *p<0.05, **p<0.01 (significantly different from mock or naive). ND, not detected; SV, Sindbis virus vector. [Figure 7d] FIG. 1: Immunity to endogenous CT26 TAAs develops during SV / LacZ treatment. CT26.WT.Fluc tumors were inoculated iv into naive mice (N) and SV / LacZ-treated tumor-cured mice (S) more than 30 days after the last SV / LacZ treatment. Eight days after tumor inoculation, splenocytes were extracted from each mouse and incubated with LacZ, gp70, or control peptide for 3 days. After incubation, LacZ- or gp70-specific induction of IFN-γ secretion was analyzed (mean±SEM, N=3) as described in Materials and Methods. *p<0.05, **p<0.01 (significantly different from mock or naive). ND, not detected; SV, Sindbis virus vector. [Figure 7e] Immunity to endogenous CT26 TAA develops during SV / LacZ treatment. The number of gp70-specific CD8+ T cells in splenocytes extracted in Fig. 7d was quantified by flow cytometry using gp70 tetramer (mean ± SD, N = 3). *p < 0.05, **p < 0.01 (significantly different from mock or naive). ND, not detected; SV, Sindbis virus vector. [Figure 8]Diagram of a four-step model for activation of CD8+ T cells during SV / TAA treatment. Step 1: ip injection of SV / LacZ results in transient immunogenic expression of LacZ in the mediastinal lymph node (dark blue arrow), followed by induction of T cell activation at this site and / or other sites (light blue arrow). NK cells are also activated against tumor cells (brown arrow). Step 2 (red arrow): LacZ-specific CD8+ T cell cytotoxicity leads to destruction of tumor cells with subsequent release of tumor-associated antigens (such as LacZ and gp70). Step 3 (green arrow): Antigen-presenting cells capture and present the antigen in the tumor-draining lymph node to CD8+ T cells, resulting in epitope spreading, including induction of gp70-specific CD8+ T cells that could potentially target LacZ(-) tumor cell escape mutants. Step 4 (purple arrow): Memory CD8+ T cells against various tumor-associated antigens are generated. APC, antigen-presenting cell; LN, lymph node; MLN, mediastinal lymph node; NK, natural killer cell; SV, Sindbis virus vector; TAA, tumor-associated antigen; Tc, cytotoxic CD8+ T cell; Tm, memory CD8+ T cell. [Figure 9a] FIG. 1 shows that enhanced SV / LacZ therapeutic efficacy in lung tumor-bearing mice is dependent on LacZ expression on the tumor. Tumor growth was analyzed in CT26.CL25.Fluc or CT26.WT.Fluc lung tumor-bearing mice at the indicated time points. The left panel shows representative IVIS images of two independent experiments. The right panel shows relative tumor growth at the indicated time points. Data are presented as mean±SEM. (N=4-7). *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 9b] Figure 1 shows that the enhanced therapeutic effect of SV / LacZ in lung tumor-bearing mice depends on LacZ expression on the tumor. Kaplan-Meier survival plots show the survival rates of mice bearing CT26.CL25.Fluc or CT26.WT.Fluc lung tumors (N=5-7). *p<0.05, **p<0.01. SV, Sindbis virus vector. [Figure 10a]Figure 1 shows that SV does not target CT26 tumors in the lung. Tumor-free or CT26.WT lung tumor-bearing mice were treated ip with SV / Fluc every 2 days. Whole-body bioluminescence images were generated and taken at the indicated time points after the first SV / Fluc treatment. Fluc, firefly luciferase; SV, Sindbis virus vector. [Figure 10b] SV, lungs were imaged, and then the indicated organs were extracted and imaged separately; Fluc, firefly luciferase; SV, Sindbis virus vector. [Figure 11a] Figure 1 shows that SV / Fluc and SV / GFP induce CD8+ T cell responses. Peritoneal tumor-bearing mice were treated with SV / Fluc (left panel), SV / GFP (right panel), or medium (mock). Peritoneal cells were analyzed using flow cytometry at the indicated time points. Calculated numbers of CD8+ T cells in the peritoneum are shown (mean ± SEM, N = 2-3 for each time point). Fluc, firefly luciferase; GFP, green fluorescent protein; SV, Sindbis virus vector. [Figure 11b] Figure 1 shows that SV / Fluc and SV / GFP induce CD8+ T cell responses. Representative flow cytometry plots show L-selectin expression on peritoneal CD8+ T cells from SV / GFP or mock treated mice 7 days after treatment (N=2). Fluc, firefly luciferase; GFP, green fluorescent protein; SV, Sindbis virus vector. [Figure 12a]Figure 1 shows that SV / TAA induces activation of effector and memory LacZ-specific CD8+ T cells. Left panel: LacZ-naive, tumor-free mice were injected with SV / LacZ or medium (mock). Four days later, peritoneal cells were extracted and analyzed for the presence of LacZ-specific CD8+ T cells. Right panel: Activation levels of peritoneal CD8+ T cells from mock and SV / LacZ-treated mice were compared with each other and with LacZ-specific CD8+ T cells obtained from SV / LacZ-treated mice (SV / LacZ tet+). Activated cells were defined as high NKG2D, low L-selectin cells. All plots show gated CD8+ T cells. Fluc, firefly luciferase; LacZ, β-galactosidase; SV, Sindbis virus vector; tet, tetramer. [Figure 12b] Figure 1 shows that SV / TAA induces activation of effector and memory LacZ-specific CD8+ T cells. LacZ tetramer analysis from peritoneal CT26.CL25 tumor-bearing mice treated with SV / LacZ, SV / Fluc, or medium (mock). All plots represent gated CD8+ T cells. Fluc, firefly luciferase; LacZ, β-galactosidase; SV, Sindbis virus vector; tet, tetramer. [Figure 12c] FIG. 1 shows that SV / TAA induces activation of effector and memory LacZ-specific CD8+ T cells. Splenocytes from naive mice bearing ip CT26.CL25 tumors or from SV / LacZ-treated long-term survivors (SV / LacZ survivors) were stained with anti-CD 127 (memory cell marker) and LacZ-specific tetramer to determine the presence of long-term surviving LacZ-specific memory (CD127+, tetramer+) cells. Data are representative of two samples taken more than 3 months after treatment was stopped. All plots show gated CD8+ T cells. Fluc, firefly luciferase; LacZ, β-galactosidase; SV, Sindbis virus vector; tet, tetramer. [Figure 13a]Figure 1 shows that NK cells are activated at early stages of SV treatment. The percentages of lung CD4+ T cells, CD8+ T cells, LacZ-specific CD8+ T cells, and NK (CD3-CD122+) cells within the total lung immune cell (CD45+) population from CT26.CL25.Fluc lung tumor-bearing mice were analyzed at the indicated time points after initiation of mock or SV / LacZ treatment. Data are presented as mean ± SEM (N=3). *p<0.05, **p<0.01. LacZ, β-galactosidase; NK, natural killer cells; SV, Sindbis virus vector; tet, tetramer. [Figure 13b] Figure 1 shows that NK cells are activated at an early stage of SV treatment. Expression of NKG2D on NK cells in lungs from CT26.CL25.Fluc lung tumor-bearing mice was analyzed at the indicated time points after initiation of mock or SV / LacZ treatment. Data are presented as mean ± SEM (N=3). *p<0.05, **p<0.01. LacZ, β-galactosidase; NK, natural killer cells; SV, Sindbis virus vector; tet, tetramer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The term "about" or "approximately" usually means within an acceptable range of error for the type of measurement and method of measurement. For example, it can mean within 20%, more preferably within 10%, and most preferably within 5% of a given value or range. Alternatively, particularly in biological systems, the term "about" means within about one log (i.e., one order of magnitude), preferably within two times, of a given value.

[0014] The present invention is based on the following discoveries: (i) SV represents a potentially powerful therapeutic platform for immunogenic delivery of TAAs; (ii) the therapeutic benefit derived from SV / TAA does not necessarily require direct targeting of tumor cells; (iii) SV / TAA therapy includes transient and early delivery of the TAA to lymph nodes draining the injection site, particularly the MLN in the case of ipSV injection; and (iv) SV / TAA therapy induces potent TAA-specific CD8 + (v) SV / TAA treatment results in epitope spreading, which may be a solution to the problem of tumor escape due to TAA loss or modification, and (vi) SV / TAA treatment ultimately results in long-term survival of tumor-bearing mice and the induction of long-lived CD8 T cells against multiple TAAs. + Leading to the generation of memory T cells.

[0015] According to the present invention, an immune response against mammalian tumors is induced using Sindbis virus vectors carrying genes encoding tumor-associated antigens (TAA). Oncolytic viruses (OV) have recently emerged as a promising strategy for the immunogenic delivery of TAAs to cancer patients. However, prior to the present invention, a safe and effective OV / TAA therapy had not yet been established. It was previously demonstrated that Sindbis virus (SV)-based vectors can target tumor cells, inhibit tumor growth, and activate the innate immune system of mice. It has now been unexpectedly discovered that parenterally administered SV vectors carrying genes encoding tumor-associated antigens (TAA) result in a dramatic enhancement of therapeutic efficacy in mice bearing subcutaneous, intraperitoneal, and lung cancers. Surprisingly, SV / TAA efficacy was characterized by the transient expression of TAA in lymph nodes draining the injection site, independent of tumor cell targeting. Early T cell activation at this site was followed by antigen-specific cytotoxic CD8 +A robust influx of NKG2D expressing T cells into the tumor site followed, leading to the generation of long-term surviving memory T cells. Such cells were protective against rechallenge with TAA-positive and TAA-negative tumor cells. As described herein, a combination of in vivo imaging, flow cytometry, cytotoxicity / cytokine assays, and tetramer analysis revealed the relationship between these events. As a result, CD8 + A model of T cell activation and a method of treating a mammal bearing a tumor by inducing an immune response against the tumor are provided.

[0016] SV / TAA may be combined with chemotherapy, as it has previously been shown that SV and chemotherapy can elicit synergistic effects (see, e.g., U.S. Patent Application No. 13 / 133,680). This includes, but is not limited to, chemotherapy that stimulates the immune system, inhibits suppressor elements in the immune system, or affects tumor cells to make them more susceptible to T-cell (or other immune cell) cytotoxicity. For example, certain chemotherapies may enhance SV / TAA immunostimulation by suppressing immune suppressive cells, thus facilitating SV / TAA therapy. There are also reports in the literature suggesting that chemotherapy enhances tumor cell susceptibility to T-cell mediated cytotoxicity. See, e.g., Ramakrishnan et al., Journal of Clinical Investigation, 120(11): 4141-4154, 2010.

[0017] In the methods of the present invention, a patient suffering from a tumor is examined to identify a TAA associated with the tumor. Examples of solid tumors that may be treated according to the present invention include sarcomas and carcinomas, such as, but not limited to, the following items: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland cancer, and sarcoma. Carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, glioma, and retinoblastoma.

[0018] Hematological malignancies may also be treated according to the present invention, provided that a specific TAA can be identified.

[0019] According to the present invention, the tumor and SV must express either the same TAA or a similar but non-identical TAA that immunologically cross-reacts with the TAA expressed by the SV / TAA. TAAs are well known in the art. For example, Cheevers et al. (Clin Cancer Res 15: 5323-5337, 2009) disclosed 75 representative TAAs for comparison and ranking by collecting information based on predefined criteria for specific antigens and ranking these antigens based on predefined and preweighted criteria. Any TAA expressed by the tumor may be utilized. However, only a portion of TAAs can potentially induce a strong response (immunodominant TAAs), and the variability of the efficacy of different TAAs is expected; which ones are exactly preferred can be determined using routine research well known to those skilled in the art.

[0020] TAAs expressed by a patient's tumor can be identified from a biopsy, or from a blood test if a biopsy is not possible. Serological analysis of expression cDNA libraries (SEREX) has previously been used to identify human TAAs. Alternative methods may be used.

[0021] After a relevant TAA is identified, a Sindbis virus vector carrying the gene encoding the TAA is constructed using techniques well known in the art, such as those described in Materials and Methods below. Nucleotide sequences encoding the TAA are also well known in the art and can be readily obtained from the literature. For example, the sequence of NY-ESO-1, a testis antigen aberrantly expressed in human cancers, was published in 1997 (http: / / www.pnas.org / content / 94 / 5 / 1914.full Yao-Tseng Chen*†‡, Matthew J. Scanlan†, Ugur Sahin§, Ozlem Tureci§, Ali O. Gure†, Solam Tsang†, Barbara Williamson†, Elisabeth Stockert†, Michael Pfreundschuh§, and Lloyd J. Old† PNAS 1997), and the carcinoembryonic antigen sequence was published in 1987 (http: / / mcb.asm.org / content / 7 / 9 / 3221.short Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen. N Beauchemin, S Benchimol, D Coumoyer, A Fuks, and CP Stanners, Molecular and Cellular Biology 1987).

[0022] Any Sindbis viral vector may be used in the present invention, including replication-competent (e.g., as described in U.S. Patent No. 8,282,916) and replication-deficient (e.g., as described in U.S. Patent Nos. 7,303,898, 7,306,792, and 8,093,021). To prevent infection of healthy tissue, replication-deficient vectors are preferred for use in the present invention.

[0023] According to the present invention, a single ip injection of a therapeutically effective amount of SV / TAA sufficient to infect cells in the mediastinal lymph nodes (MLNs) results in rapid immunogenic delivery to the MLNs. Such therapeutically effective amounts vary widely from about 10 million vector particles to about 100 billion vector particles. In mice, a single ip injection of SV / TAA results in detectable CD8 expression to tumors. + Although sufficient to induce a mediated immune response, other regimens may be required to achieve a maximal response. For example, preferably one to about eight ip injections over a period of one to several weeks may be administered for maximal effect, allowing for one or more booster injections a year or more later.

[0024] MLNs have previously been shown to drain the peritoneum [27, 28], providing an environment in which antigens delivered by SV vectors (e.g., TAAs) can potentially be processed by antigen-presenting cells (APCs) and presented to T cells under SV viral danger signals such as double-stranded (ds) RNA

[22] . One of the primary functions of lymph nodes is to promote the induction of adaptive immune responses. Viral danger signals are components of viruses (or infected cells) that stimulate the immune system. Double-stranded RNA is such a danger signal because it is not normally found in cells and is associated with viral infection. MLNs express CD8 against TAAs. + Provide a venue for the induction of T cell-mediated immune responses. Consistent with this finding, the number of T cells in MLNs was significantly increased 24 hours after SV / TAA treatment using LacZ as a model antigen.

[0025] It is also possible to use two (or more) different vectors, including injection at different times of different vectors carrying different cytokines to facilitate the induction and progression of an enhanced immune response to the TAA(s).

[0026] CD8 + In addition to T cells, SV / TAA treatment also enhanced humoral immune responses as well as CD4 + It may also activate additional immune (or non-immune) cells, including but not limited to T cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils and other cells. + Not only T cells, but also CD4 + It may also occur in T cells. As seen in Example 2, tumor cell targeting is not required for effective SV / TAA therapy, suggesting that immune cell activation during SV / TAA therapy may occur far from the tumor site (in this case the lung), for example in lymph nodes draining the SV injection site.

[0027] As shown in Example 3, flow cytometry was used to detect high numbers of CD8 + T cell influx was observed 7 days after the first SV / TAA injection. This peritoneal CD8 + T cells were activated. Activated CD8 + In addition to the robust influx of T cells, small numbers of high NKG2D, low L-selectin CD8 + T cells could also be seen in the lungs of mice bearing pulmonary CT26.CL25 tumors treated with SV / TAA. LacZ-specific CD8 +We found that a subset of T cells eventually develops into memory T cells. We analyzed splenocytes from SV / LacZ-treated long-term survivor mice bearing ipCT26.CL25 tumors. Using LacZ tetramers in combination with the memory marker CD127, we identified LacZ-specific CD127 T cells in these mice. + CD8 + The memory T cell population (CD8 + Approximately 1% of the T cell splenocyte population was identified more than 3 months after the last SV / LacZ injection, thus indicating that treatment according to the present invention led to long-term maintenance of antitumor activity.

[0028] The use of the methods of the present invention causes epitope spreading. One of the limitations of prior art cancer vaccine strategies is the inherent heterogeneity and genomic instability of tumor cell populations, which, combined with the selective pressure induced by the treatment, results in tumor evasion by losing or modifying the TAAs used in the vaccine [38, 39]. In this context, an important aspect of the present invention is the induction of epitope spreading, i.e., the amplification of antitumor T cell responses

[32] that incorporate new TAAs endogenous to the tumor but not delivered by the vector during SV / TAA treatment. Clinical trials are increasingly including the analysis of epitope spreading

[40] , and in some cases have shown a positive correlation between the induction of epitope spreading and therapeutic efficacy

[25] . As shown in Example 7, SV / TAA treatment of CT26.CL25 tumors caused epitope spreading, leading to the development of immunity against other unrelated antigens expressed on CT26 tumors.

[0029] In an alternative embodiment of the invention, a dual expression SV vector is utilized that carries and delivers a gene encoding a TAA together with a gene encoding an immune stimulating cytokine suitable for providing optimal conditions in the lymph node for T cell stimulation. Such immune stimulatory cytokines include, but are not limited to, IL-12 (disclosed in http: / / www.jimmunol.org / content / 146 / 9 / 3074.short Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. SF Wolf, PA Temple, M Kobayashi, D Young, M Dicig, L Lowe, R Dzialo, L Fitz, C Ferenz and RM Hewick the Journal of Immunology) and CCL17 (disclosed in http: / / www.jbc.org / content / 271 / 35 / 21514.short Molecular Cloning of a Novel T Cell-directed CC Chemokine Expressed in Thymus by Signal Sequence Trap Using Epstein-Barr Virus Vector* Toshio Imai‡, Tetsuya Yoshida, Masataka Baba, Miyuki Nishimura, Mayumi Kakizaki and Osamu Yoshie. The Journal of biological Chemistry).

[0030] Additional immune stimulating cytokines include, but are not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17. Additional cytokines include IL-18-IL-36. In addition to CCL17, other chemokines may be used, including, but are not limited to, CCL1-CCL27 and other CC chemokines, CXCL1-CXCL13 and other CXC chemokines, C chemokines, and CX3C chemokines. Cytokine or chemokine receptors and soluble receptors may be used. Additional immune modulators that may be used include TGF-β and TNFα. Different combinations of the above (or alternative) cytokines may also be used.

[0031] Moreover, because MLN TAA expression is transient and re-inducible (unpublished results), different cytokines can be delivered at different stages of SV / TAA treatment to further modify the antitumor immune response, for example, SV / IL12 can be delivered at an early stage of SV / TAA treatment to stimulate Th1 cytotoxic T cell responses, and SV / CCL17 can be delivered later to increase epitope spreading by enhancing cross-priming of additional TAAs.

[0032] SV vectors carrying the IL-12 gene have previously been demonstrated to enhance therapeutic efficacy in tumor-bearing mice

[16] and promote IFN-γ-dependent activation of M1-type macrophages

[19] . However, the effect of IL-12 delivery to MLNs has not been specifically studied prior to the present invention.

[0033] In another alternative embodiment, SV vectors are used to target and / or deliver payloads to mediastinal masses, such as those resulting from certain neurogenic tumors.

[37] Because tumors often metastasize to lymph nodes, including mediastinal lymph nodes, and SV may naturally target certain lymph nodes, including mediastinal lymph nodes, SV may be used to deliver antigens, cytokines, or other payloads directly to the site of tumor growth.

[0034] Multiple TAAs can be used by using one Sindbis vector expressing multiple TAAs or by using multiple Sindbis vectors expressing different TAAs, and the route of administration is parenteral, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, intraorbital, intranodal, and intratumoral injection.

[0035] A model of the method is shown below:

[0036] Step 1: ip injection of SV / TAA induces transient immunogenic expression of the TAA in the mediastinal lymph nodes, followed by induction of T cell activation at this site and / or other sites; NK cells are also activated against tumor cells. Step 2: TAA-specific CD8 + T cell cytotoxicity leads to the destruction of the tumor cells and the subsequent release of tumor-associated antigens. Step 3: Antigen-presenting cells capture the antigen in the tumor-draining lymph nodes and express the CD8 + TAA-specific CD8 that can present to T cells, resulting in epitope spreading and potentially targeting TAA(-) tumor cell escape mutants + Step 4: Induction of memory CD8 T cells against various tumor-associated antigens + T cells are generated.

[0037] The present invention is further illustrated below in working examples intended to further explain the invention without limiting its scope.

[0038] Materials and Methods cell lineage Baby hamster kidney (BHK), CT26.WT, and LacZ-expressing CT26.CL25 cells were obtained from the American Type Culture Collection. Firefly luciferase (Fluc)-expressing CT26 cells (CT26.WT.Fluc and CT26.CL25.Fluc) for non-invasive bioluminescence imaging were generated by stable transfection of Fluc-expressing plasmid into CT26.WT and CT26.CL25 cells. Fluc-expressing plasmid was constructed by introducing SV40 promoter sequence into the multiple cloning site of pGL4.20 vector (Promega, WI).

[0039] cell culture BHK cells were maintained in minimal essential a-modified medium (a-MEM) (Mediatech, VA) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA). CT26.WT, CT26.CL25, CT26.WT.Fluc, and CT26.CL25.Fluc cells were maintained in Dulbecco's modified essential medium (DMEM) containing 4.5 g / L glucose (Mediatech) supplemented with 10% FBS. All basal media were supplemented with 100 mg / mL penicillin-streptomycin (Mediatech) and 0.5 mg / mL amphotericin B (Mediatech). For culturing CT26.CL25 and CT26.CL25.Fluc cells, 0.4 mg / mL G418 sulfate (Mediatech) was added to the basal media. For culturing CT26.WT.Fluc and CT26.CL25.Fluc cells, 5 mg / ml puromycin (Sigma-Aldrich, MO) was added to the basal medium.

[0040] SV / TAA Production SV / LacZ was used as the immunogenic SV / TAA agent, and SV / Fluc and SV / GFP were used as control vectors. SV / Fluc was also used in imaging experiments (see below). Vectors were produced as previously described

[16] . Briefly, plasmids carrying replicons (SinRep5-LacZ, SinRep5-GFP, or SinRep5-Fluc) or DHBB helper RNA (SinRep5-tBB) were linearized with XhoI (for SinRep5-LacZ, SinRep5-GFP, and SinRep5-tBB) or PacI (for SinRep5-Fluc). In vitro transcription was performed using the mMessage mMachine RNA Transcription Kit (Ambion, TX). Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37°C in -MEM supplemented with 10% FBS. After 12 hours, the medium was replaced with OPTI-MEM I (Invitrogen, CA) supplemented with CaCl2 (100 g / mL) and the cells were incubated at 37° C. After 24 hours, the supernatants were collected, centrifuged to remove cell debris, and frozen at −80° C. Vector titers were determined as previously described

[15] .

[0041] Mice and tumor inoculation Female BALB / c mice aged 4 to 8 weeks were purchased from Taconic (Germantown, NY). For the sc tumor model, 0.5 × 10 6 Or 1×10 6 CT26.WT or CT26.CL25 cells were injected sc into the right flank of each mouse. For the ip tumor model, 2.5 × 10 cells in 0.2 mL PBS were 4 Or 5 x 10 4 CT26.CL25 cells were injected i.p. into each mouse. For lung tumor model, 0.3 × 10 cells in 0.2 ml PBS were injected i.p. 6 CT26.WT.Fluc or CT26.CL25.Fluc cells were injected iv into each mouse.

[0042] Treatment Efficacy In the sc tumor model, tumor volumes of 40 mm 3 Treatment was initiated after the tumor volume exceeded 100 μg / mL (volume = width × width × length / 2). In the ip tumor model, treatment was initiated on day 4 after tumor cell inoculation. In the lung tumor model, treatment was initiated on day 3 after tumor cell inoculation. SV / LacZ, SV / GFP, or SV / Fluc (approximately 10 μg / mL in 0.5 mL of OPTI-MEM I) were injected into the mouse. 7 Plaque forming units (PFU) and mock treatment (0.5 mL of OPTI-MEM I supplemented with 100 mg / L CaCl2) were administered ip, four times per week for 2 weeks for a total of eight treatments. Treatment efficacy was monitored in three ways: tumor volume (for sc tumors, measured by mechanical caliper), tumor luminescence (for lung tumors), and survival (for ip and lung tumors). Noninvasive bioluminescence imaging was performed using an IVIS Spectrum imaging system (Caliper Life Sciences, MA) as previously described

[16] , and tumor growth was quantified using Living Image 3.0 software (Caliper Life Sciences). Survival was monitored and recorded daily.

[0043] Bioluminescence imaging of SV / Fluc Tumor-bearing and tumor-free mice were treated with SV / Fluc (approximately 10 in 0.5 mL of OPTI-MEM I). 7 Plaque forming units (PFAs) were intraperitoneally injected into the mouse. After treatment, bioluminescence signals were detected by IVIS at the indicated time points as previously described

[16] .

[0044] Ex vivo cytotoxicity assay Seven days after SV treatment was initiated, lung lymphocytes or splenocytes from tumor-bearing mice were harvested. Lung lymphocytes (1 × 10 5 / ml) or splenocytes (2 × 10 6 CT26.WT.Fluc cells (2 × 10 4 / ml) or CT26.CL25.Fluc cells (2 × 104 Cells were co-cultured with 100 μl of M-PER Mammalian Protein Extraction Reagent (Pierce, IL) per well for 1 h. The cells were then ...

[0045] IFN-γ secretion assay. CT26 tumor cells (2 × 10 4 Lung lymphocytes (1 × 10 5 / ml) or splenocytes (2 × 10 6 Cells were stimulated with 100 µg / ml of IFN-γ secretion medium. The peptides used were the LacZ peptide TPHPARIGL

[43] , the gp70 peptide SPSYVYHQF

[44] , or the PIA peptide LPYLGWLVF (as a negative control)

[45] . After stimulation, IFN-γ levels in the medium were measured using a mouse IFN-γ Quantikine ELISA kit (R&D systems, Minneapolis, MN). TPHPARIGL- and SPSYVYHQF-mediated increases in IFN-γ secretion were calculated by subtracting IFN-γ levels in control (LPYLGWLVF-stimulated) samples from IFN-γ levels in TPHPARIGL- and SPSYVYHQF-stimulated samples.

[0046] Flow cytometry. Anti-mouse antibodies anti-CD8a eFluor® 450 and eFluor® 650NC, anti-CD4 PE-Cyanine 7, anti-CD69 PE, anti-CD314 (NKG2D) PE-Cyanine7, anti-CD62L (L-selectin) FITC and Alexa Fluor® 700, and anti-CD45 eFluor® 450 were purchased from eBioscience (San Diego, CA). PE-labeled LacZ tetramers were obtained from NYU Vaccine and Cell Therapy Core (New York, NY), and APC-labeled gp70 tetramers were obtained from NIH Tetramer Core Facility (Atlanta, Georgia). For flow cytometry analysis of lung lymphocytes and splenocytes, mice were euthanized and lungs and spleens were extracted. The extracted lungs were cut into small pieces and incubated with a digestion mixture [collagenase I (50 μg / ml), collagenase IV (50 μg / ml), hyaluronidase V (25 μg / ml), and DNAse I (20 units / ml)] at 37°C for 30 min. The extracted spleen and digested lungs were then mashed through a 70-100 μm cell strainer and treated with 1× RBC lysis buffer (eBioscience) to exclude red blood cells. Peritoneal cells were collected from the peritoneal exudate as previously described

[19] . Cells were then stained with various Abs, washed twice with HBSS (Mediatech), and analyzed using an LSR II machine (BD biosciences, CA). Data were analyzed using FlowJo (Tree Star, San Carlos, CA).

[0047] CD8 + T cell depletion. CD8 T cell depletion was detected using anti-CD8 antibody (clone 2.43) (Bio X cell, Lebanon, NH). + T cells were depleted by injecting each mouse with 0.4 mg antibody in 0.2 mL PBS, starting 1 day before the first SV treatment and every 2-3 days for 2 weeks. Control mice were injected with PBS.

[0048] statistics For flow cytometry, IVIS imaging, ELISA, tumor growth, and survival experiments, Student's t-test (two-tailed), analysis of variance (ANOVA) followed by Dunnett's test, or Kaplan-Meier log-rank test were performed using Prism® 4 for Macintosh (GraphPad Software, La Jolla, CA). EXAMPLES

[0049] SV / LacZ inhibits the growth of LacZ-expressing tumors in immunocompetent mice. To evaluate the use of SV vectors carrying TAA for cancer therapy, a LacZ-expressing mouse colon cancer cell line (CT26.CL25) was used as a model tumor TAA system. First, we tested the efficacy of SV / TAA (SV / LacZ) in subcutaneous (sc) tumor-bearing mice. As seen in Fig. 1a, SV / LacZ significantly inhibited the growth of LacZ-expressing CT26.CL25 tumors, whereas the control vector SV / Fluc had no observable therapeutic effect (Fig. 1a, left panel). On the other hand, both SV / LacZ and SV / Fluc had little effect on the growth of LacZ-negative CT26.WT tumors (Fig. 1a, right panel). These results demonstrate that SV / LacZ has a potent antigen-dependent therapeutic effect in mice bearing scCT26 tumors.

[0050] To examine the efficacy of SV / LacZ in a physiologically relevant colon cancer model, CT26.CL25 cells were injected intraperitoneally to mimic peritoneal carcinomatosis

[26] . The therapeutic efficacy in this model was evaluated by monitoring the survival of mice. As in the sc model, SV / LacZ was found to have a strong therapeutic effect on this tumor, whereas the control vector (SV / Fluc) had only a mild therapeutic effect (Fig. 1b). Next, we examined the efficacy of SV / LacZ against tumors growing in the lung. To supplement the survival data in this model, we constructed Fluc-expressing CT26 cell lines (CT26.CL25.Fluc and CT26.WT.Fluc), which could be used to noninvasively monitor tumor growth using the IVIS imaging system

[16] . Intravenous injection of Fluc-expressing CT26.CL25 cells resulted in lung tumors, and it was found that SV / LacZ induced complete tumor remission and long-term survival in this model, whereas the control vector SV / GFP only delayed tumor growth and did not result in long-term survival (Fig. 1c). As in the sc tumor model, the enhanced therapeutic effect obtained from SV / LacZ in the lung tumor model depended on the expression of TAA (LacZ) from the vector and tumor cells, since LacZ-negative CT26.WT tumor growth was only slightly inhibited by SV / LacZ (Fig. 9). Taken together, these results demonstrate that SV vectors carrying TAA induce a strong therapeutic effect in mice bearing TAA-expressing CT26 tumors, regardless of the site of tumor growth. EXAMPLES

[0051] Mediastinal lymph nodes transiently express antigens delivered by SV vectors and are the site of early T cell activation during SV treatment. It has been previously shown that SV vectors have oncolytic potential and can target specific tumors in vivo

[16] . To evaluate the role of tumor cell targeting in the therapeutic effects observed in the CT26 tumor model, tumor-bearing mice were treated with SV / Fluc vectors. This can be used to monitor vector localization in mice

[16] . Even after multiple injections, SV vectors were found not to target sc-grown CT26.CL25 tumors (Fig. 2a, left panel). Similarly, the vectors did not target lung tumors; instead, SV / Fluc was found in the peritoneal fat of tumor-bearing mice 24 h after the first injection and in the liver 5 days later (Fig. 10). This general pattern was not dependent on the presence of tumor cells and occurred in tumor-free mice as well (Fig. 10). These results are consistent with other studies demonstrating that CT26 cells are not infected in vitro by SV

[20] and suggest that the strong therapeutic effects obtained from SV / LacZ are not dependent on tumor cell targeting.

[0052] Interestingly, by focusing on very early time points after SV / Fluc injection, we noticed that a transient Fluc signal could be seen in the upper body as early as 3 hours after ipSV / Fluc injection (Fig. 2a and Fig. 10). By extracting and imaging the mediastinal lymph node (MLN) separately, we determined that the signal in the upper body originated from this lymph node (Fig. 2a, right panel). Notably, the transient Fluc expression in the MLN occurred in both tumor-bearing and tumor-free mice (Fig. 10).

[0053] MLNs have previously been shown to drain the peritoneum [27, 28], providing an environment in which antigens delivered by SV vectors (such as Fluc, LacZ, or other TAAs) can potentially be processed and presented to T cells by antigen-presenting cells (APCs) under the SV viral danger signal, such as double-stranded (ds) RNA

[22] . Thus, MLNs provide a place where immune responses to SV / TAAs can be induced. Consistent with this hypothesis, the number of T cells in MLNs was significantly increased 24 h after SV / LacZ treatment (Fig. 2b). As a control lymph node, we used inguinal lymph nodes (ILNs), which do not directly drain the peritoneal cavity

[29] and were not targeted by ip injection of SV / Fluc (Fig. 2a and further data not shown). Unlike MLNs, there was no increase in T cells in ILNs 24 h after SV / LacZ injection (Fig. 2b). In addition to the clear influx of T cells into the MLNs, expression of CD69, an early activation marker of T cells, increased with increasing expression of CD8+ / -T cells in the MLNs 24 hours after SV / LacZ treatment. + In contrast, CD8 T cells derived from control ILNs were strongly induced by ILN-associated ... + T cells were significantly less activated, although a slight increase in CD69 expression was observed in these cells (Fig. 2c). Taken together, Fig. 2 demonstrates that tumor cell targeting is not required for effective SV / LacZ therapy and suggests that immune cell activation during SV / LacZ therapy may occur far from the tumor site, for example in lymph nodes draining the SV injection site. EXAMPLES

[0054] SV / LacZ treatment inhibited CD8 + Induce robust activation of T cells. CD8 in lymph nodes draining the SV injection site + T cell activation was observed, so activated CD8 + It was anticipated that T cells could then migrate into the injection site in the peritoneum. Using flow cytometry, we found that numerous CD8 T cells were present in the peritoneum up to 7 days after the first SV / LacZ injection. +The influx of T cells was confirmed (Figure 3a). + T cells were activated, as evidenced by upregulation of NKG2D

[30] and downregulation of the lymph node homing receptor L-selectin

[31] (Figure 3b). + In addition to the robust influx of T cells, small numbers of high NKG2D, low L-selectin CD8 + T cells could also be seen in the lungs of SV / LacZ-treated mice bearing pulmonary CT26.CL25 tumors (FIG. 3b). EXAMPLES

[0055] SV / LacZ treatment inhibited the expression of LacZ-specific effector and CD8 + Induce memory T cells. The fact that SV therapeutic efficacy depends on expression of LacZ from both vector and tumor cells (Fig. 1 and Fig. 9), together with the robust activation of CD8+ T cells observed during SV / LacZ treatment (Fig. 2 and Fig. 3), supports the notion that CD8 + These results suggest that T cells may be involved in the antitumor effect of SV / LacZ in this model. Nevertheless, CD8 + T cell activation also occurred during SV / GFP and SV / Fluc treatment (Figure 11). However, the therapeutic efficacy of these vectors was significantly lower (Figure 1). What distinguishes SV / LacZ from other vectors is the expression of LacZ-specific CD8 T cells that can then target LacZ-expressing tumors. + It was hypothesized that the ability to directly stimulate T cells was the key to this. To prove this concept, SV / LacZ was injected into LacZ-naive tumor-free mice, and robust LacZ-specific CD8 T cells were detected after 4 days in the peritoneum. + T cell responses were observed (Figure 12a). LacZ-specific CD8 +Expansion of T cells was observed in the spleens of SV / LacZ-treated sc tumor-bearing mice (Fig. 4a and 4b), in the peritoneum of ip and lung tumor-bearing mice (Fig. 4c), and in the lungs of lung tumor-bearing mice (Fig. 4d, left panel). + T cells were fewer (Fig. 12b). As expected, LacZ-specific CD8 T cells from SV / LacZ-treated mice + T cells were characterized by an activated (high NKG2D, low L-selectin) phenotype (Fig. 4d, right panel, and Fig. 12a, right panel). Taken together, these results suggest that SV / LacZ treatment enhanced the expression of LacZ-specific CD8 + We demonstrated that this leads to potent activation of T cells, which may be the mechanism for the LacZ-dependent efficacy seen in FIG.

[0056] LacZ-specific CD8 generated during SV / LacZ treatment + To determine whether a subset of T cells ultimately develops into memory T cells, splenocytes from SV / LacZ-treated long-term survivor mice bearing ipCT26.CL25 tumors were analyzed. LacZ tetramers were used in combination with the memory marker CD127 to identify LacZ-specific CD127 T cells in these mice. + CD8 + The memory T cell population (CD8 + Approximately 1% of the T cell splenocyte population was identified more than 3 months after the last SV / LacZ injection, whereas control splenocytes from naive mice had only background levels of this population (<0.1%) (Fig. 12c). EXAMPLES

[0057] SV / LacZ treatment induces lymphocyte cytotoxicity against CT26.CL25 tumor cells. As shown in FIG. 4d, LacZ-specific CD8 +T cells appeared to be activated. To examine the function of these cells, ex vivo cytotoxicity assays were performed using lung lymphocytes from SV / LacZ (or SV / GFP)-treated lung tumor (CT26.CL25)-bearing mice. As shown in Figure 5a, the viability of CT26.CL25 tumor cells was significantly lower when cocultured with lung lymphocytes from SV / LacZ-treated mice compared to when cocultured with lymphocytes from mock- or SV / GFP-treated mice. Notably, lung lymphocytes from SV / LacZ-treated mice did not affect the viability of LacZ-negative CT26.WT tumor cells, demonstrating the antigen-specific nature of the immune response in the lung. Consistent with this result, only lung lymphocytes from SV / LacZ-treated mice cocultured with LacZ-expressing CT26.CL25 tumor cells showed increased IFN-γ production (Figure 5b). EXAMPLES

[0058] CD8 + T cells are required to enhance the antigen-specific therapeutic effect of SV / LacZ. The results of cytotoxicity and IFN-γ secretion assays (Fig. 5) confirm the in vivo observation that SV / LacZ has a significantly stronger therapeutic effect against CT26.CL25 tumors than the control vector (Fig. 1), and that SV / LacZ induces strong LacZ-specific CD8 expression in tumor-bearing mice. + These results are consistent with the observation that CD8 T cell responses were induced by SV / TAA therapy (Figure 4). Taken together, these results support the contribution of CD8 T cell responses to the antigen-specific benefits of SV / TAA therapy. + This strongly suggests the involvement of T cells. + To directly determine the role of T cells, we analyzed CD8 T cells in mice bearing sc (Fig. 6a), peritoneal (Fig. 6b), and lung (Fig. 6c) tumors. + Depleting T cell populations to reduce CD8 + We found that in the absence of T cells, the therapeutic efficacy of SV / LacZ was greatly reduced in all three models. EXAMPLES

[0059] SV / LacZ treatment induces epitope spreading. Surprisingly, unlike lung lymphocytes, splenocytes from SV / LacZ-treated tumor-cured mice were found to acquire cytotoxicity not only against CT26.CL25 cells, but also against LacZ-negative CT26.WT cells (Fig. 7a). Consistently, when the splenocytes were cocultured with CT26.WT cells, an increase in IFN-γ production was observed. However, the extent of production was lower than when they were cocultured with CT26.CL25 cells (Fig. 7b). Based on these results, we hypothesized that SV / LacZ-treated tumor-cured mice might have acquired resistance to LacZ-negative CT26.WT tumors. To determine whether this was the case, we injected CT26.WT cells iv (Fig. 7c) or ip (data not shown) into SV / LacZ-treated tumor-cured mice and found that tumors did not grow. In contrast, tumor growth was readily observed in control (naive) mice. These results suggest that an immune response to endogenous CT26 tumor antigen may have developed as a result of SV / LacZ treatment, a concept alternatively known as epitope spreading, antigen spreading, determinant spreading, or antigen cascade

[32] . To confirm that epitope spreading occurred during SV / LacZ treatment, we focused on gp70, an endogenous CT26 TAA. As shown in Figure 7d, an increase in IFN-γ secretion from splenocytes taken from SV / LacZ-treated tumor-cured mice was observed after culturing the cells with either gp70 or LacZ peptide, whereas neither peptide induced IFN-γ secretion from naive splenocytes. These results indicate that splenocytes from SV / LacZ-treated tumor-cured mice were able to respond to endogenous CT26 TAAs such as gp70 in addition to LacZ. Consistent with this observation, flow cytometry analysis using gp70 tetramers revealed that gp70-specific CD8 +We demonstrated that the number of T cells was increased in the spleens of SV / LacZ-treated tumor-cured mice compared to naive mice (Fig. 7e). Taken together, these results indicate that SV / LacZ treatment of CT26.CL25 tumors induced epitope spreading, which led to the development of immunity against other antigens expressed on CT26 tumors.

[0060] Using the murine cancer TAA system disclosed herein, the use of SV vectors carrying TAAs for cancer therapy was examined, with the following key observations: (i) SV represents a potentially powerful therapeutic platform for immunogenic delivery of TAAs; (ii) the therapeutic benefit derived from SV / TAA does not necessarily require direct targeting of tumor cells; (iii) SV / TAA therapy includes transient and early delivery of the TAA to lymph nodes draining the injection site, particularly the MLN in the case of ipSV injection; and (iv) SV / TAA therapy induces potent TAA-specific CD8 + (v) SV / TAA treatment results in epitope spreading, which may be a solution to the problem of tumor escape due to TAA loss or modification, and (vi) SV / TAA treatment ultimately results in long-term survival of tumor-bearing mice and the induction of long-lived CD8 T cells against multiple TAAs. + Leading to the generation of memory T cells.

[0061] Based on these findings, CD8 + A four-step model for the activation of T cell-mediated antitumor immunity (induction, cytotoxicity, epitope spreading, and memory) is presented.

[0062] Over the last decades, various methods have been developed for the immunogenic delivery of TAAs, including the use of vectors targeted to antigen-presenting cells (APCs)

[33] or injected directly into lymph nodes

[34] . As disclosed herein, we demonstrated that a single ip injection of SV / TAA resulted in rapid immunogenic delivery of TAA to MLNs. TAA expression in MLNs was transient and would likely have remained unnoticed without the use of sensitive IVIS imaging systems. ip injections are frequently used in animal studies and are becoming increasingly common in the clinic

[35] . Thus, the observation of transient TAA expression and subsequent T cell activation at this site (Figure 2) may have broad implications for the development of cancer immunotherapy. In this context, Hsu et al. recently demonstrated that ip-injected cytomegalovirus inhibited the uptake of CD169 in MLNs. + demonstrated that macrophage depletion leads to productive infection of SV-derived heterologous proteins in MLNs

[28] . Consistent with this, macrophage depletion substantially reduced the expression of SV-derived heterologous proteins in MLNs (unpublished results). However, notably, anti-TAA CD8 + The induction of T cell immunity was not significantly inhibited in macrophage-deficient mice treated with SV / LacZ (unpublished results). This difference may be due to the fact that while both macrophages and dendritic cells (DCs) express viral antigens in the draining lymph nodes, only DCs express these antigens and only naive CD8 + This may be resolved by the observation that MLNs efficiently present TAA to T cells

[36] . Another possible explanation is the fact that additional lymph nodes other than MLNs also drain the peritoneal cavity. Indeed, transient heterologous protein expression was also observed in the peritoneal cavity of SV-treated mice (Figure 10, upper panel). Further studies are necessary and ongoing to clarify the role of TAA delivery to MLNs during SV / TAA treatment.

[0063] In addition to the activation of T cells in the MLN, CD8 +T cells appear to redistribute throughout the body. Various tissues, including the peritoneum (Fig. 11a) and lungs (Fig. 13), express CD8 T cells during the first 1-2 days after SV / TAA injection. + The SV / TAA-treated mice showed a decrease in CD8 T cells. A clear efflux of T cells from these tissues coincides with their influx into the MLNs (Fig. 2B). It is interesting to note that during this early phase, lung tumors in SV / TAA-treated mice already appear to shrink (Fig. 1c). Moreover, this early therapeutic effect was observed in mice treated with a control vector that does not express TAA (Fig. 1c), in SV-treated mice bearing tumors that do not express TAA (Fig. 9), and in CD8 T cells. + This was also observed in T cell-depleted SV / TAA-treated mice (Fig. 6c). One possible explanation for this is the activation of natural killer (NK) cells by SV. It has previously been shown that SV treatment induces robust NK cell responses in tumor-bearing mice

[19] . In the CT26 lung model, a rapid influx of NKG2D-expressing NK cells into the lungs was observed as early as 2 days after SV injection, in response to TAA-specific CD8 + This was observed several days before the maximum influx of T cells (Figure 13).

[0064] One of the limitations of cancer vaccine strategies is that the inherent heterogeneity and genomic instability of tumor cell populations, combined with treatment-induced selection pressure, may lead to tumor evasion by losing or modifying the TAAs used in the vaccine [38, 39]. In this context, an interesting and therapeutically significant observation is the induction of epitope spreading, i.e., the amplification of antitumor T cell responses that incorporate new TAAs endogenous to the tumor but not delivered by the vector during SV / TAA treatment

[32] (Figure 7). Clinical trials are increasingly including the analysis of epitope spreading

[40] and in some cases have shown a positive correlation between the induction of epitope spreading and therapeutic efficacy

[25] . These developments may represent a paradigm shift in the design of cancer vaccines, which will focus on the induction of strong and diversified T cell responses that could potentially be effective even against tumors expressing a variety of different antigens.

[0065] In summary, this application provides a method for the use of SV / TAA for cancer therapy and offers valuable insight into the mechanisms underlying SV / TAA efficacy. In accordance with the present invention, the use of SV vectors carrying TAA not only greatly enhances SV efficacy but also paves the way for a broader application of SV anticancer therapy by eliminating the need for tumor cell targeting, which was previously a prerequisite for effective oncolytic SV therapy. The present findings complement and expand on previous studies on the use of SV nucleic acid

[41] and replicon particle

[42] vaccines, as well as previous studies on the oncolytic potential of SV [15, 16], and demonstrate the versatility of SV anticancer therapy.

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[0067] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

[0068] It should further be understood that all values ​​are approximate and are provided for purposes of illustration.

[0069] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated by reference in their entireties for all purposes.

Claims

1. 1. An immunogenic composition formulated for parenteral delivery comprising a Sindbis virus vector carrying a polynucleotide encoding a tumor-associated antigen (TAA) NY-ESO-1 or a TAA that is immunologically cross-reactive with said TAA, the parenteral delivery is selected from intraperitoneal delivery or intravenous delivery; the Sindbis viral vector is present in an amount sufficient to elicit an anti-tumor immune response in lymph nodes and cause epitope spreading when administered to a subject; the Sindbis viral vector does not directly target tumor cells in the subject; composition.

2. The immunogenic composition of claim 1, wherein the parenteral delivery is intraperitoneal delivery.

3. The immunogenic composition of claim 1, wherein the parenteral delivery is intravenous delivery.

4. The induced anti-tumor immune response is characterized by CD8 + The immunogenic composition of any one of claims 1 to 3, which is a T cell mediated immune response.

5. 4. The immunogenic composition of any one of claims 1 to 3, wherein the Sindbis viral vector infects lymph nodes followed by induction of T cell activation.

6. 4. The immunogenic composition of any one of claims 1 to 3, wherein the Sindbis virus vector is replication-defective.

7. 4. The immunogenic composition of any one of claims 1 to 3, wherein the Sindbis virus vector is replication competent.

8. The immunogenic composition according to any one of claims 1 to 3, wherein the subject is a human.

9. The immunogenic composition according to any one of claims 1 to 3, wherein the tumor is a solid tumor.

10. The immunogenic composition of any one of claims 1 to 3, wherein the Sindbis virus vector is a dual-expression Sindbis virus vector having a polynucleotide encoding NY-ESO-1 and a polynucleotide encoding an immunostimulatory cytokine selected from interleukin-12 (IL-12) or CCL17.

11. Interleukin-1 (IL-1) to interleukin-36 (IL-36), chemokine (CC motif) ligand 1 (CCL1) to chemokine (CC motif) ligand 27 (CCL27), chemokine (CXC motif) ligand 1 (CXCL1) to chemokine (CXC motif) ligand 13 (CXCL13), and chemokine (CX 3 -C motif) (CX 3 The immunogenic composition according to any one of claims 1 to 3, further comprising a polynucleotide encoding a cytokine selected from the group consisting of C).

12. The immunogenic composition of claim 9, wherein the solid tumor is an ovarian tumor.

13. 4. The immunogenic composition of any one of claims 1 to 3, further comprising a pharma- ceutically acceptable excipient, vehicle or diluent.

14. The immunogenic composition of claim 13 for inducing an anti-tumor immune response in a subject.

15. The immunogenic composition of claim 14 for treating a tumor.

16. The immunogenic composition of claim 14, wherein the tumor is an ovarian tumor.

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