TREATMENT OF CANCER

Abstract

A method of treating cancer in a subject is disclosed, the method comprising administration of an oncolytic herpes simplex virus and administration of lymphocyte cells modified to express a chimeric antigen receptor (CAR) or modified to express a T cell receptor (TCR).

Claims

1. A method of treating cancer in a subject, the method comprising administration of an oncolytic herpes simplex virus and administration of human lymphocyte cells modified to express a chimeric antigen receptor (CAR) or modified to express a T cell receptor (TCR).

2. The method of claim 1, wherein the lymphocyte cells are T-cells.

3. The method of claim 1, wherein the T-cells are cytotoxic T-cells, CD8+ T cells or CD4+ T cells.

4. The method of claim 1, wherein the cancer is a solid tumor.

5. The method of claim 1, wherein the CAR or TCR targets an antigen selected from the group consisting of GD2, CD44v7/8, DNAM-1 (DNAX accessory molecule-1), EGP-40 (epithelial glycoprotein-40), EpCAM (endothelial cell adhesion molecule), FBP (folate-binding protein), FR, GD3, VEGFR2, LMP-1 (latent membrane protein 1), MUC1 (mucin 1), PSCA (prostate stem cell antigen), α-folate receptor, CD171, CAIX, Her2, IL13Rα2, IL13R, IL3RA, CEA, CD19, CD20, Lewis-Y, CD33, CD38 (also known as cyclic ADP ribose hydrolase), CD123, gp100, MART1, CEA, CAIX, Her2//Neu, MAGE-A3/A19/A12, MAGE-A3/titin, CD19, GD2, NY-ESO-1, CTAG1B, MAGE-A1, MAGE-C1, SSX2, MAGE-A2B, Brachyury, NY-BR1, BCMA, KRAS (e.g. KRAS G13D, KRAS G12V, KRAS G12R, KRAS G12D, KRAS G12C), KIT, PD-L1, EGFRviii, HPV 16 E6, HPV 16 E7, HPV18 E6, HPV18 E7 and other tumor associated antigens

6. The method of claim 1, wherein the administration of the oncolytic herpes simplex virus and lymphocyte cells is simultaneous or sequential.

7. The method of claim 1, wherein the oncolytic herpes simplex virus is administered to the blood.

8. The method of claim 1, wherein the oncolytic herpes simplex virus is administered by intratumoral injection.

9. The method of claim 1, wherein the administration of human lymphocyte cells is part of a method of autologous therapy.

10. The method of claim 1, wherein the oncolytic herpes simplex virus does not express, or is not modified to express, a cytokine or chemokine.

11. The method of claim 1, wherein the oncolytic herpes simplex virus does not contain, or is not modified to contain, nucleic acid encoding at least one copy of a polypeptide that is heterologous to the virus.

12. The method of claim 1, wherein the oncolytic herpes simplex virus is an HSV-1 strain 17+ or mutant thereof.

13. The method of claim 1, wherein the oncolytic HSV is HSV1716.

14. A method of increasing the efficacy of adoptive cell therapy in a subject by administering an oncolytic herpes simplex virus to a subject in need thereof.

15. A kit comprising at least one container having a predetermined quantity of oncolytic herpes simplex virus, and at least one container having a predetermined quantity of human lymphocytes modified to express a chimeric antigen receptor (CAR) or T cell receptor (TCR).

16. The kit of claim 15, wherein the oncolytic herpes simplex virus and lymphocytes are in separate containers.

17. The kit of claim 15, wherein the kit comprises a container having a mixture of a predetermined quantity of oncolytic herpes simplex virus and predetermined quantity of human lymphocytes.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0176] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0177] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0178] FIGS. 1A and 1B. Most mouse tumor cell lines do not endogenously express GD2. FIG. 1A Mouse Fibroblasts (3T3), Neuroblastoma (DFCI-331), Melanoma (B16), and RMS (HGF-116, 76-9, and M3-9-M) Cells were stained for cell surface GD2 and analysed by flow cytometry. FIG. 1B Mouse tumor cell lines were modified to express GD2 by either being fused to dorsal root ganglion cells (NXS2) or by the over-expression of GD2 and GD3 synthases (B78D14, B16/GD2, HGF-116/GD2, and 76-9/GD2) then stained for cell surface GD2 and analysed by flow cytometry.

[0179] FIG. 2. Soluble 14G2a scFv binds specifically to GD2-expressing Tumor cells. Mouse cell lines were incubated with soluble 14G2a scFv fused to rat CH.sub.2CH.sub.3. Binding of soluble 14G2a was detected using anti-rat F(ab′)2 and analysed by flow cytometry.

[0180] FIGS. 3A-3D. GD2-28z CAR T cells are functionally active in the presence of antigen positive targets. FIG. 3A Time line for generating GD2-28z. FIG. 3B Day 4 T GD2-28z CAR T cells were stained for CD4 and GD2 CAR and analysed by flow cytometry. FIG. 3C Tumor Cells were labelled with Chromium-51 for 1-2 Hours then co-cultured with GD2-28z or mock Transduced T cells for 6 hrs and Chromium-51 release was measured using a scintillation counter. FIG. 3D GD2-28z or Mock transduced T cells were co-cultured in the presence or absence of tumor cells for 24 hrs at a 1:1 ratio and secreted cytokines were measured using CBA beads.

[0181] FIG. 4. GD2-28z CAR T Cells persist in vivo and delay tumor growth.

[0182] Mice Received 2×10.sup.6 B78D14 Cells on day 0, 500cGy TBI on day 6, and 4×10.sup.6 GD2-28z CAR T cells or mock T cells on day 7. CD8+CAR Persistence (A), tumor size (B), and percent survival (C) was measured.

[0183] FIG. 5. RMS cell lines express HSV1716 entry receptors and are susceptible to oncolytic lysis. A) RNA Was extracted from three RMS cell lines (M3-9-M, 76-9, And HGF-116GL) and two melanoma lines (B16 and B78D14) And Nectin 1 Expression levels were determined via real-time PCR. (B) Mouse cell lines were infected with HSV1716 at MOI: 0.01, 0.1, 1.0, 10, And 100. Percent survival by MTS assay is shown relative to mock-infected control 2 days post-infection.

[0184] FIG. 6. HSV1716 Increases GD2 CAR Persistence in melanoma models. Mice received 500cGy TBI on day 0, 2×10.sup.6 B78D14 (A) or B16/GD2 (B) Cells on day 1, 500cGy TBI on day 6, and 3×10.sup.6 GD2-28z CAR T cells or mock T cells on day 7. HSV1716 was administered on days 3, 5, 7, 10, and 13. CAR persistence, tumor size, and percent survival was measured weekly.

[0185] FIGS. 7A-7D. HSV1716 and T cells synergize to delay tumor growth and enhance T cell persistence. Mice received 500cGy TBI On day 0, 2×10.sup.6 76-9GL/GD2 cells on day 1, and 5×10.sup.6 GD2-28z CAR T cells or mock T cells on day 7. HSV1716 was administered on days 3, 5, 7, 10, and 13. Adoptively Transferred T cell persistence FIG. 7A, CAR persistence FIG. 7B, tumor size FIG. 7C, and percent survival FIG. 7D were measured over time.

[0186] FIG. 8. Cytokine and chemokine profiles of tumor models in response to oHSV infection in vitro. Human Ewing sarcoma model A673 and human neuroblastoma models SK-N-AS and SK-N-BE(2) were cultured with oHSV at a multiplicity of infection (MOI) of 10 and gene expression analysis was performed at 12 hours post infection by RT-PCR.

[0187] FIG. 9. Baseline GD2 expression in human Ewing sarcoma xenograft model A673 and human neuroblastoma xenograft models SK-N-AS and SK-N-BE(2) in vitro by flow cytometry analysis. Results represent averages of 3 replicates, each with triplicate samples.

[0188] FIG. 10. Migration of human GD2-directed CAR-T cells toward human Ewing sarcoma A673 and neuroblastomas SK-N-AS and SK-N-BE(2) with and without oHSV infection by transwell assay. Human Ewing sarcoma A673 and human neuroblastoma SK-N-AS and SK-N-BE(2) cells were cultured with Seprehvir at a multiplicity of infection (MOI) of 1 for 24 hours and red fluorescent PKH23-stained GD2-directed human CAR-T cells were added into 5 um pore transwell inserts above the cell culture at an E:T ratio of 2:3 for 2 hours. Media alone served as a negative control, while media containing 75 ng/ml CXCL-10 (IP-10) and media containing 10 ng/ml CCL-5 (RANTES) served as positive controls. Cells were quantified through microscopic visualization. Results represent averages of 5 replicates for each sample.

[0189] FIG. 11. Survival curve of Ewing sarcoma tumor-bearing mice treated with human GD2-directed CAR-T cells with and without Seprehvir. Athymic nude mice were subcutaneously inoculated with human Ewing sarcoma xenograft A673 cells and tumors were allowed to reach volumes of 200-250 mm.sup.3. Mice were treated with either PBS or Seprehvir at a dose of 1e7 PFU intra-tumorally on days 0, 3, and 5. Mice received either PBS or 2.5 mg cyclophosphamide (CPM) intraperitoneally on day 3 as lymphodepletion. On day 6, 1.2e7 total GD2-directed human CAR T-cells (83% CAR positivity) were injected intravenously. Tumor growth and overall survival were monitored for 85 days after the initial treatment.

[0190] FIG. 12. No significant change in GD2 expression with oHSV1716 infection in hNBL cell lines SK-N-AS and SK-N-BE2, but increased GD2 expression with infection in hEWS cell line A673. No significant change in GD2 expression with oHSV1716 infection in hNBL cell lines SK-N-AS and SK-N-BE2, but increased GD2 expression with infection in hEWS cell line A673

[0191] FIG. 13. No significant change in PD-L1 expression with oHSV1716 infection in pediatric solid tumor cell lines. PD-L1 expression before and after HSV1716 infection of human Ewing sarcoma xenograft model A673 and human neuroblastoma xenograft models SK-N-AS and SK-N-BE(2) in vitro. Cells were infected with HSV1716 at multiplicity of infection (MOI) of 0, 0.1, or 1 for 24 hours and then analyzed by flow cytometry. A673 displayed intermediate PD-L1 expression. SK-N-AS displayed high PD-L1 expression. SK-N-BE(2) displayed modest PD-L1 expression. HSV1716 infection did not significantly affect PD-L1 levels in any of the models.

[0192] FIG. 14. oHSV1716-Induced Chemokine/Cytokine Gene Expression in Pediatric Solid Tumor Cell Lines. Human Ewing sarcoma model A673 and human neuroblastoma models SK-N-AS and SK-N-BE(2) were cultured with HSV1716 at MOI 10 and gene expression analysis was performed at 6 hours and 12 hours post infection by RT-PCR. These data suggest that oHSV infection will increase T-cell migration to the tumor site and increase T-cell activation.

[0193] FIG. 15. oHSV1716-Induced Chemokine/Cytokine Gene Expression in hNBLSK-N-BE2 In Vivo. Athymic nude mice were implanted with SK-N-BE(2) and treated with 1×107PFU HSV1716 or PBS as control intra-tumorally on days 0, 2, and 4. Data represent average of all samples (n=2 tumors per treatment group). These data suggest that oHSV infection will increase T-cell migration to the tumor site, proliferation and activation. These data also suggest that oHSV1716 induces expression of T-cell inhibitory ligands, and that the addition of a PD-1 inhibitor to oHSV1716 may be of benefit. Human data shown in left bar with mouse data in adjacent right bar.

[0194] FIG. 16. Flow scheme for transwell assays.

[0195] FIG. 17. Negative controls.

[0196] FIG. 18. Positive controls.

[0197] FIG. 19. SKNAS hNBL cells Negative Controls: No CARs.

[0198] FIG. 20. SKNAS hNBL cells.

[0199] FIG. 21. SKNBE2 hNBL cells Negative Control: No CARs.

[0200] FIG. 22. SKNBE2 hNBL cells.

[0201] FIG. 23. Transwell summary. SK-N-BE2 induces more GD2 hCAR-T cell migration than SK-N-AS at baseline. oHSV1716 infection induces increased migration of GD2 hCAR-T cells toward hNBLcell lines SK-N-AS and SK-N-BE2.

[0202] FIG. 24. Historical data.

[0203] FIG. 25. Study design: SK-N-AS In Vivo #1 Efficacy of Combination of oHSV1716+GD2 CAR T-cells in Pediatric Solid Tumor Xenograft Model SK-N-AS In Vivo.

[0204] FIG. 26. SK-N-AS+/−oHSV+/−CAR-T:Tumor Growth Curves.

[0205] FIG. 27. SK-N-AS+/−oHSV+/−CAR-T:Tumor Growth Curves. In the right panel, the line extending furthest right is oHSV+CAR T.

[0206] FIG. 28. SK-N-AS+/−oHSV1716+/−CAR-T: Survival Curves. In the right panel, the line extending furthest right is oHSV+CAR T.

[0207] FIG. 29. Study design: A673 In Vivo Lymphodepletion Pilot #1 Efficacy of Combination of oHSV1716+GD2 CAR T-cells with and without Lymphodepletion in Pediatric Solid Tumor Xenograft Model A673 In Vivo.

[0208] FIG. 30. Charts showing tumor volume in A673+CAR-T cells.

[0209] FIG. 31. Summary of methods for Example 2. *pfu=plaque forming unit.

[0210] FIG. 32. In vitro results for example 3: Seprehvir infection induces T-cell attractant chemokines and T-cell activating cytokines in vitro.

[0211] FIG. 33. In vitro results for example 3: CAR T-cells express chemokine receptors (left). Tumor cells variably express GD2 and PD-L1 (right).

[0212] FIG. 34. In vitro results for example 3: Migration of CAR T cells toward SK-N-AS with and without oHSV infection.

[0213] FIG. 35. In vivo results for Example 3: Seprehvir significantly delays tumor growth and enhances anti-tumor efficacy of GD2-directed human 3.sup.rd generation CAR T-cells against the neuroblastoma xenograft model SK-N-AS.

[0214] FIG. 36: In vivo results for Example 3: Seprehvir significantly delays tumor growth and enhances anti-tumor efficacy of human GD2-directed CAR T-cells against human Ewing sarcoma xenograft model A673. Cured mice were re-challenged with tumor cells after 100 days survival.

EXAMPLES

Example 1—Evaluation of Attenuated HSV1716 in Combination with Chimeric Antigen Receptor T Cells for Solid Tumors

[0215] Neuroblastoma, osteosarcoma, and rhabdomyosarcoma are among the most prevalent childhood solid tumors. Each of these tumor types as well as melanomas exhibit increased levels of the tumor associated carbohydrate, GD2 on their cell surface making them ideal targets for chimeric antigen receptor (CAR) T cell-directed therapies. Despite the ability of GD2 CAR T Cells to target GD2-expressing tumor cells in vitro, there is great interest in improving tumor clearance in vivo, especially for solid tumors where current outcomes remain poor. We hypothesize that the immunosuppressive milieu present within the solid tumor microenvironment serves as a major factor limiting the effectiveness of GD2 CAR T cells and propose that administration of oncolytic viruses could induce inflammation within the tumor microenvironment that may enhance, rather than inhibit, the effectiveness of immune based therapies. GD2 CAR T cells composed of the 14G2a Single chain variable fragment linked to the cytoplasmic signalling domains of CD28 and CD3 zeta (GD2-28z) were expressed in murine lymphocytes and evaluated for the ability to target and lyse GD2-expressing tumor cells. Additionally GD2-28z T cells were co-cultured with tumor cells to access their ability to secrete proinflammatory cytokines IFNγ and IL-2. In order to determine the oncolytic ability of attenuated HSV1716, tumor cells were cultured in the presence or absence of HSV1716 and relative cell survival was measured. We observed specific lysis of GD2-expressing tumor cells when co-cultured with GD2-28z, but not mock T cells. Furthermore, GD2-28z T cells secrete IFNγ and IL-2 following co-culture with GD2-expressing tumor cells. Interestingly, melanoma cell lines were not susceptible to oncolytic lysis while rhabdomyosarcoma (RMS) cell lines were susceptible. Using a melanoma model, GD2-28z T cells displayed anti-tumor activity. The combination of GD2-28z and HSV1716 enhanced CAR persistence in a melanoma model. Given that the melanoma cells in our model are not susceptible to oncolytic lysis yet we observe an increased T cell persistence when used in combination with HSV1716, this supports our hypothesis that HSV1716 in inducing inflammation, which is then triggering T cell expansion.

[0216] Results are shown in FIGS. 1A to 7D.

[0217] Our results showed that GD2 CAR T cells target GD2+ tumor cells in vitro and in vivo, and delay tumor growth. HSV1716 oncolytically lyses nectin 1-expressing cells, enhances GD2 CAR persistence in vivo, and delays tumor growth.

Example 2—Oncolytic Virotherapy-Enhanced Chimeric Antigen Receptor T-Cell Therapy in Pediatric Solid Tumors

[0218] While chimeric antigen receptor (CAR) T-cell therapies have shown remarkable anticancer efficacy in patients with relapsed and refractory lymphoid leukemias, their effectiveness in patients with solid tumors has thus far been disappointing. Trials of treatment in solid tumors have shown little clinical success, with modest homing to tumors and lack of CAR persistence. These findings may be attributed to the immunosuppressive microenvironment characteristic of solid tumors. Oncolytic virotherapy is a promising platform which may potentiate the competence of CAR T-cells within solid tumors. Oncolytic viruses specifically amplify in malignant tissues and cause tumor-specific cell death not only through direct cell lysis, but also through the induction of an immunologic response. This mechanism suggests that oncolytic virotherapy may be a useful strategy to reverse the immune-escape tactics of solid tumors and augment the effects of directed T-cell therapies. We sought to determine whether the use of oncolytic Herpes Simplex virotherapy (oHSV) might enhance the efficacy of CAR T-cells in pediatric solid tumors. HSV1716 (trade name Seprehvir, Virttu Biologics, Ltd., Glasgow, U.K.) is a mutant Herpes Simplex-1 virus that lacks the RL1 gene encoding the virulence factor ICP34.5. This deletion nullifies the virus' ability to counteract host cell anti-viral responses and effectively restricts virus replication to cancer cells in which these mechanisms are absent or impaired. Seprehvir's safety has been demonstrated in multiple phase I clinical trials, including an ongoing trial for adolescents and young adults with refractory solid tumors initiated by our laboratory team (NCT00931931). We characterized the chemokine and cytokine profiles of human Ewing sarcoma and neuroblastoma cell lines before and after oHSV inoculation. We performed transwell migration assays of third-generation (containing CD28, OX40, and CD3z signaling domains) GD2-directed human CAR T-cells before and after the addition of Seprehvir in these models in vitro. We then performed in vivo survival studies using athymic nude mice and cyclophosphamide (CPM) lymphodepletion prior to CAR therapy. Our preliminary results suggest that infection of these pediatric solid tumor models with Seprehvir induces an immune response, which includes the T-cell attractant chemokines CXCL-10 (IP-10) and CCL-5 (RANTES) and T-cell activating cytokines such as IFN-γ and TNF-α while down-regulating such inhibitory cytokines as TGF-β (FIG. 8). Flow cytometry analysis revealed variable tumoral GD2 surface expression on each of these models (FIG. 9), while the CAR T-cells displayed high CXCR-3 and CCR-5 surface expression, allowing for chemotactic signaling through CXCL-10 and CCL-5, respectively (data not shown). These CAR T-cells displayed increased migration toward oHSV-infected tumor cells over non-infected cells (FIG. 10). Mice treated with combination therapy had significantly delayed tumor growth (data not shown) and prolonged survival when compared to CAR treatment alone, with 80% versus 0% of mice cured, respectively (FIG. 11). These results indicate that the addition of the oHSV construct Seprehvir is a valuable adjunct to GD2-directed CAR T-cell therapy in GD2-expressing pediatric solid tumors and should be further explored in clinical trials.

[0219] FIGS. 12 to 23 show results for experiments with α-GD2 hCAR-T cells +/−oHSV1716 for GD2-Positive Pediatric Solid Tumor Models In Vitro.

[0220] FIGS. 8 to 11 and 24 to 30 show results for experiments with α-GD2 hCAR-T cells +/−oHSV1716 for GD2-Positive Pediatric Solid Tumor Models In Vivo.

[0221] Experiment 1:

[0222] SK-N-AS In Vivo #1 Efficacy of Combination of oHSV1716+GD2 CAR T-cells in Pediatric Solid Tumor Xenograft Model SK-N-AS In Vivo (FIGS. 24 to 28).

[0223] SK-N-AS+/−oHSV+/−CAR-T study conclusions: [0224] There is no significant difference between oHSV+PBS and oHSV+Mock-T survival curves [0225] There is a significant survival advantage for oHSV+CAR-T compared to oHSV+PBS arm [0226] There is a very significant survival advantage for oHSV+CAR-T compared to PBS+CAR-T arm [0227] Lack of efficacy of PBS+CAR-T arm may be in part due to low GD2 expression of SK-N-AS [0228] Lack of significant efficacy of T-cell arms overall may be in part due to inherent mouse NK cells getting rid of T-cells

[0229] Experiment 2:

[0230] A673 In Vivo Lymphodepletion Pilot #1 Efficacy of Combination of oHSV1716+GD2 CAR T-cells with and without Lymphodepletion in Pediatric Solid Tumor Xenograft Model A673 In Vivo (FIGS. 8 to 11 and 29 to 30).

[0231] A673+/−Lymphodepletion+/−oHSV1716+GD2 CAR T-cells study conclusions: [0232] Lymphodepletion with CPPM in the absence of oHSV results in improvement of survival compared to no lymphodepletion or NK depletion with Asialo [0233] Lymphodepletion did not seem to have a significant effect on tumor growth or mouse survival when combined with oHSV [0234] All oHSV arms have superior survival benefit compared to PBS arms

Example 3—Oncolytic Virotherapy Enhances GD2-Directed Chimeric Antigen Receptor (CAR) T-Cell Therapy in GD2-Expressing Pediatric Solid Tumor Xenograft Models

[0235] High Risk Neuroblastoma (NBL) is the most common non-CNS pediatric solid tumor, requires multimodal and targeted therapy, is responsible for ˜15% total childhood cancer deaths and has <10% survival for ˜50% of children who relapse

[0236] Ewing Sarcoma (EWS) is among most prevalent solid tumor afflicting older children and adolescents, ˜30% are refractory to conventional therapy, there is ˜30% survival for patients with metastases.

[0237] GD2 is a disialoganglioside expressed on NBL and EWS, and is a strategic immunotherapeutic target.

[0238] Chimeric Antigen Receptor (CAR) T-Cells are engineered T-cells targeted against tumor antigen, have remarkable efficacy in relapsed/refractory lymphoid leukemias. CAR T cells have so far shown little clinical success against solid tumors, modest migration to tumor, lack of activation, proliferation, and persistence. These limitations are attributable to the solid tumor immunosuppressive microenvironment.

[0239] Oncolytic Herpes Simplex Virotherapy (oHSV) is tumor selective, subject of recent FDA approval with several open clinical trials. It combines two antitumor efficacy mechanisms: a direct lytic effect and induction of immune response.

[0240] While chimeric antigen receptor (CAR) T-cell therapies have shown remarkable anticancer efficacy in patients with relapsed and refractory lymphoid leukemias, their effectiveness in patients with solid tumors has been more challenging. Among the barriers thought to interfere with CAR T cell efficacy are impaired homing to tumors and poor CAR T cell persistence, likely attributable to the immunosuppressive microenvironment. Due to their pro-inflammatory effects, oncolytic viruses are strong candidates to potentiate the competence of CAR T cells within solid tumors. Seprehvir (HSV1716) is an HSV-1 attenuated by deletion of the RL1 gene encoding the neurovirulence protein ICP34.5. The virus has a long track record of safety in clinical trials and is currently being tested in adolescents and young adults with refractory solid tumors (NCT00931931, NCT02031965). We hypothesized that intra-tumoral administration of Seprehvir enhances GD2-directed CAR T cell efficacy. We characterized the chemokine and cytokine profiles of human GD2-positive Ewing sarcoma and neuroblastoma cell lines before and after oHSV inoculation. We performed transwell migration assays of third-generation (containing CD28, OX40, and CD3z signaling domains) GD2-directed human CAR T-cells before and after the addition of Seprehvir in these models in vitro. We then performed in vivo survival studies using athymic nude mice and cyclophosphamide (CPM) lymphodepletion prior to CAR therapy. Our results suggest that infection of these pediatric solid tumor models with Seprehvir induces an immune response, which includes the T-cell attractant chemokines CXCL-10 (IP-10) and CCL-5 (RANTES) and T-cell activating cytokines such as IFN-g and TNF-α, while down-regulating such inhibitory cytokines as TGF-b. Flow cytometry analysis revealed variable tumoral GD2 surface expression on each of these models, while the CAR T-cells displayed high CXCR-3 and CCR-5 surface expression, allowing for chemotactic signaling through CXCL-10 and CCL-5, respectively. The CAR T-cells displayed increased migration toward oHSV-infected tumor cells over non-infected cells. Mice treated with combination therapy had significantly delayed tumor growth and prolonged survival when compared to CAR treatment alone. Despite being athymic nude mice, the majority of mice cured by combination therapy were resistant to tumor re-challenge, suggesting the long-term persistence of CAR T cells. These results indicate that the addition of Seprehvir may be a valuable adjunct to CAR T-cell therapy and should be further explored in clinical trials.

[0241] In vitro, we: [0242] Characterized oHSV-induced chemokine/cytokine gene expression by RT-PCR [0243] Tumor cells cultured with Seprehvir at multiplicity of infection (MOI)=10×12 hours [0244] Determined tumoral GD2 expression by flow cytometry [0245] Determined CAR T-cell CXCR-3 and CCR-5 expression by flow cytometry [0246] Performed transwell migration assays: [0247] Tumor cells cultured with Seprehvir at MOI=1×24 hours [0248] Red fluorescent PKH23-stained CAR T-cells added to 5 mm pore inserts×2 hours [0249] Negative control: media alone [0250] Positive controls: media with 75 ng/ml CXCL-10 (IP-10) or 10 ng/ml CCL-5 (RANTES) [0251] Cells quantified through microscopic visualization [0252] Results represent averages of n replicates for each sample

[0253] In vivo: [0254] Athymic nude mice with subcutaneous flank tumors [0255] PBS or Seprehvir was administered intra-tumorally (i.t)×3 (FIG. 31) [0256] Intra-peritoneal (i.p.) PBS or cyclophosphamide (CPM)×1 prior to CAR treatment [0257] Intravenous (i.v.) PBS or CAR T-cells×1

[0258] Results are shown in FIGS. 31 to 36.

[0259] Our results showed that oHSV infection induces release of chemokines and cytokines that promote CAR T-cell migration and activation; oHSV enhances GD2-directed human CAR T-cell antitumor efficacy against GD2-expressing pediatric solid tumors. oHSV is a promising adjunct to CAR T-cell therapy for pediatric solid tumors.

REFERENCES

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