Intratumorally delivered formulation, INT230-6, containing potent anticancer agents induces protective T cell immunity and memory

Abstract

The benefits of anti-cancer agents extend beyond direct tumor killing. One aspect of cell death is the potential to release antigens that initiate adaptive immune responses. Here, a diffusion enhanced formulation, INT230-6, containing potent anti-cancer cytotoxic agents, was administered intratumorally into large (approx. 300mm3) subcutaneous murine Colon26 tumors. Treatment resulted in regression from baseline in 100% of the tumors and complete response in up to 90%. CD8+ or CD8+/CD4+ T cell double-depletion at treatment onset prevented complete responses, indicating a critical role of T cells in promoting complete tumor regression. Mice with complete response were protected from subcutaneous and intravenous re-challenge of Colon26 cells in a CD4+/CD8+ dependent manner. Thus, immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the CD8+ T-cell AH-1 epitope. AH-1-specific CD8+ T cells were detected in peripheral blood of tumor-bearing mice and their frequency increased 14 days after treatment onset. AH-1-specific CD8+ T cells were also significantly enriched in tumors of untreated mice. These cells had an activated phenotype and highly expressed Programmed cell-death protein-1 (PD-1) but did not lead to tumor regression. CD8+ T cell tumor infiltrate also increased 11 days after treatment. INT230-6 synergized with checkpoint blockade, inducing a complete remission of the primary tumors and shrinking of untreated contralateral tumors, which demonstrates not only a local but also systemic immunological effect of the combined therapy. Similar T-cell dependent inhibition of tumor growth was also found in an orthotopic 4T1 breast cancer model.

Keywords: Endogenous vaccine; PD-1; T cells; anticancer agent; chemotherapy; combination therapy; immunogenic cell death; intratumoral delivery; tumor model.

© 2019 Taylor & Francis Group, LLC.

Figures

Figure 1.

INT230-6 efficacy is dependent on CD8+ T cell. Naïve mice were SC challenged with 1 × 106 C26 cells into the right flank. Vehicle or INT230-6 (0.5 mg/ml cisplatin, 0.1 mg/ml vinblastine, 10 mg/ml IT-006 cell penetration enhancer) were intratumorally (IT) administered into 300 mm3 (approx. 8.5 mm in diameter, 100 µl/400 mm3 volume C26 tumor) SC tumors (n = 10/group) for 5 sequential days (day 0 to 4) and tumor growth was monitored. a) Kaplan-Meier plot (left graph) and individual responses (right graphs) are displayed of a representative experiment of at least five repeats with similar or better results; vehicle (black) and INT230-6 (red) treatment are shown. The fraction 5/10 indicates the number of complete responders. The Log-rank test indicated a significant difference between the groups (p < .0001). b) Kaplan-Meier plot (top) and individual responses (below) of a representative experiment (of 3 with similar results) are shown illustrating vehicle (black) and INT230-6 with either IgG control antibody (300 µg, blue), anti-CD4 (100 µg, orange), anti-CD8 (200 µg, purple) or anti-CD4 and anti-CD8 (red) depletion antibodies. Antibodies were administered intraperitoneally (IP) on day 0, 1, 5, 8 and 15 after the initial INT230-6 dose (n = 10/group). Fractions (e.g. 8/10) indicate the number of complete responders. By log-rank test, differences were significant (p < .0001 unless stated otherwise) between vehicle and all other groups as well as INT230-6 + IgG control vs INT230-6 + anti-CD8 (p < .0006) or INT230-6 + anti-CD8/4, INT230-6 + anti-CD4 vs INT230-6 + anti-CD8 or INT230-6 + anti-CD8/4. c) Vehicle or INT230-6 (50 μl/400 mm3 C26 tumor) were administrated for 3 sequential days (day 0 to 2) and tumor growth was monitored. Kaplan-Meier plots (top) and individual responses (below) of WT BALB/c and RAG1−/- mice are shown. This experiment was performed twice with similar results. The fraction (3/10) indicates the number of complete responders. Log-rank test was significantly different between WT BALB/c + INT230-6 vs all other groups (p < .0001), WT BALB/c + vehicle vs RAG1−/- + vehicle (p < .05), WT BALB/c + vehicle vs RAG1−/- + INT230-6 (p < .01) as well as RAG1−/- + vehicle vs RAG1−/- + INT230-6 (p < .0001). d) INT230-6 (100 μl/400 mm3 C26 tumor) was administrated IT for 5 sequential days (n = 10). Untreated tumor-bearing mice as well as naïve mice (n = 5) were bled before treatment (day 0). Treated mice and naïve mice were bled on day 7, 14, 21 and 28 after the initial dose of drug. Peripheral blood was stained for tumor antigen-specific (AH-1/H-2Ld-tetramer-positive) CD8+ T cells and analyzed by flow cytometry. A representative experiment (of 2 with similar results) is shown. Percentage of AH-1/H-2Ld-tetramer-reactive CD8+ T cells amongst CD8+ T cells are shown as individual values with mean. Percentage of AH-1/H-2Ld-tetramer-reactive CD8+ T cells was significantly increased (p < .05) by Kruskal–Wallis post hoc Dunn’s test on day 14 after treatment onset. Representative flow cytometry plots of AH-1/H-2Ld-tetramer reactive populations on day 14 are shown (right panels).

Figure 2.

Mice with complete response (CR) were protected from tumor re-establishment in a T cell-dependent manner. a) Mice with complete response (CR) and naïve () mice were challenged with 1 × 106 C26 tumor cells by SC injection into the right flank (n = 10/group). Mice with CR underwent prior depletion of CD4 (), CD8 (), CD4/8 () or control IgG injections (), (150 µg anti-CD8/4, 300 µg IgG, day −1, 3, 5, 12 and 19) by IP administration. Kaplan-Meier plot is shown of a representative experiment (of 2 with similar results) and a Log-rank test indicated significant differences (p < .0001: naïve vs CR+IgG, naïve vs CR+aCD4, naïve vs CR+aCD8, CR+IgG vs CR+aCD4/8, CR+CD8 vs CR+CD4/8; p < .001: CR+CD4 vs CR+CD4/8; p < .01: naïve vs CR+aCD4, CR+IgG vs CR+aCD4). b) C26 tumor cells (1x106) were inoculated IV into mice with CRs (solid line) and naïve mice (dashed line, n = 10/group) to produce lung metastases. Kaplan-Meier plot is shown of a representative experiment (of 3 with similar results) and a log-rank test showed a significant difference (p < .0001) between groups. c) C26 tumor cells (1x106) were inoculated SC into the right flank and 2.5 × 105 C26 tumor cells were inoculated IV 7 days later. An additional 10 mice received IV tumor cells only (dotted line). Vehicle (dashed line) or INT230-6 (solid line) were administered IT into 275mm3 (50µl/400mm3 C26 tumor) SC tumors (n = 10/group) for 5 sequential days (day 0 to 4) starting on day 14 and tumor growth was monitored. Kaplan-Meier plot of a representative experiment (of 2 with similar results) is shown (top left), and a log-rank test indicated significant differences (p < .01: C26 iv only vs vehicle; p < .0001: C26 iv only vs INT230-6, vehicle vs INT230-6). Quantification of lung nodules at the time of death (n = 9–10/group) is presented (bottom left). Median test was applied for vehicle vs INT230-6 treatment groups. Representative lungs that had been injected with 15% black India ink are shown on the right. Healthy lung tissue is black and smooth whereas tumor nodules appear white and nodular.

Figure 3.

PD-1hi, CD44hiCD69+ AH-1-specific CD8+ T cells were induced by growth of untreated C26 tumors. a) Mice with bulky (1000mm3) C26 tumors (untreated) as well as naïve controls (n = 4–6/group) were bled and euthanized to harvest and prepare single cell suspensions of tumors, spleen and draining lymph nodes (LN). Lymphocytes were stained for AH-1/Ld-tetramer reactive populations (top panel), activation markers (CD44hiCD69+CD62−, middle panel) and PD-1 expression (lower panel) on CD8+ T cells and analyzed by flow cytometry. Individual responses and mean values are shown of pooled data from two experiments. Kruskal–Wallis with Tukey post hoc test was performed and indicated that AH-1 specific CD8+ T cells are significantly enriched (p < 0.01) in the tumor compared to the periphery (blood, spleen or lymph nodes) of tumor-bearing or naive mice. Total tumor CD8+ T cells and AH-1 specific CD8+ T cells were significantly more activated (p < 0.0001) and expressed higher levels of PD-1 (p < 0.0001) than in the periphery of tumor-bearing or naïve mice. b) Splenocytes, harvested from mice with CR, were stimulated for one week with high (1 μM, left graph) or low (1 nM, right graph) AH-1 peptide concentration with syngeneic BALB/c splenocytes as antigen-presenting cells. Lymphocytes were stained for AH-1/Ld-tetramer reactive CD8+ T populations. c) Lactate dehydrogenase (LDH) cytotoxicity assay was performed with expanded splenocytes. For this, C26 cells were applied as AH-1 expressing target (T) cells (solid line). A20 cells were utilized as an AH-1 negative target cell line (dashed line). These cells were incubated with different ratios of CD8+ effector (E) T cells for 4 h and LDH assay was performed to calculate cytotoxicity. Mean ± standard deviation is shown of three pooled experiments. Two-way ANOVA with Sidak’s multiple comparison test was performed and showed significant difference (**p < 0.01) at the 25:1 ratio. Representative flow cytometry plots (bottom) illustrate the expansion of AH-1 specific CD8+ T cells.

Figure 4.

CD8+ T cells infiltrate increases 11 days after treatment onset. Top panel shows representative images of histology of C26 tumors before treatment (day 0) and after treatment (day 8, 11–12 and 14). H&E staining is depicted in the top row, CD8+ T cells are presented in the middle and bottom row at different magnifications (see scale bar). The bottom graph is a quantification of CD8+ T cells per mm2 of tumor tissue (n = 6 per time point). Each point represents a different mouse tumor, as mice has to be euthanized to harvest tumors at each time point. The 12 specimens at days 11 + 14 differ from those at the earlier time points (p = 0.04) by a Kruskal–Wallis test.

Figure 5.

Contralateral tumor response can be induced by INT230-6 in combination with anti-CTLA-4 but not with anti-PD-1. a) Illustration of INT230-6 and anti-PD-1 treatment regimen (top). C26 (1x106) were inoculated into the right flank (day −14). Contralateral tumors were inoculated 11 days after primary tumors (day −3). Primary tumors were treated with INT230-6 (50 μl/400 mm3 tumor, 5 sequential days) starting on day 0. Average primary tumor volume on day 0 was 290mm3 and average contralateral tumor volume was 42mm3. Anti-PD-1 treatment (100 μg) was given on day 0, 3, 7 and 10. b) Kaplan-Meier plot (below illustration) and individual responses of c) treated (ipsilateral) (middle) and d) contralateral flank (bottom) are shown of vehicle (black), anti-PD-1 (purple), INT230-6 (red) and anti-PD-1+ INT230-6 (blue) treatment as well as contralateral tumor only control (orange, n = 10/group). Fractions (e.g. 3/10) indicate the number of mice that completely lost tumor that the indicated flank. Log-rank test was significantly different between vehicle and INT230-6 (p < 0.0001), vehicle and anti-PD-1+ INT230-6 (p < 0.0001), anti-PD-1 and INT230-6 (p < 0.01) and anti-PD-1 and anti-PD-1 + INT230-6 (p < 0.0001). Two-way ANOVA with Sidak’s multiple comparison test of growth curves showed that untreated contralateral tumors only were significantly different (p < 0.0001) from all groups on secondary site. Furthermore, vehicle was significantly different (p < 0.0001) from INT230-6 and INT230-6+ anti-PD-1 on day 8 and 11 on the primary site. Vehicle was significantly different (p < 0.05) from INT230-6+ anti-PD-1 on day 11 on the secondary site. INT230-6 was significantly different anti-PD-1 or INT230-6+ anti-PD-1 on day 16 (p < 0.01) and day 18 (p < 0.001) on the primary tumor site. INT230-6 was significantly different (p < 0.01) from INT230-6 + anti-PD-1 on day 18 on the contralateral site. Anti-PD-1 was significantly different (p < 0.0001) from INT230-6+ anti-PD-1 on day 8 and 11 at the primary site only. e) Illustration of INT230-6 and anti-CTLA-4 treatment regimen (top). C26 (1x106) tumor cells were inoculated into the right flank (day −15). Contralateral tumors were inoculated 7 days after primary tumors (day −8). Primary tumors were treated with INT230-6 (50 μl/400 mm3 tumor, 5 sequential days) starting on day 0. Average primary tumor volume on day 0 was 250mm3 and average contralateral tumor volume was 60mm3. Anti-CTLA-4 treatment (100 μg) was given on day 0, 3 and 6. f) Kaplan-Meier plot (below illustration) and individual responses of g) treated (ipsilateral) (middle) and h) contralateral flank (bottom) are shown of vehicle (black), anti-CTLA-4 (purple), INT230-6 (red) and anti-CTLA-4+ INT230-6 (blue) treatment as well as contralateral tumor only control (orange, n = 10/group). Fractions (e.g. 4/10) indicate the number of mice that completely lost tumor in the indicated flank. Log-rank test was significantly different between vehicle and all other groups (p < 0.0001), INT230-6 and anti-CTLA-4+ INT230-6 (p < 0.0001) and anti-CTLA-4 and anti-CTLA-4+ INT230-6 (p < 0.0001). All experiments were performed twice. Two-way ANOVA with Sidak’s multiple comparison test of growth curves showed that untreated contra-later tumors were significantly different (p < 0.0001) from all other groups on the contralateral site. Vehicle was significantly different (p < 0.0001) from INT230-6, anti-CTLA-4 and anti-CTLA-4+ INT230-6 on day 6 and 8 at the primary (ipsilateral) site. Vehicle was significantly different from anti-CTLA-4 (day 8: p < 0.0001) and anti-CTLA-4+ INT230-6 (day 6: p < 0.001, day 8: p < 0.0001) at the contralateral site. INT230-6 was significantly different from anti-CTLA-4 at the primary site (day 8: p < 0.05, day 10: 0.001) and secondary site (day 8: p < 0.01, day 9: p < 0.001, day 10: p < 0.0001). INT230-6 was significantly different from anti-CTLA-4 + INT230-6 at the contralateral site only (day 6: p < 0.05, day 8–10: p < 0.0001). Anti-CTLA-4 was significantly different from anti-CTLA-4 + INT230-6 at the primary site (day 8: p < 0.01, day 9: p < 0.05, day 10: p < 0.0001) and at the secondary site (day 6: p < 0.05).

Figure 6.

Intratumoral administration of INT230-6 delays tumor growth rate and improves overall survival, in a T cell-dependent manner, in 4T1 orthotopic breast carcinoma tumor models: a & b) 4T1 tumor growth curve of individual mice when treated with either INT230-6 (a) or vehicle (b). The INT230-6 treated 4T1 mammary tumors grew at a significantly slower rate than the vehicle-treated tumors (n = 15, **p < 0.003). c) Kaplan-Meier survival curve shows a significant difference between the vehicle-treated 4T1 mammary tumor-bearing mice as compared to INT230-6 treated ones (n = 15, *p < .03). d & e) Vehicle treated 4T1 tumor grow at a similar rate with (d) or without (e) CD4/CD8 depletion. f) Kaplan-Meier survival curve depicting the differences in survival probabilities of mice carrying 4T1 mammary tumor upon treatment with either INT230-6 or vehicle, with or without depletion of CD4 and CD8 cells. g-h) The CD4/CD8 depleted INT230-6 group (g) shows a significantly faster tumor growth rate compared to INT230-6 & IgG treated (h) ones(***p < 0.0003). There was a significant difference in survival probability of INT230-6 + IgG (red curve) treated mice (n = 22) compared to INT230-6 + anti-CD4/CD8 treated mice (purple curve) (n = 22, **p < 0.008). No significant difference, either in survival or tumor growth rate, was observed in vehicle-treated groups, with (e) or without (f) CD4/CD8 depletion. There was a significant difference in overall survival of vehicle versus INT230-6 treated tumor-bearing mice when both were T-cell depleted (panel f, blue vs purple curve **p < 0.001).