Neoadjuvant Intratumoral Immunotherapy with TLR9 Activation and Anti-OX40 Antibody Eradicates Metastatic Cancer

Abstract

The combination of the synthetic TLR9 ligand CpG and agnostic OX40 antibody can trigger systemic antitumor immune responses upon co-injection into the tumor microenvironment, eradicating simultaneous untreated sites of metastatic disease. Here we explore the application of this in situ immunotherapy to the neoadjuvant setting. Current neoadjuvant checkpoint blockade therapy is delivered systemically, resulting in off-target adverse effects. In contrast, intratumoral immunotherapy minimizes the potential for toxicities and allows for greater development of combination therapies. In two metastatic solid tumor models, neoadjuvant intratumoral immunotherapy generated a local T-cell antitumor response that then acted systemically to attack cancer throughout the body. In addition, the importance of timing between neoadjuvant immunotherapy and surgical resection was established, as well as the increased therapeutic power of adding systemic anti-PD1 antibody. The combination of local and systemic immunotherapy generated an additional survival benefit due to synergistic inhibitory effect on tumor-associated macrophages. These results provide a strong rationale for translating this neoadjuvant intratumoral immunotherapy to the clinical setting, especially in conjunction with established checkpoint inhibitors.

Significance: This work demonstrates the ability of neoadjuvant intratumoral immunotherapy to target local and distant metastatic disease and consequently improve survival.

Trial registration: ClinicalTrials.gov NCT03410901 NCT03831295 NCT01347034 NCT00499083 NCT00365872 NCT04025879.

Conflict of interest statement

Conflict of Interest: Dr. Ronald Levy serves on the advisory boards for Five Prime, Corvus, Quadriga, BeiGene, GigaGen, Teneobio, Nurix, Dragonfly, Abpro, Apexigen, Spotlight, 47 Inc, XCella, Immunocore, Walking Fish.

The other authors have declared that no conflict of interest exists.

©2022 American Association for Cancer Research.

Figures

Figure 1.

A-F, H Groups of BALB/c WT mice (n = 10) were inoculated with 5x105 CT26-Luc colorectal carcinoma tumor cells into tail vein followed a day later with inoculation of 5x105 CT26-Luc tumor cells subcutaneously into the right side of the abdomen to simulate metastatic and local disease respectively. Treatment started once local (subcutaneous) tumor reached 0.7cm in diameter. A. Schematic illustrating general experimental timeline. B-D. Groups of mice were treated with intratumoral administration of neoadjuvant immunotherapy (3X injections, 50ug CpG and 8ug aOX40 antibody per injection vs CpG alone vs aOX40 alone vs vehicle (PBS)) followed 4 days later by resection. B. Individual primary tumor growth curves for each treatment group. C. Average local tumor growth in different treatment groups. Data presented as mean tumor size +/− SEM. D. Kaplan-Meier curves for overall survival of each group shown. P values were calculated using the log-rank test (Mantel-Cox). p

Figure 2.

Efficacy of neoadjuvant immunotherapy dependent on CD4+ and CD8+ antitumor T cell activity. Groups of BALB/c WT mice (n = 10) were injected with either depleting anti-CD4, anti-CD8, anti-CD4 and anti-CD8 or no depleting antibodies one day prior to subcutaneous tumor inoculation and again on days 7 and 17 after tumor inoculation. Mice were inoculated with CT26-Luc tumors as described in Figure 1. All groups of mice were treated with intratumoral administration of neoadjuvant immunotherapy (3X injections, 50ug CpG and 8ug aOX40 antibody per injection). Neoadjuvant treatment started once local (subcutaneous) tumor reached 0.7cm in diameter. All groups underwent resection of the primary subcutaneous tumor four days after the last neoadjuvant intratumoral injection. A. Schematic illustrating general experimental timeline. B. Local tumor growth and recurrence in different groups. Resection occurred on day 20 following subcutaneous tumor inoculation. Also shown is metastatic dissemination following T cell depletion via BLI, images representing 5/10 mice in each group. C. Survival was monitored and investigated by Kaplan–Meier analysis. P values were calculated using the log-rank test (Mantel-Cox). p

Figure 3.

Neoadjuvant immunotherapy decreases local recurrence and improves survival in aggressively metastatic 4T1-Luc tumor model. A-C, Groups of BALB/c WT mice (n = 10) were inoculated with 7.5x104 mammary carcinoma 4T1-Luc tumor cells orthotopically into the right mammary fat pad. Groups of mice were treated with local administration of neoadjuvant immunotherapy (3X injections, 50ug CpG and 8ug aOX40 antibody per injection), immunotherapy alone (3X injections of 50ug of CpG and 8ug of aOX40 antibody per injection), or neoadjuvant vehicle. Treatment started once local (subcutaneous) tumor reached 0.7cm in diameter. Groups that underwent resection did so on day 15 (blue arrow) following subcutaneous tumor inoculation. A. Schematic illustrating experimental setup. B. Average local tumor growth in different treatment groups. Date of resection indicated by blue arrow. Data presented as mean tumor size +/− SEM. Differences in primary tumor growth between neoadjuvant vehicle followed by resection, CpG/aOX40 without resection and neoadjuvant CpG/aOX40 followed by resection were significant by unpaired t test. p = 0.008 (Neoadjuvant CpG/aOX40 + Resection vs CpG/aOX40 Alone). p = 0.04 (Neoadjuvant CpG/aOX40 + Resection vs Neoadjuvant Vehicle + Resection). C. Kaplan-Meier curves for overall survival of each group shown. P values were calculated using the log-rank test (Mantel-Cox). p = 0.006 (Neoadjuvant CpG/aOX40 + Resection vs CpG/aOX40 Alone). p = 0.002 (Neoadjuvant CpG/aOX40 + Resection vs Neoadjuvant Vehicle + Resection). D-E. In a separate experiment, groups of BALB/c WT mice were inoculated with tumors as described above in A (n = 10). Mice from each group were euthanized at specific time points (2 days post resection, 1 week post resection, 2 weeks post resection and 3 weeks post resection) and their lungs and liver imaged ex vivo to evaluate systemic disease burden (n = 2 at each time point). The differences between the groups were statistically significant, p = 0.09 between neoadjuvant CpG/aOX40 vs Neoadjuvant Vehicle + Resection by paired t test.

Figure 4.

Tumor specific immunity in long term survivors of neoadjuvant intratumoral CpG and aOX40. A. Schematic illustrating experimental setup. B. Average local tumor growth in different treatment groups. Date of resection indicated by blue arrow. Data presented as mean tumor size +/− SEM. Differences in primary tumor growth between neoadjuvant vehicle followed by resection, CpG/aOX40 without resection and neoadjuvant CpG/aOX40 followed by resection were significant by unpaired t test. p = 0.004 (No Depletion vs CD4 Depletion). C. Metastatic dissemination following T cell depletion via BLI, images representing 5/10 mice in each group. D. Kaplan-Meier curves for overall survival of each group shown. P values were calculated using the log-rank test (Mantel-Cox). p = 0.02 (No Depletion vs CD4 Depletion). p

Figure 5.

Locally activated antitumor T cells circulate systemically to eradicate systemic disease A-C. Mice were implanted with either CT26 or 4T1 tumor cells as previously described in Figure 1 and 3 respectively and treated with either vehicle or intratumoral CpG and aOX40. Splenocytes from the indicated groups harvested on day 4 after treatment and cocultured with either media, CD3 and CD28 antibodies, A20 cells (unrelated control tumor), or homologous tumor cells for 24 hours. A. Schematic illustration of experiments. B. For 4T1, analyzed population was enriched for CD3+ cells. Intracellular IFN-γ was measured in CD8+ T cells by flow cytometry as a percentage of CD44hi (memory CD8) T cells shown in dot plots and bar graph n=3 mice/group, ns=not significant **p = 0.0018 (4T1) *p=0.044 (CT26), unpaired t test. C. Intratumoral injection of aOX40+ CpG induces production of Granzyme B and proliferation of CD8+ T cells in lungs and draining lymph nodes of treated mice by day 4 post treatment. Single cell suspensions of draining lymph nodes (dLN) and lungs were stained for Ki67 and Granzyme B, highlighted are percentages of the double positive cells. n=3 mice/group, ***p = 0.000557, *p = 0.0137 (4T1) *p=0.0489, ns=not significant (CT26), unpaired t test.

Figure 6.

Duration between neoadjuvant immunotherapy and removal of primary tumor impacts long-term survival. A. Groups of BALB/c WT mice (n = 10 per group) were inoculated with 7.5x104 mammary carcinoma 4T1-Luc tumor cells orthotopically into the right mammary fat pad. As indicated on the schematic, one group of mice was treated intratumorally with 3X CpG (50ug/per injection) and aOX40 (8ug/injection) on days 7, 9, and 11, followed by resection of the primary tumor 4 days later on day 15. One group received 1X intratumoral injection of CpG (150ug) and aOX40 (24ug) on day 7 followed immediately by resection of the primary tumor. On group received 1X intratumoral injection of CpG (150ug) and aOX40 (24ug) on day 7 followed by resection of the primary tumor 4 days later on day 11. B. Growth curves of primary tumors in mice from each of the treatment groups. C. Metastatic dissemination following T cell depletion via BLI, images representing 5/10 mice in each group. D. Kaplan–Meier overall survival curve. P values were calculated using the log-rank test (Mantel-Cox). p = 0.03 (1X CpG/aOX40 (150ug/24ug) before resection vs 1X CpG/aOX40 (150ug/24ug) at resection).

Figure 7.

Combination of systemic and local neoadjuvant immunotherapy strengthens antitumor response. A. Intratumoral injection of CpG+aOX40 upregulates PD-1 on macrophages, T cells and CD11b cDCs. Groups of BALB/C WT mice (n = 3) were orthotopically inoculated with 4T1-Luc tumors as described in Figure 3. Mice were randomized into the following treatment groups: no treatment, and CpG/aOX40, (3X injections, 50ug CpG and 8ug aOX40 antibody per injection). Lungs were harvested 4 days after therapy and analyzed by flow cytometry. Histogram plots of the indicated populations for vehicle control (grey) or CpG/aOX40 (blue) shown here with data presented as a bar graph of n=3 mice, unpaired t test * p=0.04 macrophages, ** p=0.032 T cells, ** p=0.0021 CD11b cDCs. B. Addition of aPD-1 to the treatment combination resulted in increased T cell activation. Neoadjuvant treatment of aPD-1 (3X intraperitoneal injects, 10mg/kg per injection) was added to the CpG/aOX40 treatment and draining lymph nodes were analyzed by flow cytometry. Dot plots demonstrate Granzyme B and Ki67 of the CD8+ population, bar graph summarizing n=3 mice/group, unpaired t test. C. Suppression assays, MDSCs (top) and T regulatory cells (bottom) were incubated with VTD labeled T cells in 1:1 ratio for 72h. Dot plots describing VTD dilution of CD4 or CD8+ populations, graph bars summarizing n=3, unpaired t test. D-F. Groups of BALB/C WT mice (n = 10) were orthotopically inoculated with 4T1-Luc tumors. Mice were randomized into the following treatment groups: no treatment, neoadjuvant treatment of aPD-1 (3X intraperitoneal injects, 10mg/kg per injection), neoadjuvant CpG/aOX40 (3X injections, 50ug CpG and 8ug aOX40 antibody per injection) and neoadjuvant CpG/aOX40/aPD-1. D. Average local tumor growth and recurrence in different groups. Resection occurred on day 15 (blue arrow) following subcutaneous tumor inoculation. Data presented as mean tumor size +/− SEM. E. Average systemic bioluminescent signal in different groups. Data presented as mean whole body BLI +/− SEM. Systemic disease as denoted by systemic BLI was significantly different between No Treatment and triple therapy groups (p = 0.0001) at 2 days post-resection, and at 2 weeks post resection (p = 0.02) by unpaired t test. Systemic disease was also significantly lower in triple therapy group compared to aPD-1 alone (p = 0.01). F. Kaplan-Meier curves for overall survival of each group shown. P values were calculated using the log-rank test (Mantel-Cox). p = 0.02 between triple therapy vs CpG/OX40. p