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. 2019 Nov 20;7(1):311.
doi: 10.1186/s40425-019-0786-7.

Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism

Affiliations

Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism

Jaina M Patel et al. J Immunother Cancer. .

Abstract

Background: TNF receptor family agonists and checkpoint blockade combination therapies lead to minimal tumor clearance of poorly immunogenic tumors. Therefore, a need to enhance the efficacy of this combination therapy arises. Antigen-presenting cells (APCs) present antigen to T cells and steer the immune response through chemokine and cytokine secretion. DRibbles (DR) are tumor-derived autophagosomes containing tumor antigens and innate inflammatory adjuvants.

Methods: Using preclinical murine lung and pancreatic cancer models, we assessed the triple combination therapy of GITR agonist and PD-1 blocking antibodies with peritumoral injections of DRibbles-pulsed-bone marrow cells (BMCs), which consisted mainly of APCs, or CD103+ cross-presenting dendritic cells (DCs). Immune responses were assessed by flow cytometry. FTY720 was used to prevent T-cell egress from lymph nodes to assess lymph node involvement, and MHC-mismatched-BMCs were used to assess the necessity of antigen presentation by the peritumorally-injected DR-APCs.

Results: Tritherapy increased survival and cures in tumor-bearing mice compared to combined antibody therapy or peritumoral DR-BMCs alone. Peritumorally-injected BMCs remained within the tumor for at least 14 days and tritherapy efficacy was dependent on both CD4+ and CD8+ T cells. Although the overall percent of tumor-infiltrating T cells remained similar, tritherapy increased the ratio of effector CD4+ T cells-to-regulatory T cells, CD4+ T-cell cytokine production and proliferation, and CD8+ T-cell cytolytic activity in the tumor. Despite tritherapy-induced T-cell activation and cytolytic activity in lymph nodes, this T-cell activation was not required for tumor regression and enhanced survival. Replacement of DR-BMCs with DR-pulsed-DCs in the tritherapy led to similar antitumor effects, whereas replacement with DRibbles was less effective but delayed tumor growth. Interestingly, peritumoral administration of DR-pulsed MHC-mismatched-APCs in the tritherapy led to similar antitumor effects as MHC-matched-APCs, indicating that the observed enhanced antitumor effect was mediated independently of antigen presentation by the administered APCs.

Conclusions: Overall, these results demonstrate that peritumoral DR-pulsed-BMC/DC administration synergizes with GITR agonist and PD-1 blockade to locally modulate and sustain tumor effector T-cell responses independently of T cell priming and perhaps through innate inflammatory modulations mediated by the DRibbles adjuvant. We offer a unique approach to modify the tumor microenvironment to benefit T-cell-targeted immunotherapies.

Keywords: Antigen presenting cells; Dendritic cells; GITR; PD-1; Peritumoral injection; Tumor microenvironment.

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Conflict of interest statement

HMH is a cofounder of UbiVac, a biotech company that has licensed the DRibbles intellectual property. The other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Peritumoral BMC vaccination enhances the survival of GITR agonist and PD-1 blockade treated tumor-bearing mice. a, Experimental schematic. b, Line-1-tumor bearing mice individual tumor growth curves and overall survival. Pooled data from 5 independent experiments are shown. c, Panc02-tumor bearing mice individual tumor growth curves and overall survival. Pooled data from 2 independent experiments are shown. d, Line-1 tumor bearing mice individual tumor growth curves and overall survival. Representative data from 2 independent experiments are shown (n = 5)
Fig. 2
Fig. 2
Peritumorally injected BMCs remain in the tumor of tritherapy-treated mice. a, Treated mice were euthanized 7 days after peritumoral injection of PKH67-labeled BMCs. Representative dot plots of PKH67-labeled cells from tumors of one experiment (n = 5) are shown. b, PKH67-labeled cells in the tumor, LN or spleen of mice 7 days after peritumoral vaccination of PKH67-labeled BMCs. Representative data (mean ± SD) of 5 mice in the antibody and tritherapy group and of 4 mice in the untreated and BMC only group from one independent experiment is shown. c, (Top row) Percent of CellVue+ cells found in the tumors, LNs and spleens of antibody treated mice injected with or without peritumoral CellVue-labeled BMCs 1, 3, 7 or 14 days after peritumoral BMC vaccination. Data represents the mean ± SEM of 3 mice in the tritherapy group and mean ± SEM of 2 mice in the antibody therapy group from one experiment. (Bottom row) Percent of PKH67+ cells found in the tumors, LNs and spleens of antibody treated mice injected with or without peritumoral PKH67-labeled BMC 1, 3, 7 or 10 days after peritumoral BMC vaccination. The data represents the mean ± SEM of 3 mice in the antibody and tritherapy group and 5 mice in the untreated group on days 1,3, and 10 whereas on day 7, mean ± SEM of 4 mice for each group is displayed from one experiment
Fig. 3
Fig. 3
CD8+ and CD4+ T cells are required for tritherapy efficacy. a, Tritherapy-treated mice were depleted of CD4+ and/or CD8+ cells 1 day before beginning anti-GITR antibody administration. Survival was assessed. Pooled data from 2 independent experiments is shown. b, Tumors from line-1-tumor bearing mice were harvested 7 days after p.t. DR-BMC injections and analyzed by flow cytometry for total CD8+ T-cell (left) and AH1-tetramer-specific CD8+ T-cell (middle) infiltration into the tumor. Tumors from Panc02-SIY tumor bearing mice were harvested 10 days after p.t. DC vaccination and analyzed by flow cytometry for SIY-specific CD8+ T cells (right). Line-1 tumor CD8+ T-cell data demonstrates pooled data from 6 independent experiments whereas mean ± SEM from one independent experiment each is shown for AH1 + CD8+ T cells (n = 4) and pSIY+CD8+ T cells (n = 3). c, Same experimental setup as b, but Line-1 tumor CD4+ T cells were assessed. Pooled data from 6 independent experiments is shown for total CD4+ T cells, from 2 independent experiments for Tbet+CD4+ Th1 cells, and from 4 independent experiments for CD4+ Teffs and Tregs. d, Same experimental setup as c, but the ratios of CD8+ T cells:Tregs and CD4+ Teffs:Tregs in the tumor were assessed. Pooled data from 5 independent experiments is shown. e, Same experimental setup as c-d, but intracellular Ki-67 staining was assessed in tumors by flow cytometry. Pooled data from 3 independent experiments is shown. b-e One-Way ANOVA
Fig. 4
Fig. 4
Increased effector function of CD8+ and CD4+ T cells in tumors of tritherapy-treated mice. a, Representative flow plots and b, graphical representation of intracellular GzA and surface CD107a expression on CD8+ T cells in tumors harvested 7 days after p.t. DR-BMC injections. Pooled data from 3 independent experiments is shown for GzA + CD8+ T cells, and from 2 independent experiments for CD107a + and GzA + CD107a + CD8+ T cells. One-Way ANOVA. c, 10 days after DR-BMC p.t. injections, Line-1 tumors were harvested 4.5 h after BFA i.v. injections and intracellular cytokine staining on CD8+ T-cells was performed. Data shown here is representative of 2 independent experiments. d, Same as c but representative flow plots of tumor-infiltrating CD4+ T cells. e, Same as c-d but cytokine production by tumor CD4+ T cells is shown. Data shown here is representative of 2 independent experiments. One-Way ANOVA
Fig. 5
Fig. 5
Increased T cell activation in lymph nodes of tritherapy-treated mice. a, LNs were harvested and analyzed by flow cytometry 7 days after p.t. DR-BMC administration. Pooled data from 2 independent experiments are shown here. b, Same as a but activation markers on CD8+ T cells and c, CD4+ T cells were analyzed. Shown is representative data of 4 independent experiments for ICOS expression, 2 independent experiments for CD69 expression and one experiment for TIGIT expression. d, Same as a-c, but activation markers on effector FoxP3-CD4+ T cells versus FoxP3 + CD4+ Tregs are shown. Representative data from 2 independent experiments are shown. e, Same as a-d, but cytolytic potential of CD8+ T cells from the lymph nodes are shown. Pooled data from 2 independent experiments are shown. b-e One-Way ANOVA
Fig. 6
Fig. 6
Tritherapy promotes tumor regression locally within the TME after initial lymph node T cell priming. a, Individual tumor growth curves and survival of antibody therapy and tritherapy-treated mice with and without daily FTY720 administered starting at day 0 before tumor inoculation. Shown is data from one independent experiment (n = 5). b, Individual tumor growth curves and survival of antibody therapy and tritherapy-treated mice with and without daily FTY720 administered starting 1 day before p.t. DR-BMC vaccination. Pooled data from 3 independent experiments are shown. c, Mice were treated as in b but sacrificed 7 days after peritumoral BMC vaccination. Line-1 tumors were harvested and analyzed by flow cytometry. d, Mice were treated as in b-c but LNs were harvested and analyzed by flow cytometry. c-d data shown is mean ± SD of one experiment with n = 5. One-Way ANOVA
Fig. 7
Fig. 7
Tritherapy efficacy is independent of antigen presentation by peritumorally administered APCs. a, Line-1-tumor bearing mice were treated i.p. with anti-GITR antibody on days 5 and 8 and anti-PD-1 antibody on days 10, 12, and 14. Line-1 cell derived DRibbles were peritumorally administered on day 12. Individual tumor growths and overall survival is shown. Representative data from 1 experiment is shown (n = 5). b, Panc02-tumor bearing mice were treated as in a except DRibbles were derived from the Panc02 cell line. Representative data from 1 experiment is shown (n = 5). c, Washed unpulsed and DRibbles-pulsed day 8 BMCs were cultured for 24 h after which the supernatant was collected and analyzed by ELISA for IL-1beta, IL-6 or IL-12p40. Type I IFN presence in the supernatant was analyzed using B16Blue-IFNa/b cells. Data (mean ± SD) from one independent experiment performed in triplicate wells for IL-1β and IL-6 or duplicate wells for IL-12p40 and Type I IFNs is shown. d, Line-1 tumor bearing BALB/c mice were treated with BMC-tritherapy using BMCs derived from either syngeneic BALB/c mice or allogeneic C57BL/6 mice bone marrows. BMCs were pulsed with Line-1 cell-derived DRibbles before peritumoral administration. Representative data from 1 experiment is shown (n = 5). e, Same as d however mice were treated with syngeneic BALB/c or allogeneic C57BL/6 CD103+ DCs pulsed with DRibbles derived from Line-1 cells. Representative data from 1 experiment is shown (n = 5)

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