Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy

Abstract

Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4(+) and CD8(+) T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8(+) and CD4(+) effector but not regulatory T cells, and was dependent on CD8(+) cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic.

Figure 1. NDV increases distant tumor lymphocyte infiltration and delays tumor growth

(A) Treatment scheme. (B) Representative flow cytometry plots of percentages of tumor-infiltrating CD45+ and CD3+ cells. (C) Absolute numbers of CD45+ cells/g tumor. (D) Absolute numbers of innate immune cells/g tumor. (E) Tumor sections from distant tumors were stained with H&E (upper panels) or labeled for CD3 and FoxP3 (bottom panels) and analyzed by microscopy. Areas denoted by arrows indicate areas of necrosis and inflammatory infiltrates. Scale bars represent 200 μm. (F) Representative flow cytometry plots of percentages of CD4+FoxP3+ (Treg) and CD4+FoxP3− (Tconv) cells. (G) Absolute numbers of conventional and regulatory CD4+ cells and CD8+ cells/g tumor calculated from flow cytometry. (H) Relative percentages of Tregs out of CD45+ cells. (I) Calculated Tconv/Treg and CD8+/Treg ratios. (J,K) Upregulation of ICOS, Granzyme B, and Ki-67 on tumor-infiltrating Tconv (J) and CD8+ cells (K). (L) Growth of NDV-injected and distant tumors. (M) Overall animal survival. Data represent cumulative results from 3 (B–K) or 2 (L–M) independent experiments with n=3–5 per group. Mean +/− SEM is shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2. NDV induces infiltration of tumor-specific lymphocytes and facilitates tumor inflammation

(A) Treatment scheme. (B) Representative luminescence images from animals treated with NDV and adoptively-transferred Trp1-Fluc lymphocytes. (C) Quantification of average luminescence from the tumor sites. (D) The area under the curve (AUC) calculated from the data in panel (C). (E) Absolute number of Pmel lymphocytes from distant tumors calculated from flow cytometry. (F) Representative flow cytometry plots of percentages of CD45+ and CD3+ cells infiltrating distant tumors of animals treated per treatment scheme in panel (A). (G) Experimental scheme for serum transfer from animals treated intratumorally with single injection of NDV or PBS. (H) Representative flow cytometry plots of percentages of CD45+ and CD3+ cells infiltrating serum-injected tumors. (I) Absolute numbers of the indicated cell subsets in serum-injected tumors calculated from flow cytometry. Data for panels B–E represent 1 of 3 experiments with n=4–5 per group. Data for panels G–I represent pooled data from 2 independent experiments with n=5 per group. Mean +/− SEM is shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. NDV and CTLA-4 blockade synergize to reject local and distant tumors

(A) Treatment scheme. (B) Growth of virus-treated (right flank) B16-F10 tumors. (C) Growth of distant (left flank) B16-F10 tumors. (D) Long-term survival in the B16-F10 model. (E) Surviving animals were injected with 1×10⁵ B16-F10 cells in right flank on day 90 and followed for survival. Data represent cumulative results from 3 experiments with n=6–11 per group. (F) Growth of virus-treated (right flank) and distant (left flank) TRAMP C2 tumors. (G) Long-term survival in the TRAMP C2 model. (H) In vitro sensitivity of B16-F10 and TRAMP C2 cells to NDV-mediated lysis at different multiplicities of infection (MOI's). (I–K) Upregulation of MHC I, MHC II, CD80, and CD86 in B16-F10 and TRAMP C2 cells infected with NDV. Representative flow cytometry plots from B16-F10 cells (I) and calculated average median fluorescent intensities (MFI) for B16-F10 (J) and TRAMP C2 (K) cells are shown. Mean +/− SEM is shown. Data represent results from 1 of 3 (B–E), or 1 of 2 (F,G) independent experiments with n=5–10 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4. Systemic anti-tumor effect is restricted to the injected tumor type

(A) Animals were injected i.d. in right flank with B16-F10 melanoma, MC38 colon carcinoma, or PBS, and in the left flank with B16-F10 cells and treated as outlined in the scheme. (B,C) Growth of distant tumors (B) and overall survival (C) of animals that received right B16-F10 or no right flank tumors. Data show representative results from 1 out of 2 independent experiments with 5–10 mice/group. (D,E) Growth of distant tumors (D) and overall survival (E) of animals that received right B16-F10 or MC38 tumors. Data represent results from 1 out of 2 independent experiments with n=10 per group. **p<0.01, ****p<0.0001.

Figure 5. Combination therapy with NDV and CTLA-4 blockade induces inflammatory changes in distant tumors

Animals were treated per schema in Fig. 3A. Tumors were harvested on day 15 and analyzed for infiltrating immune cells. (A) Tumor sections from distant tumors were stained with H&E (upper panels) or for CD3 and FoxP3 (lower panels) and analyzed by light and fluorescence microscopy, respectively. Areas denoted by arrows indicate necrosis and inflammatory infiltrates. Scale bars represent 200 μm. (B) Absolute number of tumor-infiltrating CD45+ and CD3+ cells/g tumor calculated from flow cytometry. (C) Representative flow cytometry plots of percent of tumor-infiltrating CD4+ and CD8+ cells gated on CD45+ population. (D) Absolute numbers of Tconv, Treg, and CD8+ cells per gram of tumor. (E) Relative percentages of tumor-infiltrating Tregs out of CD45+ cells. (F) Calculated Tconv/Treg and CD8+/Treg ratios. (G–I) Upregulation of ICOS, Granzyme B, and Ki-67 on tumor-infiltrating CD8+ and Tconv lymphocytes. Representative flow cytometry plots (upper panels) and cumulative results (bottom panels) are shown. (J) TILs were restimulated with DC's pulsed with B16-F10 tumor lysates, and IFNγ production was determined by intracellular cytokine staining. Representative flow cytometry plots (left panel) and cumulative results (right panel) are shown. Data represent cumulative results from 5 (A–I) or 2 (J) independent experiments with n=3–5 per group. Mean +/− SEM is shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. Anti-tumor activity of NDV combination therapy depends on CD8+ and NK cells and type I and type II interferons

(A–C) Animals were treated as described in Fig. 3A with or without depleting antibodies for CD4+, CD8+, NK cells, or IFNγ. (A) Growth of injected tumors. (B) Growth of distant tumors. (C) Long-term survival. (D–F) IFNAR−/− or age-matched C57BL/6 mice (BL/6) were treated as described in Fig. 3A and monitored for tumor growth. (D) Growth of injected tumors. (E) Growth of distant tumors. (F) Long-term survival. Data for all panels represent cumulative results from 2 independent experiments with n=3–10 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.