Timing of PD-1 Blockade Is Critical to Effective Combination Immunotherapy with Anti-OX40

Abstract

Purpose: Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to affect patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T-cell costimulatory receptors, such as OX40, remains a critical question.Experimental Design: We utilized an anti-PD-1-refractory, orthotopically transplanted MMTV-PyMT mammary cancer model to investigate the antitumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. As PD-1 naturally aids in immune contraction after T-cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1.Results: The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type I and type II serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4⁺ T-cell proliferation at day 13, but at day 19, both CD4⁺ and CD8⁺ T-cell proliferation was significantly reduced compared with untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors, sequential combination was dependent on both CD4⁺ and CD8⁺ T cells and completely regressed tumors in approximately 30% of treated animals.Conclusions: These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials. Clin Cancer Res; 23(20); 6165-77. ©2017 AACRSee related commentary by Colombo, p. 5999.

Figure 1. Concurrent administration of anti-PD-1 and anti-OX40 reduces anti-tumor effect of anti-OX40 alone

(A) Representative flow cytometric quantification of OX40 and PD-1 on conventional CD4⁺FoxP3⁻, CD8⁺, and CD4⁺FoxP3⁺ T cells in the draining lymph node, spleen, and tumor of pretreatment 7 day MMTV-PyMT tumor-bearing mice. (B) Frequency of OX40⁺ T cells. n = 4–5, one representative of two independent experiments. (C) Frequency of PD-1⁺ T cells. (D) Treatment schedule of MMTV-PyMT tumor-bearing mice. Mice were treated on days 7, 9, and 11 with either 100 μg. anti-OX40, 250 μg. anti-PD-1, or both antibodies. (E) Mean tumor growth of treated tumors, n = 6, one representative of two independent experiments. (F) Kaplan-Meier survival curves of treated mice. n =10–12, combination of two independent experiments. Error bars represent SEM. ** = p < 0.01, **** = p < 0.0001

Figure 2. Concurrent combination of anti-OX40 and anti-PD-1 increases serum cytokines and inhibitory receptors on T cells

(A) Time course of serum cytokines taken from MMTV-PyMT tumor-bearing mice treated with anti OX40, anti-PD-1, or both antibodies. Black arrows (ê) indicate antibody treatment. X-axis represents time post tumor transplant. IFN-γ, TNF, IL-6, IL-10, and IL-4 were measured on days 7, 9, 11, 13, 14, 17, and 20 post tumor transplant, n = 4–14, combination of five independent experiments. (B, C) Frequency of CD4⁺ (left) and CD8⁺ (right) T cells expressing inhibitory receptors TIM-3 and CTLA-4 from the tumor (B) or spleen (C) of MMTV-PyMT tumor-bearing mice treated with anti-OX40, anti-PD-1, or both, day 13 after tumor transplant. n = 9–14, combination of two-three independent experiments. (D,E) Frequency of BTLA⁺ CD4⁺ (D) and CD8⁺ (E) T cells. n = 4–5, one representative of two independent experiments. Error bars represent SEM. Stars above single bars represent minimum significance compared to all three other groups. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

Figure 3. Concurrent combination treatment increases the frequency of proliferating T cells in the tumor, but only anti-OX40 increases total TIL

MMTV-PyMT tumor-bearing mice were treated as in Figure 1C and tumors were resected on day 13. (A) Flow cytometric quantification of the frequency of CD3⁺ T cells of the CD45⁺ cell population in treated tumors. (B) Average T cell populations as a frequency of CD45⁺CD3⁺ cells in the tumor. (C) Total conventional CD4⁺FoxP3⁻ (top), CD4⁺FoxP3⁺ T_reg (middle) and CD8⁺ (bottom) T cells in untreated and treated tumors. Normalized by mm² per tumor. (D) Frequency of Ki67⁺ proliferating conventional CD4⁺FoxP3⁻ (top), CD4⁺FoxP3⁺ T_reg (middle) and CD8⁺ (bottom) T cells in the tumor. n = 7–10, combination of two independent experiments. Error bars represent SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

Figure 4. Sequential combination treatment augments anti-OX40 therapeutic efficacy

(A) Frequency of PD-1 expression on conventional CD4⁺ (left) and CD8⁺ (right) T cells in day 13 MMTV-PyMT tumor-bearing spleens after anti-OX40 therapy. n = 5, one representative of four independent experiments. (B) Treatment schedule of MMTV-PyMT tumor-bearing mice comparing concurrent and delayed (delay, d), sequential therapy. Delayed anti-PD-1 was given on days 13, 15, and 17. (C) Mean tumor growth of treated tumors, n = 6, one representative of two independent experiments is shown. (D) Survival of combination treated mice. n = 10–12, combination of two independent experiments. (E) Mean tumor growth of tumors treated sequentially with anti-OX40 then anti-PD-1 or anti-PD-1 then anti-OX40. n = 6, one representative of two independent experiments is shown. (F) Mean tumor growth of MMTV-PyMT tumor-bearing mice treated with a combination of anti-OX40 plus 200 μg. of either concurrent (days 7, 9 and 11) or delayed (days 13, 15, and 17) anti-PD-L1, n = 5–6, one representative of two independent experiments is shown. Error bars represent SEM. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001

Figure 5. Sequential combination treatment maintains proliferating T cells and avoids increases in inhibitory receptors

MMTV-PyMT tumor-bearing mice were treated as in Figure 4B, and tumor and spleens were resected at day 19 (two days after last delayed PD-1 treatment). (A,B) Frequency of Ki67⁺ proliferating conventional CD4⁺FoxP3⁻ (A) and CD8⁺ (B) T cells from tumors. n = 9–10, combination of two independent experiments. (C,D) Frequency of BTLA⁺ CD4⁺ (C) and CD8⁺ (D) T cells from tumors. n = 4–5, one representative of two independent experiments. (E,F) Frequency of CD4⁺ (left) and CD8⁺ (right) T cells expressing TIM-3 and CTLA-4 from the tumor (E) or spleen (F). n = 8–14, combination of two (E) or three (F) independent experiments. Error bars, SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

Figure 6. Sequential combination treatment requires both CD4⁺ and CD8⁺ T cells for optimal therapeutic effect

MMTV-PyMT tumor-bearing mice were treated with anti-OX40 plus delayed anti-PD-1 (PD-1d), combined with 250 μg. anti-CD4, anti-CD8, or rat IgG on days 6 and 13 (A) Average T cell populations as a frequency of CD45⁺CD3⁺ cells in non-depleted day 19 treated and untreated tumors. n = 8–9, combination of two independent experiments. (B) Quantification of total CD8⁺ cells per mm² of tumor on day 20–21 determined via flow cytometry. n = 4, combination of two independent experiments. (C–E) Mean tumor growth of combination treated CD4-depleted (C) or CD8-depleted (D) mice. n = 9–10, one representative of two independent experiments shown. (E) Survival of CD4 or CD8-depleted mice. n = 18–20, combination of two independent experiments. Error bars represent SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001