Dronca, Roxana

Abstract Despite unprecedented successes with immune checkpoint blockade in advanced cancers, these agents provide durable clinical benefit in only a subset of patients. In addition, the clinical management of patients receiving immune checkpoint blockade remains quite challenging due to the unpredictable and kinetically heterogeneous responses which can manifest as late responses, pseudoprogression, or hyperprogression in subsets of patients. The dynamics and heterogeneity of programmed cell death protein ligand 1 (PD-L1, aka B7-H1) expression confound its use as a predictive biomarker in cancer immunotherapy, but blood-based biomarkers have the potential to predict responders and detect mechanisms of resistance to immunotherapy. We previously reported that Bim (BH3-only protein) is a downstream signaling molecule of the PD-1 pathway, and that Bim levels in circulating tumor-reactive T-cells reflected patient responses to anti-PD-1 therapy in melanoma. We also discovered the existence of soluble PD-L1 (sPD-L1) in the plasma of cancer patients and showed a correlation of sPD-L1 with cancer stages in renal cell carcinoma and with prognosis in metastatic melanoma. Here we evaluated the frequency of tumor-reactive T cells with Bim expression along with effector memory profile and sPD-L1 as biomarkers of response in a new cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD-1 therapy. We recruited 100 cancer patients treated with anti-PD-1 therapy who had peripheral blood collected at baseline and at each subsequent radiographic tumor evaluation. Frequencies of Bim+ and effector memory CD8+ T cells were measured by flow cytometry in gated tumor-reactive CD11a high PD1+ CD8+ T cells. We also measured levels of sPD-L1 in plasma at baseline and serially during treatment with ELISA. Baseline T cell markers and sPD-L1 levels and their percent changes in patients who had a radiographic response were compared to those who had progressive disease (PD). We found that patients with objective response after 4 cycles of anti-PD-1 therapy had higher frequency of Bim+ CD8+ T cells at baseline compared to patients with PD (mean 43% vs. 30%, P=0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P=0.0111). The frequency of effector memory CD8+ T cells also dramatically increased in responders in 3-6 months after anti-PD-1 therapy (mean 89% vs. 9.56%, p=0.002). High baseline sPD-L1 was associated with progression on anti-PD-1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p=0.07) or development of immune-related toxicities and the levels increased by 12 weeks in patients progressing on therapy. Conclusion: Measurements of T cell biomarkers (Bim and effector memory) and sPD-L1 levels provide a new noninvasive way to predict and monitor patient responses to anti-PD-1 monotherapy in melanoma and lung cancers. Note: This abstract was not presented at the conference. Citation Format: Roxana S. Dronca, Aaron Mansfield, Xin Liu, Susan Harrington, Elizabeth Enninga, Svetomir Markovic, Lisa Kottschade, Rob Mcwilliams, Matthew Block, Wendy Nevala, Michael Thompson, Haidong Dong. Blood-based T cell biomarkers and soluble PD-L1 predict responses and immune-related toxicity to PD-1 blockade in melanoma and lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A38.

Abstract Background: Clinical management of metastatic melanoma (MM) after PD-1 blockade failure remains a challenge and lacks a standard of care. Immunotherapy or chemotherapy alone provides limited benefit in this setting. Chemo-immunotherapy (CIT) combinations have demonstrated favorable efficacy and safety profiles in lung cancer patients. Our preclinical study in MM has shown that the addition of chemotherapy to PD-1 blockade can reshape a subset of therapy-responsive CD8+ T-cells with resultant enhanced antitumor activities, suggesting the potential clinical benefits of CIT in MM patients whose disease have progressed after anti-PD-1 therapy. Further understanding of the clinical benefits and immunoregulatory mechanisms of CIT in this setting is crucial for the development of optimal combinatorial chemo-immunotherapeutic strategies to improve clinical outcomes in patients with advanced cancer. Methods: MM patients (n=22) who have failed PD-1 blockade therapy were subsequently treated with CIT (paclitaxel and carboplatin in combination with pembrolizumab). The overall survival (OS), objective response rate (ORR), time-to-next therapy (TTNT), and toxicities were assessed. Using peripheral blood (PB) from MM patients, the phenotypic and functional changes induced by chemotherapy in therapy-responsive T-cells, in the setting of anti-PD1 therapy, were examined. The immunoregulatory effects of CIT were also examined in melanoma mouse model. Results: MM patients who have received subsequent CIT had a median OS of 5 years (95% CI: 2-NR) (median follow up of 3.9 years), with ORR of 61% (CR of 23%). The median TTNT was 8 months (95% CI: 6-15). No additional toxicities were identified. CX3CR1+CD8+ therapy-responsive T-cells are low in MM patients who have failed to respond to anti-PD-1 monotherapy. However, in MM patients who responded to subsequent CIT, this subset of therapy-responsive T-cells survived the chemotherapy with increased frequency and enhanced function. The clinical benefit of CIT is only observed in CX3CR1 wild type mice, not in KO mice, and ongoing PD-1 blockade is necessary to improve its anti-tumor activities. Conclusion: In MM patients who have failed anti-PD-1 therapy, the chemo-immunotherapy combination showed favorable clinical outcomes and an acceptable toxicity profile. CX3CR1+ CD8+ effector T-cells are responsible for the clinical benefit of CIT. This novel therapy-responsive population underlies the key cellular and molecular immunoregulatory mechanisms of chemotherapy. It serves as a meaningful marker to measure these collaborative effects and to develop the optimal chemo-immunotherapy strategy to improve clinical responses to current immune checkpoint blocking agents. Citation Format: Yiyi Yan, Haidong Dong, Roxana Dronca, Svetomir Markovic. CX3CR1+CD8+ T-cells are responsible to the clinical benefit of chemoimmunotherapy in metastatic melanoma patients after disease progression on PD-1 blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR11.

Abstract Background: Tumor surface PD-L1 (B7-H1) signals through T-cell surface receptor PD-1 to suppress antitumor immunity. PD-1 inhibitors like pembrolizumab block this pathway to prevent tumor escape from immunosurveillance. These PD-1 checkpoint inhibitors achieve objective response in 20-30% of patients with melanoma (1-3). As the majority of these patients are resistant to this treatment, there is a critical need to combat PD-1 checkpoint inhibitor resistance in melanoma. What is the mechanism of PD-1 checkpoint inhibitor resistance in melanoma? Our group has reported that soluble PD-L1 (sPD-L1, sB7-H1) is elevated in the serum of patients with PD-1 inhibitor-resistant melanoma and non-small cell lung cancer (NSCLC) (4). We have also shown that soluble PD-L1 is produced by cleavage from other tumor types and leads to apoptosis in CD8+ T-cells (5). The aim of this study is to confirm the source of sPD-L1 in human melanoma and its relation to outcomes. Methods: B7H1-expressing transgenic melanoma cells (Mel-B7H1) were treated with an array of protease inhibitors over 24 hours. Cells were then pelleted and supernatants were analyzed by ELISA for sPD-L1 (sB7-H1) production. Tumor serum samples were also analyzed by ELISA for sPD-L1 production.Tumor pathology slides from patients with melanoma (serum donors from the above study) were stained by immunohistochemistry with antibodies against proteases ADAM10 or ADAM17. Slides were visualized by microscopy for the presence of these antigens. Activated human CD8+ T-cells were cultured with recombinant human B7-H1 (PD-L1) protein, sPD-L1-rich, or sPD-L1-depleted supernatants from a tumor cell line in the presence of placebo or PD-1 checkpoint inhibitor antibodies. Cell survival was measured at 48 hours by flow cytometry.Results: ADAM10 inhibitor GI254023X, ADAM17 inhibitor TAPI-0, and broad metalloprotease inhibitor TAPI-2 inhibited Mel-B7H1 production of sB7H1 (p<0.001, 0.095, and 0.001 respectively). Specific inhibitors of other metalloproteases did not significantly alter sB7H1 production. PD-L1 transcription levels were not significantly altered. Tumor pathology slides from patients with high serum sPD-L1 exhibited high ADAM10 and/or ADAM17 staining. Conversely, tumors from patients with low-sPD-L1levels tended to exhibit low ADAM10 and/or ADAM17 staining. Recombinant PD-L1 and PD-L1-rich supernatants induced cell death of CD8+ T-cells, whereas PBS control solution and PD-L1-depleted supernatants did not. Supraphysiologic doses of pembrolizumab decreased cell death in PD-L1-rich supernatant-treated CD8+ T-cells. Conclusions: Matrix metalloprotease inhibitors ADAM10 and ADAM17 mediate sPD-L1 production from melanoma cells. In samples from patients with melanoma, immunohistochemical staining of these proteases in tumor tissue correlates with serum sPD-L1 levels. As we have shown previously, sPD-L1 induces apoptosis of activated CD8+ T-cells, which may explain PD-1 inhibitor resistance in most patients with melanoma.We posit that sPD-L1 outcompetes PD-1 inhibitors to suppress tumor immunosurveillance. Our results suggest that the combination of ADAM10/17 inhibitor and PD-1 checkpoint inhibitor may overcome this mechanism of PD-1 resistance in melanoma and other cancers. Future studies will determine (A) how a combined ADAM10/17 inhibitor and PD-1 inhibitor may function in clinical use, (B) how tumor-derived sPD-L1 signals downstream to cause apoptosis in T-cells, and (C) whether surface ADAM10/17 predict surface PD-L1 staining in melanoma. References: 1. Ribas et al., Lancet Oncol 2015;16(8):908–18. 2. Hamid et al., Ann Oncol 2016;27:6. 3. Weber et al., Lancet Oncol 2015:16(4):375–84. 4. Dronca et al., J Clin Oncol 2017:35(15):11534. 5. Orme et al., AACR Adv Malig Lymphoma 2018. Citation Format: Jacob J. Orme, Khalid Jazieh, Susan Harrington, Matthew Ball, Tariq U. Azam, Xin (Cindy) Liu, Tiancheng Xie, Aaron Mansfield, Roxana S. Dronca, Haidong Dong. Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor immunity – A resistance mechanism to PD-1 checkpoint blockade in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A210.

11534 Background: To date, there are no validated blood-based biomarkers of predicting response to PD-1 blockade. We previously reported that Bim is a downstream signaling molecule of the PD-1 pathway, and that measurement of Bim levels in circulating T-cells may predict and monitor responses to anti–PD-1 therapy in melanoma. We have identified the existence of sPD-L1 in cancer patients and showed that the sPD-L1 is biologically active and capable of triggering apoptosis in activated T-cells. Here we evaluated T cell Bim and sPD-L1 in the peripheral blood (PB) as biomarkers of response in a cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD1 therapy. Methods: 60 pts treated with anti-PD-1 had PB collected at baseline and at radiographic tumor evaluation. Frequencies of Bim+ T cells and Bim median fluorescence intensity (MFI) were measured by flow cytometry in gated tumor-reactive CD11ahighPD1+ CD8+ T cells. We also measured levels of sPD-L1 at baseline and serially during treatment with sPD-L1 ELISA. Baseline Bim and sPD-L1 levels and percent change in Bim levels in patients (pts) who had a radiographic response (CR/PR) were compared to those who had progressive disease (PD) at 12 wks. Results: Similarly to previously reported preliminary data, pts with objective response (CR/PR, 15/60) after 4 cycles of anti-PD1 therapy had higher frequency of Bim T cells at baseline compared to pts with PD (16/60) (mean 43% vs. 30%, P = 0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P = 0.0111). High baseline sPD-L1 were associated with progression on anti-PD1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p = 0.07, n = 13) and the levels increased by the first tumor assessment in patients resistant to anti-PD-1. Conclusions: Measurements of Bim and sPD-L1 levels may help to select patients who are likely to benefit from anti-PD1 monotherapy versus combinatorial strategies, and provide a new non-invasive way to monitor response to anti-PD-1 blockade. A larger validation study is underway.