Liu, Xin

Abstract B7-H1 (also named PD-L1) is up-regulated on activated CD8 T cells at the late stage of antigen stimulation, and is required for T cell survival during contraction phase. Since more anti-B7-H1 (PD-L1) antibody has been used in clinical trials of treatment of human cancers, it is necessary to know whether antibody ligation of B7-H1 expressed by T cells would affect T cell function and survival. After screening several anti-B7-H1 antibodies, we found that some antibody triggered the activation of p38 MAPK in activated CD8 T cells. In addition, the levels of activated p38 MAPK increased in T cells isolated from B7-H1 knockout mice compared to wild type mice. Inhibition of DNA-PKcs activation increased the activation of p38 MAPK in wild type T cells but not in B7-H1 KO T cells, suggesting B7-H1 may negatively regulate p38 MAPK through DNA-PKcs pathway. To that end, we identified an association of B7-H1 with DNA-PKcs in activated human T cells but not in resting T cells. Since p38 MAPK inhibition rescued activated CD8 T cells from apoptosis, the pro-survival function of T cell intrinsic B7-H1 may be mediated by suppression of p38 MAPK. However, ligation of B7-H1 by agonist antibody may disrupt B7-H1’s pro-survival function by increasing p38 MAPK activation. Our results identified a link of B7-H1 with p38 MAPK pathway and may help to define agonist function of anti-B7-H1 antibody prior to clinical application.

Abstract Background: CLL patients (pts) treated with novel targeted therapies continue to experience disease progression. Approximately 7-15% of relapsed CLL treated with ibrutinib or venetoclax developed Richter's syndrome (RS), an aggressive high grade lymphoma transformation. Standard chemotherapy has limited efficacy in RS. We hypothesized that checkpoint blockade would re-establish anti-tumor immune response in progressive CLL/RS and here we aim to update the clinical data and correlative analysis of MC1485: a phase 2 study of PD-1 blocking antibody pembrolizumab in relapsed/refractory CLL and RS. Methods: Relapsed/refractory CLL including RS enrolled in the CLL arm of MC1485 trial was reported here. The primary end point of this study is overall response rate (ORR). Pts with prior allogeneic stem cell transplant were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks. NCI CTCAE v4.0 and IWCLL 2008 criteria were used for non-hematological adverse events (AE) and for CLL grade hematological AE. Tumor expression of PD-1 and PD-L1 were assessed with standard immunohistochemical (IHC) staining. Percentage expressions of individual antigens were analyzed with whole-slide scanning followed by image analysis with Image-Pro software (Media Cybernetics). Fluorescence in situ hybridization (FISH) was performed on available CLL/RS tissue sections to assess copy number of chromosome 9p region that contains PD-L1 and PD-L2. Results: 25 pts including 16 CLL and 9 RS (biopsy-proven large cell lymphoma) were enrolled in CLL arm. The median age was 69 years (46-81). Twelve (48%) pts had del(17p) or monosomy 17 or TP53 mutation. The median number of prior therapies was 4 (1-10). 96%, 72% or 48% of pts had received alkylator and anti-CD20 antibody, purine analog or anthracycline. Fifteen (60%) pts had prior ibrutinib and 12 (48%) progressed clinically while receiving ibrutinib, including 6 who progressed to RS and 6 who developed progressive CLL. The median number of pembrolizumab doses that pts received was 3 (1 to 21), administered over a median treatment duration of 11 weeks (1 to 56). Drug-related AE occurred in 21 pts (88%) with the most common ones being neutropenia in 9 (37%), cough in 7 (29%) and dyspnea in 6 (25%) pts. Drug-related grade 3 or above AE occurred in 9 (38%) pts with most common ones being thrombocytopenia (5, 21%), dyspnea (2, 8%), and fatigue (2, 8%). The most common immune-related AE was liver enzyme elevation (12%, 8% G3) and was reversible with therapy interruption or steroid therapy. Based on investigator assessment using the Revised Response Criteria for Lymphomas and IWCLL 2008 criteria, one RS pt had complete response (CR, 4%), 3 RS had partial responses (PR, 12%). Among 9 RS pts, 1 CR (11%), 3 PR (33%), 3 SD (33%) and 2 PD (22%) were observed. Thus the ORR in RS was 44%. No CLL pts achieved a CR/PR, 3 had SD and 9 had progressive disease (PD). The ORR of all pts was 16%. Of the 4 RS who had CR/PR, responses occurred early after 2 cycles of therapy and median duration of therapy for CR/PR pts was 8.3 months with data cut-off by June 30th 2016 (Figure 1A). The causes for therapy discontinuation in RS were PD (3, 33%), alternate therapy (2, 22%), and thrombocytopenia due to increased marrow CLL (1, 11%). Increased marrow CLL was observed in 2 RS pts with PR to RS phase of disease assessed by PET, thus the protocol was amended to allow addition of idelalisib/ibrutinib to control the underlying marrow CLL. For RS pts who had a CR/PR or SD, variable nodal responses were observed (Figure 1B). In particular, 6 RS who had prior ibrutinib experienced CR or PR or SD with nodal responses. After a median follow-up of 10.2 (1.9 to 16.1) months, the median overall survival (OS) for all pts was 10.7 months (95% confidence interval [CI], 5.4 to not reached). The 6 month OS rate for RS and CLL pts were 73% and 59%, respectively. Biomarker assessment using IHC analysis on 10 pts with available tumor/nodal tissues (6 RS and 4 CLL) showed an increased expression of PD-L1 (p = 0.02) and a trend for an increased expression of PD-1 (p = 0.1) in the group of pts with CR/ PR versus pts in the group with no clinical response. One out of 10 tested showed polysomic for chromosome 9p. Conclusion: Pembrolizumab had an acceptable safety profile in CLL and RS patients. We confirmed that pembrolizumab has substantial therapeutic activity in RS. Single-agent pembrolizumab does not appear to have clear activity in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Introduction: IL-15 has been used in clinical trials with PD-1 antibody to overcome cancer resistance to PD-1 monotherapy in cancer patients. However, it is not clear what T cell population would be expanded to mediate tumor control in IL-15 in combination with PD-1 antibody. We recently found CX3CR1+ cytotoxic CD8+ T cells, which increased in responders compared with non-responders after anti-PD-1 in melanoma patients, demonstrated an increased transcription of CD122 (IL2RB). Since CD122 is a subunit of IL-15 receptor, we hypothesized that IL-15 in combination with anti-PD-1 therapy may have the ability to expand CX3CR1+ cytotoxic CD8+ T cells for tumor control. Methods and findings: To examine whether IL-15 plus anti-PD-1 expand CX3CR1+ cytotoxic CD8+ T cells in tumor tissues, we treated tumor-bearing mice with IL-15/IL-15Rα complexes alone or with PD-1 antibody and measured the frequency of CX3CR1+Granzyme B+ CD8+ T cells. As only the combination therapy induced dramatic tumor regression, we found the highest increase of CX3CR1+Granzyme B+ CD8 T cells in tumor tissues in the group treated with IL-15 plus anti-PD-1 (28.87±3.19% vs. 7.91±1.32%, p<0.01) compared with groups treated with either IL-15 alone or anti-PD-1 alone. In contrast to wild type mice, IL-15 plus anti-PD-1 did not suppress tumor growth in CX3CR1-KO mice. To determine whether IL-15 alone can also expand human CX3CR1+ cytotoxic CD8+ T cells, we cultured peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 5) with IL-15 for 24 hours in vitro. We found IL-15 (5 ng/mL) only modestly increased the frequency of CX3CR1+ Granzyme B+ CD8+ T cells than those cultured with no IL-15 (39.9±6.44% vs. 27.56±4.98%). To test whether this increase is due to enhanced proliferation of this subset of CD8+ T cells, we measured the expression of Ki-67, a cell proliferative marker, in CX3CR1+ CD8+ T cells by intranuclear staining. We found IL-15 modestly induced more proliferating CX3CR1+ CD8+ T cells in vitro culture compared with control group without IL-15 (6.25±0.54% vs. 0.91±0.25%). Summary: Taken together, we found IL-15 combined with anti-PD-1 therapy has the ability to significantly expand CX3CR1+ cytotoxic CD8+ T cells, however IL-15 alone may have limited ability to significantly expand this effector cell population. Our studies provide a new rational for combination therapy of IL-15 and PD-1 antibody to overcome cancer resistance for patients who did not respond to initial PD-1 therapy. Citation Format: Henan Zhang, Xin Liu, Siyu Cao, Lingling Chen, Susan Harrington, Ying Li, Aaron Mansfield, Sean Park, Yiyi Yan, Roxana Dronca, Haidong Dong. Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4080.

Key Points Pembrolizumab was first shown to be clinically active in CLL patients with RT. PD-1 and PD-L1 expression in tumor microenvironment are promising biomarkers to select RT patients for PD-1 blockade.