Zhenyu Mu

Immunologic Correlates of the Abscopal Effect in a Patient with Melanoma

The abscopal effect is a phenomenon in which local radiotherapy is associated with the regression of metastatic cancer at a distance from the irradiated site. The abscopal effect may be mediated by activation of the immune system. Ipilimumab is a monoclonal antibody that inhibits an immunologic checkpoint on T cells, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). We report a case of the abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy. Temporal associations were noted: tumor shrinkage with antibody responses to the cancer-testis antigen NY-ESO-1, changes in peripheral-blood immune cells, and increases in antibody responses to other antigens after radiotherapy. (Funded by the National Institutes of Health and others.).

Enhancement of tumor-reactive cytotoxic CD4 + T cell responses after ipilimumab treatment in four advanced melanoma patients

Using archived blood samples from 4 NY-ESO-1-seropositive patients with advanced melanoma who were treated with the CTLA-4-blocking monoclonal antibody ipilimumab, Kitano and colleagues analyzed changes in antigen-specific CD4+ T cells during cancer immunotherapy. They characterized a novel consequence of the CTLA-4 blockade, the induction or expansion of tumorreactive cytotoxic CD4+ T cells after ipilimumab treatment. CD4+ T cells provide help to enhance and sustain cytotoxic CD8+ T-cell responses. A direct lytic role for this cell population in mouse models further supports the use of tumor-reactive CD4+ T cells for cancer immunotherapy. CTLA-4 blockade has been shown to expand antigen-specific cytotoxic CD4+ T cells in mouse models. We took advantage of spontaneous immunity to the NY-ESO-1 cancer-testis antigen to investigate quantitative and qualitative changes in antigen-specific CD4+ T-cell responses after ipilimumab (anti-CTLA-4 monoclonal antibody) treatment in patients with advanced melanoma. Four patients with NY-ESO-1 seropositive melanoma were chosen upon the availability of suitable blood specimens for characterizing the functions of NY-ESO-1 antigen-specific CD4+ T-cell response by enzyme-linked immunospot (ELISPOT), intracellular cytokine staining (ICS), and cytotoxicity assays. Multiple NY-ESO-1 antigen-specific CD4+ T-cell responses with TH1 dominance were induced or enhanced after ipilimumab treatment in peripheral blood in all four patients. NY-ESO-1 antigen–specific CD4+ T-cell lines established from all four patients after ipilimumab treatment recognized naturally processed NY-ESO-1 protein in antigen-presenting cells, expressed master transcription factor Eomesodermin (Eomes), and secreted perforin and Granzyme B. Finally, we showed that these NY-ESO-1 antigen-specific CD4+ T-cell lines directly lysed autologous melanoma cell lines expressing NY-ESO-1 in an MHC class II restricted manner. Our results show that antigen-specific cytotoxic CD4+ T-cell responses are induced after ipilimumab therapy in human cancer patients. Ipilimumab may induce the expression of lytic granules on antigen-specific cytotoxic CD4+ T cells via Eomes, revealing a novel consequence of immunologic checkpoint blockade. Cancer Immunol Res; 1(4); 235–44. ©2013 AACR.

Immunologic Response to Xenogeneic gp100 DNA in Melanoma Patients: Comparison of Particle-Mediated Epidermal Delivery with Intramuscular Injection

Purpose: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8+ T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED). Experimental Design: Human leukocyte antigen (HLA)-A*0201+ disease–free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 μg or 2,000 μg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201–restricted gp100 epitopes [gp100209-217 (ITDQVPFSV) and gp100280-288 (YLEPGPVTA)]. Results: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer+CD8+ T cells, all carrying the CCR7loCD45RAlo effector-memory phenotype. Five of 27 patients generated IFN-γ+CD8+ T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-γ+CD8+ T-cell generation (P = 0.07). Conclusion: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection. Clin Cancer Res; 16(15); 4057–65. ©2010 AACR.

Abstract P2-15-01: Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts)

Background: In pts with ESBC, cryo combined with cytotoxic T-lymphocyte antigen 4 blockade was well tolerated and did not delay standard-of-care mastectomy. As observed in mice, cryo+ipi may liberate tumor-associated antigens, synergistically activate tumor-reactive T-cells, and confer long-term anti-tumor immunity. Because singular biomarkers of response to immunotherapy have not been well defined, we conducted an integrated immunologic assessment to explore potential predictors of immune activation and response. Methods: Serial blood and pre-/post-treatment tumor tissue were collected from 18 pts treated with cryo (6 pts), single-dose ipi at 10mg/kg (6 pts), or cryo + ipi (6 pts). A Meso Scale Discovery platform was used to measure plasma cytokine interferon gamma (IFNγ). Multiparameter flow cytometry was used to evaluate peripheral and intratumoral T-cell and myeloid cell quantity, T-cell phenotype (effector versus regulatory), proliferation state (Ki67), and activation state (inducible costimulator [ICOS] expression). Finally, a DNA deep sequencing platform was used to conduct T-cell repertoire analysis of peripheral T-cells and TILs. Results: Sustained >2-fold elevations (1 month post-treatment) in plasma IFNγ were observed in the majority (4/6) of pts receiving cryo/ipi (median 6-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6, median 0-fold). Similarly, sustained >2 fold elevations in ICOS expression in peripheral CD3+CD4+ T-cells, a known pharmacodynamic marker of ipi, were observed in the majority (5/6) of pts receiving cryo/ipi (median 4-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6 ipi; median 1-fold). No trends were observed in peripheral myeloid derived suppressor cells. Analysis of TILs by flow cytometry identified increased numbers of proliferating CD8+ T-cells (CD8+Ki67+) in ipi and cryo/ipi groups relative to cryo alone; furthermore, the ratio of proliferating (CD8+Ki67+) to regulatory (CD4+CD25+FoxP3+) cells was enhanced in the cryo/ipi group. Finally, analysis of T-cell repertoire in TILs demonstrated that cryo/ipi generated an influx of novel T-cell clones, with select clones surging dramatically in predominance and circulating within the periphery. Conclusions: Utilizing an integrated assessment, we identified evidence of immunologic synergy with combination cryo/ipi versus either therapy alone. Of the tested parameters, peripheral CD4+ ICOS expression, plasma IFNγ, Ki67-gated TIL effector/regulatory ratios, and clonal repertoire analysis were identified as promising biomarkers of immune activation. These findings will inform a prospective assessment of potential immunologic biomarkers of immune response and clinical benefit in a phase 2 study of cryo-immunotherapy in ESBC. Citation Format: David Page, Jianda Yuan, Adi Diab, Zhiwan Dong, Arielle Ginsberg, Phillip Wong, Ryan Emerson, David Redmond, Brian Blum, Zhenyu Mu, Chunjun Zhao, Christopher Comstock, Elizabeth Morris, Elizabeth Comen, Alan Kotin, Janice Sung, Edi Brogi, Monica Morrow, Stephen Solomon, Virgilio Sacchini, Majid Maybody, Deirdre Neville, Harlan Robins, Sujata Patil, Jedd Wolchok, Clifford Hudis, Larry Norton, James Allison, Padmanee Sharma, Heather McArthur. Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-15-01.

Presence of myeloid-derived suppressor cells (MDSC) in patients with metastatic castration-sensitive and castration-resistant prostate cancer.

222 Background: MDSC contribute to an immune suppressive environment and have been implicated in cancer progression. Measurement (identification and enumeration) is challenged by the lack of analytically valid assays limiting data interpretation between groups. This impacts our understanding of rationale immune targets and design of clinical trials. We employed a novel biomarker based assay in whole blood to enumerate MDSC from patients with metastatic castration sensitive (CSPC) and castration resistant prostate cancer (CRPC). Methods: Whole blood was collected in Cyto-Chex (Streck) tubes. A published computational algorithm-based approach (developed by Memorial Sloan Kettering Cancer Center and commercialized by Serametrix) was employed to determine the %MDSC-monocytic and coefficient of variance (CV=ratio of SD and geometric mean fluorescence intensity) to assess HLA-DR spread on CD14+CD11b cells. Samples were performed in duplicate. Clinical variables including clinical state, past and current treatme...

Matched T cell repertoire analysis of peripheral blood and tumor-infiltrating lymphocytes (TILs) in early stage breast cancer (ESBC) patients (pts) treated with pre-operative cryoablation (cryo) and/or Ipilimumab (Ipi)

Meeting abstracts Cryo plus anti-CTLA-4 therapy induces antigen-specific clonal T cell expansion, enhanced survival, and long-term anti-tumor immunity in mice [[1][1]]. We recently demonstrated that pre-operative cryo and/or anti-CTLA-4 therapy with Ipi is well tolerated and clinically feasible in