Yanyun Li

Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma

BACKGROUND Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).

Agonist Anti-GITR Monoclonal Antibody Induces Melanoma Tumor Immunity in Mice by Altering Regulatory T Cell Stability and Intra-Tumor Accumulation

In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These “unstable” Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR−/−, and the protective effects of DTA-1 were reduced in reconstituted RAG1−/− mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer.

Induction of tumoricidal function in CD4+ T cells is associated with concomitant memory and terminally differentiated phenotype

OX40 engagement induces a cytotoxic CD4+ T cell subpopulation to eradicate advance melanomas

CTLA-4 blockade increases antigen-specific CD8+ T cells in prevaccinated patients with melanoma: Three cases

BackgroundAnti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.MethodsTo determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100209–217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100209–217 (ITDQVPFSV), tyrosinase369–377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.ResultsFollowing vaccination, patients generated weak to no CD4+ or CD8+ T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7loCD45RAlo) tetramer+CD8+ T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.ConclusionVaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially “boosting” the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient

Summary We present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy. Video Abstract

Improved tumor immunity using anti-tyrosinase related protein-1 monoclonal antibody combined with DNA vaccines in murine melanoma.

Passive immunization with monoclonal antibody TA99 targeting melanoma differentiation antigen tyrosinase-related protein-1 (Tyrp1; gp75) and active immunization with plasmid DNA encoding altered Tyrp1 both mediate tumor immunity in the B16 murine melanoma model. We report here that TA99 enhances Tyrp1 DNA vaccination in the treatment of B16 lung metastases, an effect mediated by immunologic mechanisms as Tyrp1 has no known role in regulating tumor growth. TA99 is shown to increase induction of anti-Tyrp1 CD8+T-cell responses to DNA vaccination against Tyrp1 as assessed by IFN-gamma ELISPOT assays. Immunohistochemistry studies reveal that TA99 localizes rapidly and specifically to B16 lung nodules. Augmentation of T-cell responses is dependent on the presence of tumor as well as on activating Fc receptors. Furthermore, TA99 enhances DNA vaccination against a distinct melanoma antigen, gp100(pmel17/silver locus), improving antitumor efficacy, augmenting systemic CD8+ T-cell responses to gp100, and increasing CD8+ T-cell infiltration at the tumor site. Epitope spreading was observed, with CD8+ T-cell responses generated to Tyrp1 peptide in mice receiving gp100 DNA vaccination in the presence of TA99. Finally, we show that TA99 improves therapeutic efficacy of DNA vaccination combined with adoptive T-cell transfer in treatment of established subcutaneous B16 melanoma. In conclusion, TA99 enhances DNA vaccination against both the target antigen Tyrp1 and a distinct melanoma antigen gp100 in an Fc receptor-dependent mechanism, consistent with enhanced cross-presentation of tumor-derived antigen. Monoclonal antibodies should be tested as vaccine adjuvants in the treatment of cancer.

Detection of Intra-Tumor Self Antigen Recognition during Melanoma Tumor Progression in Mice Using Advanced Multimode Confocal/Two Photon Microscope

Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response “functions” in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFNγ, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.

Multimodal microscopy of immune cells and melanoma for longitudinal studies

Intravital microscopy of cancer is a well established tool that provides direct visualization of the tumor cycle. It traditionally involves one of several strategies: invasive subcutaneous (SC) implantation of tumors followed by surgical opening of skin flaps for imaging, techniques utilizing skin fold chambers and implanted optical windows or intradermal injections under 200μm from the skin surface. All of these techniques allow the use of fluorescent proteins as markers for biologically significant constituents. However, observation methods utilizing skin-flaps, skin-fold chambers and optical windows are invasive and tend to alter the immune environment of the tissue and/or limit the duration of studies that can be performed. If implanted correctly, intradermally injected tumors can be minimally invasive, will not require biopsies or surgical intervention to observe and are accessible for direct transdermal imaging with a number of in vivo modalities. We present our work in the development of a small animal intravital microscopy workstation that allows the acquisition of different contrast imaging modalities: reflectance confocal, wide field epifluorescence, multiphoton and second harmonic generation (SHG). The images are acquired pair-wise simultaneously and sequentially in time. The aim of our instrumentation is to gather all information generated by the single probing beam via the reflected or back-scattered signal, SHG signal and various fluorescence signals. Additionally, we also present our development of a microscopic tissue navigation technique to mark, label and track sites of interest. This technique enables us to revisit sites periodically and record, with different imaging contrasts, their biological changes over time.

Abstract CT018: Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial

Background: The tumor necrosis factor receptor GITR is expressed at high levels on regulatory T cells (Tregs) and up-regulated on activated T cells. Evidence from preclinical studies has shown the dual activity of GITR stimulation to counteract Tregs functionally and quantitatively and enhance T-cell effector functions, thus providing the rationale to develop GITR agonist antibodies for cancer immunotherapy. These agents have demonstrated potent therapeutic effects associated with both Treg reduction and modulation in tumor-bearing mice. Here we investigate Treg modulation as a biomarker of activity of the fully humanized agonist anti-GITR antibody TRX-518 in advanced solid cancer patients and the potential mechanisms underlying this effect. Methods: Patients were accrued to 9 cohorts (up to 6 patients/cohort) to receive a single dose of TRX518 (dose range: 0.0001-8 mg/kg). Pharmacodynamic analyses included flow cytometric evaluation of frequency and phenotype of circulating T cells at different time points up to 12 weeks after treatment. Relevant changes observed with these analyses were monitored in pre- and post-treatment tumor biopsies by immunofluorescence staining. Effects of GITR stimulation with TRX-518 on human Tregs were studied in in vitro cultures with CD4+ T cells from healthy donors. Results: Analysis of peripheral blood mononuclear cells from 37 advanced refractory solid cancer patients treated with >=0.005 mg/kg TRX-518 (cohorts 3-9) revealed frequent reduction in circulating Tregs after treatment with TRX-518. Proliferation potential of Tregs was not altered by TRX-518, as indicated by their persistent Ki67 expression. We then asked whether a subset of Tregs was preferentially affected and found that GITR+ Tregs and CD45RA-Foxp3hi effector Tregs (eTregs) were specifically targeted by TRX-518 in a dose-dependent manner. Accordingly, we observed that eTregs express GITR at higher levels than naive CD45RA+Foxp3+ Tregs. In a subset of patients for whom pre- and post-treatment tumor biopsies were available (n=8; 3 melanoma, 2 lung, 2 colorectal and 1 bladder cancer patients), we tested whether intra-tumor Tregs, which are typically enriched in eTregs, were also affected. Noteworthy, we observed that changes in intra-tumor and circulating Tregs after TRX-518 were positively correlated. As a potential mechanism responsible for this effect, our initial in vitro analyses indicate that GITR stimulation with TRX-518 destabilizes Treg phenotype by down-regulating Foxp3 and enhancing expression of the prototype Th1-lineage transcription factor T-bet. This suggests that TRX-518 may favor Treg to effector-T-cell conversion, thus explaining the effect of Treg loss observed in patients. Conclusions: Circulating Treg reduction is a potential pharmacodynamic biomarker of TRX518 biological activity. This parameter may allow predictive correlation with changes in intratumoral Treg infiltration. We plan to further investigate this effect and its relevance for the association with clinical responses in our recently opened TRX518 multi-dose study. Clinical trial information: NCT01239134. Citation Format: Roberta Zappasodi, Yanyun Li, Mohsen Abu-Akeel, Jingjing Qi, Philip Wong, Cynthia Sirard, Michael Postow, David A. Schaer, Walter Newman, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd D. Wolchok, Taha Merghoub. Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT018. doi:10.1158/1538-7445.AM2017-CT018

Abstract B09: Heterogeneous fates of metastatic lesions linked to immune escape in an ovarian cancer patient

The effect of chemotherapy on tumor heterogeneity and activation of the immune system in cancer is poorly understood. We present an unusual case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited spontaneous regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. The primary tumor was resected before chemotherapy treatment started. After 5 years of chemotherapy without response, treatment was stopped, the patient transitioned to best supportive care and was followed clinically with regular CA125 biomarker evaluation. During the course of 2 years of clinical follow up, CA125 decreased and computerized tomography (CT) scans revealed one regressing (right upper quadrant), one stable (liver), and two progressing (spleen and vaginal cuff) metastases. Due to abdominal discomfort, metastases were simultaneously resected. We performed: a) CT scan-based radiomics to anlayze tumor heterogeneity, b) exome sequencing to identify somatic mutations, predict neoepitopes, and estimate neoepitope depletion, and c) immunofluorescence staining to analyze immune cell infiltration on the primary and four metastatic tumors. We found that chemotherapy increases tumor heterogeneity, and metastases that developed after multi-line chemotherapy have a higher somatic mutation rate, more neoepitopes, and mutations in immune recognition molecules compared to metastases that appeared earlier. The right upper quadrant regressing and the liver stable metastases were infiltrated by CD4+ and CD8+ T cells. The vaginal cuff progressing metastasis was characterized by immune cell exclusion, while the spleen progressing metastasis was infiltrated only by CD8+ T cells. Finally, we detected neoepitope depletion only in the right upper quadrant regressing metastasis, potentially indicating an active immunoediting process. These findings indicate that multiple distinct tumor immune microenvironments can co-exist within a single individual, evidencing potential clinical challenges for the appropriate application of immunotherapy and chemotherapy combinations to overcome tumor heterogeneity at the genetic and tumor-microenvironment levels. Citation Format: Alejandro Jimenez-Sanchez, Harini Veeraraghavan, Yanyun Li, Hebert Alberto Vargas, Michael B. Gill, Kay J. Park, Oliver Zivanovic, Jason Konner, Jacob Ricca, Dmitriy Zamarin, Carol Aghajanian, Jedd D. Wolchok, Taha Merghoub, Evis Sala, Alexandra Snyder, Martin L. Miller. Heterogeneous fates of metastatic lesions linked to immune escape in an ovarian cancer patient. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B09.