Faculty Bibliography

Background Efficient ex-vivo generation of functional tumor-specific T cells with memory phenotype remains a significant hurdle for the broad application of adoptive cell transfer (ACT) protocols for the treatment of cancer. Genetically engineered T cells represent one option, but genetic manipulation of T cells presents significant challenges in terms of complexity and generation time. Here, we describe a novel nanoparticle-based approach for generating tumor- specific T cells at clinical grade and scale from the endogenous T cell repertoire using artificial antigen presenting cells (aAPC). Methods Our aAPC consist of a paramagnetic nanoparticle to which humanized HLA-A2-Ig dimer-molecules and anti-CD28 antibodies are covalently linked. aAPC are loaded with multiple HLA-A2 restricted peptides and used to magnetically enrich and expand tumor-specific CD8+ T cells. Using peptide loaded aAPC, a fully enclosed, semi- automated, GMP T cell expansion platform has been developed that consistently generates clinically relevant numbers of tumor-specific, central and effector memory CD8+ T cells in 14 days, providing an alternative to genetic manipulation of T cells. Results Starting from a healthy donor leukopak, CD8+ T cells were generated using an aAPC cocktail loaded with 5 HLA-A2 epitopes from AML tumor antigens WT1, PRAME and cyclin A1. On average (n>20), 1-2 x109 T cells were generated that were 90% memory T cells with about 50% central memory and 40% effector memory CD8+ T cells. AML-specific T cells were expanded 500 to >5000-fold from low frequency precursor populations. These T cells were fully functional, as demonstrated by intra-cellular cytokine analysis and tumor cell killing. The system was also used to generate Mart-1 specific T cells of the same quality from cryopreserved PBL from melanoma patients. Additional data analyzing the TCR repertoire of the expanded AML and melanoma-specific T cells will be presented. Conclusions AIM ACT is a novel nanoparticle-based T cell expansion platform for the rapid, streamlined generation of clinically-relevant numbers of tumor-specific, central and effector memory CD8+ T cells from donor and patient PBMC in 14 days. The results reported here describe a platform that will be used in a multi-institution phase I clinical trial of adoptive T cell transfer for the treatment of AML patients pre- and post-allogeneic hematopoietic stem cell transplant. The flexibility of the AIM ACT system, as demonstrated using both AML and melanoma antigens, shows the potential for clinical application in other heme and solid tumors. Additionally, the system can be used for targeting both known and neo-epitopes

Background: Approximately 40% of metastatic cutaneous melanoma (CM) patients do not respond to the current immunotherapy (IT) regimens, pointing to other, yet unknown factors conferring IT resistance. In addition, > 60% of patients from single-line or combined treatment (COMBO) regimens present severe immune related adverse events (irAEs). In this study we have developed a novel genomic approach interrogating expression quantitative trait loci (eQTLs) to explore weather germline genetic variation can serve as novel personalized determinant of immunotherapy response and toxicity.
Method(s): By interrogating the genome wide expression data and SNP array datasets of healthy twin cohort (MuTHER), we have identified 85 eQTLs most significantly associated with the expression of 265 immune genes. Using the MassARRAY system, the 85 SNPs were genotyped in 138 anti-CTLA-4 treated patients, 87 PD-1 treated patients, and 69 patients from combined (COMBO) treatments, collected from multi-institutional collaborations. To test the association of SNPs with IT response and irAEs, logistic regression analyses were performed for each treatment group adjusting by demographic and clinical covariates.
Result(s): We found significant associations with COMBO IT resistance for and eQTL in IL10/IL19 (OR = 4.249, p = 0.0167), which we have recently identified for association with melanoma survival and which, interestingly, is an established locus associated with the risk of several autoimmune diseases. Additionally, we also identified eQTLs that are associated with IT sensitivity; IL1-beta with resistance to anti-CTLA-4 and SPI1 with resistance to anti-PD-1. Interestingly, genomic scan of 85 eQTLs has identified novel loci predictive of severe autoimmunity and site specific irAEs in patients treated with COMBO or single-line anti- CTLA4 IT.
Conclusion(s): In this study, we report that eQTLs from IL19/IL10 locus, previously shown to predict autoimmunity risk and CM survival, is also a surrogate marker of response to COMBO IT, indicating a strong relationship between interleukin pathways and tumor immunogenicity. Novel loci have been found as predictive markers for autoimmune toxicity, in patients treated with COMBO and anti-CTLA4 IT. This is a first evidence that immunomodulatory pathways modulated by germline genetic variation can impact susceptibility to irAEs as well as IT efficacy. Currently, a large scan is underway using genome-wide genetic screens to further test the functional validity of these findings in a large collaborative setting

Immune-checkpoint blockade (ICB), in particular PD-1 inhibition, has rapidly changed the treatment landscape and altered therapeutic paradigms across many tumor types, with unprecedented rates of durable clinical responses in a number of cancers. Despite this success, only a subset of patients responds to ICB and, as a result, predictive biomarkers would be useful to guide the selection of patients for these therapies. This article highlights currently used biomarkers, as well as several promising novel candidates, and also discusses the challenges involved in establishing their analytic validity and clinical utility. Progress is being evaluated in melanoma and non-small cell lung cancer, for which PD-1 ± CTLA-4 inhibitors have become standard therapy, to other malignancies for which PD-L1 inhibitors remain investigational. Although single biomarkers have substantial limitations, a combination of biomarkers that reflect the interaction of host and tumor will likely be needed to provide a reproducible surrogate for the benefit of checkpoint modulation. Cancer Immunol Res; 6(10); 1122-8. ©2018 AACR.

In this perspective, we identify emerging frontiers in clinical and basic research of melanocyte biology and its associated biomedical disciplines. We describe challenges and opportunities in clinical and basic research of normal and diseased melanocytes that impact current approaches to research in melanoma and the dermatological sciences. We focus on four themes: (1) clinical melanoma research, (2) basic melanoma research, (3) clinical dermatology, and (4) basic pigment cell research, with the goal of outlining current highlights, challenges, and frontiers associated with pigmentation and melanocyte biology. Significantly, this document encapsulates important advances in melanocyte and melanoma research including emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, epidemiology, pigment biophysics and chemistry, and evolution.

PURPOSE/OBJECTIVE:PD-1 blockade induces durable responses in patients with metastatic melanoma and prolongs relapse-free survival in patients with resected melanoma; however, current biomarkers do not consistently associate with patient responses. In this study we investigated the impact of nivolumab therapy on peripheral blood Tregs and its relation to patient outcomes. EXPERIMENTAL DESIGN/METHODS:Peripheral blood Tregs and conventional CD4+ T-cells from patients with resected high-risk melanoma treated with adjuvant nivolumab were assessed for gene expression changes by RNA-seq. Percentages of circulating Tregs and phosphorylated-STAT3 (pSTAT3) expression levels were assessed by flow cytometry and validated in an independent cohort of active disease patients. Suppressive function of Tregs was assessed in allogeneic mixed lymphocyte reactions. RESULTS:Tregs from non-relapse patients had increased expression of proliferation associated genes . An increase in the proportion of circulating Tregs and pSTAT3 expression and a reduction in Treg suppressive capacity were observed in non-relapsing, but not relapsing patient samples 13 weeks after starting treatment. In vitro blockade of PD-1 increased Treg percentages and pSTAT3 expression, and reduced Treg suppressive function. PD-1 blockade also led to IL-10 production by T-cells, resulting in higher Treg proliferation. The addition of a STAT3 inhibitor ameliorated the increase in Tregs, enhanced suppressive function, and decreased T-cell IL-10 production in vitro. CONCLUSIONS:These results demonstrate that induction of pSTAT3, reduced suppressive function, and a paradoxical increase in Treg proliferation are novel correlates of patient benefit from PD-1 blockade.

Combination anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1) therapy promotes antitumor immunity and provides superior benefit to patients with advanced-stage melanoma compared with either therapy alone. T cell immunity requires recognition of antigens in the context of major histocompatibility complex (MHC) class I and class II proteins by CD8⁺ and CD4⁺ T cells, respectively. We examined MHC class I and class II protein expression on tumor cells from previously untreated melanoma patients and correlated the results with transcriptional and genomic analyses and with clinical response to anti-CTLA-4, anti-PD-1, or combination therapy. Most (>50% of cells) or complete loss of melanoma MHC class I membrane expression was observed in 78 of 181 cases (43%), was associated with transcriptional repression of HLA-A, HLA-B, HLA-C, and B2M, and predicted primary resistance to anti-CTLA-4, but not anti-PD-1, therapy. Melanoma MHC class II membrane expression on >1% cells was observed in 55 of 181 cases (30%), was associated with interferon-γ (IFN-γ) and IFN-γ-mediated gene signatures, and predicted response to anti-PD-1, but not anti-CTLA-4, therapy. We conclude that primary response to anti-CTLA-4 requires robust melanoma MHC class I expression. In contrast, primary response to anti-PD-1 is associated with preexisting IFN-γ-mediated immune activation that includes tumor-specific MHC class II expression and components of innate immunity when MHC class I is compromised. The benefits of combined checkpoint blockade may be attributable, in part, to distinct requirements for melanoma-specific antigen presentation to initiate antitumor immunity.