Identification of Small Molecule Enhancers of Immunotherapy for Melanoma

Christopher Dextras,Suman Vodnala,Ajit Jadhav,Zhiya Yu,Nicholas P Restifo,Janani Sundaresan,Juan J Marugan,Bryan Chim,Anju Singh,Marc Ferrer,Myagmarjav Dashnyam,Nicolas Acquavella,Noel Southall,Chyi-Chia Richard Lee,Lesley A Mathews Griner,Xin Hu

doi:10.1038/s41598-020-62369-1

Abstract

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

Highlights

Melanoma remains the most frequent cause of skin cancer related death in young Caucasian adults[1]
Using a highly immunogenic epitope that can be recognized by a high percentage of melanoma reactive tumor infiltrating lymphocytes[24], we have developed a 1536 well compatible co-culture cell-based assay system to identify compounds that can be used synergistically to further enable T cell recognition of melanoma tumor cells
Our screening assay is based on the principle of immune recognition of melanoma cells by antigen-specific CD8+ T cells

Summary

Introduction

Melanoma remains the most frequent cause of skin cancer related death in young Caucasian adults[1]. The cells are infused back into the patient following a preparative lymphodepletion regimen This process can produce profound and even ‘curative’ antitumor responses that can be durable due to lasting memory of the adaptive immune system. The absence of cell-based assay models for drug screening and immune recognition has hampered research in this area Towards this goal, we developed a novel cell-based high throughput screening (HTS) assay to identify compounds that would enhance immune recognition in a BRAF mutant melanoma cell model. We developed a novel cell-based high throughput screening (HTS) assay to identify compounds that would enhance immune recognition in a BRAF mutant melanoma cell model This in vitro two cell assay system measures interferon gamma (IFNγ) as a quantifiable readout which is released after the co-culture of antigen specific CD8+ T-cells with melanoma cells. Four lead compounds were identified which upon confirmation in ACT animal models could serve as a potential adjuvant therapy to ACT treatment

Methods

Results

Conclusion