Identification of Novel Immune-Checkpoint Molecules on Multiple Myeloma Cells Using a High-Throughput Discovery Platform

Introduction:

Multiple myeloma (MM) is a B-cell malignancy, characterized by accumulation of plasma cell clones in the bone marrow. While novel therapeutic agents like immunomodulatory drugs and proteasome inhibitors have improved overall survival of MM patients, the disease remains incurable in most patients. Several studies showed that immune-checkpoint molecules are expressed by myeloma cells and induce tumor-related immune suppression. Despite the promising results achieved by blocking CTLA4 and the PD-1/PD-L1 axis in the treatment of various solid tumors and Hodgkin's lymphoma, targeting these checkpoints did not induce objective responses in Phase I/II trials in MM patients. Therefore, identification of novel immune-checkpoints and defining the subsequent molecular mechanisms of inhibition are essential for further improvement.

Methods:

Our main goal is to identify novel MM-related immune-checkpoint molecules by taking advantage of a high-throughput (HT) RNAi screen and sequentially validate the role of candidate molecules, whose blockade could potentially induce anti-tumor immunity in MM patients.

Methods:

High-throughput RNAi screens offer a possibility to systemically search for immune-checkpoint molecules. Therefore, we established a high-throughput screening system to discern candidate molecules and evaluate their use as potential targets for multiple myeloma immunotherapy. We established a luciferase based read-out system by generating a stable luciferase expressing MM cell line (KMM-1-luc). To test the effect of immune-checkpoint molecules, KMM-1-luc cells were transfected with a siRNAs library targeting 2514 genes encoding for cell surface proteins, kinases and GPCRs. Transfected tumor cells were subsequently co-cultured with patient-derived HLA-matched Myeloma Infiltrating T Lymphocytes (MILs) and the effect of gene knock-down on T-cell mediated tumor lysis was measured.

Results:

Based on our primary HT-screening, we have identified 132 candidate molecules (hits) whose knockdown increased T-cell mediated killing more efficiently than the established checkpoint genes CCR9. To confirm the hits and the robustness of the screening, we re-tested the identified candidates in a secondary screening. Among these potential immune-checkpoints we selected 10 hits for further validation. So far, we were able to confirm expression of the hits at mRNA level and to validate siRNAs on-target effect by qPCR and luciferase-based cytotoxicity assay. Detailed results will be presented at the meeting.

Conclusion:

Altogether we optimized a high-throughput RNAi screen to discover novel immune-checkpoints that are potential immunotherapeutic targets for the treatment of multiple myeloma. We are currently investigating the mode of action of the candidate hits in vitro. Further in vivo validation of these immune-checkpoint molecules is still required for clinical studies.