Identifying new immunotherapy targets for pediatric leukemia using cell surface proteome profiling.

Overview: Immunotherapies that direct cytotoxic immune cells to tumor targets, such as CAR-Ts, have shown tremendous clinical success for certain B-cell malignancies(1). However, effective CAR-T strategies for pediatric T cell lymphoblastic leukemia/lymphoma (T-LBLL) are limited by the lack of tumor-specific antigens targeting malignant progenitor T cells while preserving healthy mature T cells(2). Here we apply cell-surface proteomic profiling of T-LBLL patient samples, Patient-Derived Xenografts (PDXs), cell lines and healthy-donor mature T cells to identify new tumor-specific antigens for T-LBLL CAR-T therapy. Furthermore, we perform method optimization to improve plasma-membrane protein enrichment in surfaceome profiling.

Methods: Sialylated cell surface glycoproteins were labeled with biotin followed by streptavidin enrichment and on-bead digestion(3). Samples were analyzed by LC-MS using DIA acquisition on an Exploris-480. Flow cytometry was used to isolate progenitor T cell populations in healthy thymocytes for validating expression profiles of candidate immunotherapy targets.

Results: Direct surface proteome profiling of pediatric patient samples resulted in the quantification of approx. 1500 proteins per patient, with strong enrichment for plasma membrane proteins (Fig. 1A-D). Patient samples displayed both distinct and overlapping profiles with T-LBLL PDX and cell-line models. From our identification list, 233 putative T-LBLL surface protein immune therapy targets were selected based on their conserved enrichment across patient samples and absence in the surface profiles of healthy-donor mature T cells (Fig. 1E). We further filtered our list to exclude proteins with high RNA expression profiles in healthy tissues and identified 80 candidate immune therapy targets. We validated the expression of one putative T-TBLL antigen for CAR-T therapy, CD1b, that displays higher expression across patient samples (Fig. 1F) and greater specificity to immature T-cells than the current leading T-TBLL targets in clinical trials(4-6) (Fig. 1G). Additionally, by implementing enrichment steps for cell viability prior to surface protein labeling, we significantly improve plasma membrane protein enrichment in our surfaceome profiling.

Conclusions: MS-based surfaceome proteomic profiling is a powerful technique to elucidate new targets for cell therapy. By improving surface proteome enrichment in our optimized workflow, we can confidently identify more surface proteins with higher sensitivity and specificity. Our data elucidates an exciting new potential CAR-T target for T-TBLL treatment, addressing an unmet clinical need in pediatric oncology.

References:

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