P2RX5-Directed CAR-T Cells for Genotype-Guided Treatment of Lymphoid and Plasma Cell Malignancies in Patients of African Ancestry

The use of chimeric antigen receptors (CARs) for cancer immunotherapy is limited by the paucity of lineage- and tumor-restricted surface antigens. While lymphoid and plasma cell neoplasms can be treated with CD19- and BCMA-directed CAR-T cells, respectively, there is currently no universal marker for these developmentally related malignancies. By mining publicly available bulk and single-cell RNA-sequencing data, we discovered that most B-cell lymphomas, multiple myelomas, and a subset of T-cell acute lymphoblastic leukemias express high levels of purinergic receptor 5 (P2RX5) mRNA. In contrast, almost no P2RX5 expression was detected in normal tissues aside from mature B-cells. This B lineage-restricted expression could make P2RX5 an attractive second-line immunotherapy target for patients that have relapsed after CD19- and BCMA-directed therapies.

In principle, as an ATP-gated cell-surface ion channel, P2RX5 should be readily accessible to CAR-T cells. However, due to the two deleterious single nucleotide polymorphisms rs3215407 (nonsense mutation in exon 3) and rs891746 (splice site mutation in intron 10)­, most people of European, Asian, and Latin American descent express allelic variants of P2RX5 that are truncated and short-lived, to the point of being undetectable. The majority of people of African descent, by contrast, express the full-length ancestral variant, dubbed P2RX5-FL. Using lymphoma and myeloma lines derived from patients of African ancestry, we detected robust cell-surface expression of endogenous P2RX5-FL via live-cell biotinylation and protease treatment assays and in flow cytometry experiments following FLAG-tagging of the endogenous P2RX5 loci. Furthermore, using site-direct mutagenesis and treatment with protein deglycosylases, we identified two N-glycosylation sites within P2RX5-FL. This was consistent with the surface localization P2RX5-FL as N-glycans are acquired in the endoplasmic reticulum en route to the plasma membrane.

To further credential P2RX5-FL as a CAR-T cell target, we generated one camelid nanobody and a series of mouse monoclonal antibodies (mAb) against this isoform. Two noncompeting mAbs, 1D9 and 2F4, stained endogenously expressed P2RX5-FL on the surface of live human lymphoma and myeloma cell lines, in a manner that was not dependent on N-glycosylation and showed no cross-reactivity with other ubiquitously expressed members of the P2RX family. P2RX5 expression levels remained stable after treatment with common chemo- and targeted therapy agents (vincristine, doxorubicin, acalabrutinib, duvelisib, ruxolutinib, lenalidomide). It was similarly unaffected by CRISPR-Cas9 mediated knockout of CD19, CD20, CD79B, CD38 and BCMA; nor were expression levels of these B-cell markers affected by P2RX5 knockout. Additionally, uniform staining of neoplastic cells by both mAbs was observed in primary samples obtained from African-American patients with T- and B-lymphoblastic leukemia/lymphomas and multiple myeloma. This suggested that P2RX5-directed immunotherapies could be effective against relapsed/refractory disease and prior immunotherapy escape variants, and that its use as a first-line immunotherapy would not preclude targeting of the already established antigens.

Finally, we tested a series of P2RX5-directed second-generation CARs based on the single-chain variable fragments (scFv) of 1D9 and 2F4. After optimizing their architecture, the 1D9 and 2F4 CARs displayed potent cytolytic activity against several P2RX5-FL-positive cell lines, including but not limited to Raji (endemic Burkitt lymphoma) and MM.1s (multiple myeloma), but not Ramos (sporadic Burkitt lymphoma arising in a Caucasian patient). Notably, relative to BCMA-directed 11D5-based CARs which are in clinical use, the most potent P2RX5-directed CARs displayed greater cytotoxic activity against MM.1s cells. This cytolytic activity was also retained against CD19- and BCMA-knockout cells, justifying the use of P2RX5-directed CARs the immunotherapy relapse setting.

In summary, we have credentialled P2RX5 as a novel mature B-/plasma cell marker and a promising CAR-T cell target for the genotype-guided treatment of lymphoma and multiple myeloma patients of African ancestry. If effective in clinical studies, P2RX5-directed CAR-T-cells could reduce the pervasive inferiority in treatment outcomes affecting many patients of African ancestry.