P2RX5-Directed CAR-T Cells for Genotype-Guided Treatment of Lymphoid and Plasma Cell Malignancies in Patients of African Ancestry
Authors: Ang Zhiwei , Annette Castro , Schimdt Carolin , Hayer Katharina , Hasanali Zainul S. , Torres-Diz Manuel , King Sainos Patricia , Kwok Christopher , Ji Kayla , Krohl Patrick , Fine Justyn , Sehgal Priyanka , Martinez Dan , Vogl Dan T. , Spangler Jamie B. , Allman David , Thomas-Tikhonenko Andrei 
Published: December 2024

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.