Approximately 30-60% of relapses after CD19 CAR T therapy for B-acute lymphoblastic leukemia (B-ALL) occur with loss of CD19 expression (CD19-), often with persistent CAR T. Strategies under investigation to reduce this risk include co-targeting of CD22. Modulation of CD19 and CD22 expression and changes in the leukemia transcriptome upon exposure to CD19 CAR T and in the milieu of CAR T proliferation and activation are not well explored and are important to understanding emergence of CD19 resistance.
CAR T were generated with T cells isolated from human peripheral blood mononuclear cells (PBMC) transduced with lentiviral vector encoding anti-CD19 scFv (FMC63, BPS Bioscience) and expanded in the presence of IL-2 and irradiated CD19+ feeder cells as previously described (Prommersberger, et al., 2020). Transduction efficiency was 15-20% measured by FMC63 expression on CD3+ T cells by flow cytometry. Four-hour cytotoxicity assay confirmed effective dose dependent CD19 specific killing. Leukemia cell lines expressing CD19 and CD22 (Nalm6, Raji, RS4:11) were used for in vitro coculturing experiments treated with either CD19 CAR T or untransduced T cells at an 1:1 ratio for 4, 24, and 96 hours. Surface antigen expression were assessed using flow cytometry, gating on viable leukemia cells and compared to untreated cells. Cocultured samples with unlabeled Raji and Nalm6 were FACs sorted to separate the CD19- and CD19+ compartments, gating on CD3-CD10+ cells. RNA isolated from sorted CD19-, CD19+, and untreated Raji cells in 3 biological replicates were used for bulk sequencing on the Ilumina HiSeq 4000 instrument. Sequencing and data processing were completed by the Northwestern Sequencing Core. Normalized data was used to generate principal component analysis (PCA) and heatmap visualizing the hierarchical clustering. Three pairwise comparisons (CD19- vs CD19+, CD19- vs untreated, CD19+ vs untreated) were used to generate lists of differentially expressed genes (DEG). Genes were considered significant when the Benjamini-Hochberg corrected adjusted p-value <0.05. Gene set enrichment analysis (GSEA) performed on a ranked list of all DEG is ongoing.
We first aimed to assess changes to CD19 expression after exposure to CD19 CAR T. Leukemia cells of all 3 cell lines, cultured with CAR T, yielded dramatic reduction in the proportion of live leukemia cells expressing CD19 after 4 hours. After 96 hours, less than 10-20% of the few remaining leukemia cells expressed CD19. Notably, an off target decrease of CD22 was observed after 24 hours for Nalm6 and Raji and 96 hours for RS4:11. CD20, another highly expressed B cell antigen in Raji cells, was similarly decreased at 24 hours, suggesting a more general change to the B cell phenotype. We next sought to determine if the observed changes were permanent. When sorted CD19- Nalm6 cells were re-incubated in the absence of CAR T, re-expression of CD19 and CD22 occurred after 24 hours to levels equivalent to untreated Nalm6. Bulk RNA sequencing was used to determine if the reduction in B-cell antigen expression were reflective of a more global reprogramming. The PCA demonstrated discrete clustering of samples into CD19-, CD19+, and untreated groups with the first 2 principal components explaining 83% and 9% of the total variance in the data. Hierarchical clustering of the top 500 most variable genes unsurprisingly showed CD19+ and untreated Raji cells were more closely related in gene expression compared to the CD19- group. The comparison we were most interested in was between the CD19- and CD19+ groups. We used a Venn diagram to determine the set of significant genes that overlapped with the CD19- vs untreated comparison. This resulted 2921upregulated and 2724 downregulated genes that were differentially expressed in CD19- cells. Further work is ongoing to complete pathway analysis of the narrowed list of overlapping significant genes as well as GSEA on the ranked list of all DEG in the CD19- v CD19+ comparison.
Conclusion
We found rapid and early CD19 modulation after exposure to CAR T followed by reduction in other B cell antigens. The reversibility of these changes when the immune pressure is removed suggests that the distinct transcriptional profile we found in CD19- cells may not yet be permanent. We are currently investigating gene expression pathways that are important to this reprogramming.
No relevant conflicts of interest to declare.
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