Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells

Abstract

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

Early induction of cytokine release syndrome by rapidly generated CAR T cells in a preclinical mouse model

“…Only upon further cultivation and activation CAR becomes cell-surface expressed and vector particles dissapear, demonstrating that transduction is not fully completed in stCAR T cells. Previous studies did not track the presence of vector components, while CAR transferred as protein from packaging cells to transduced cells has been studied and detected, 7, 17, 18 which however, did not mediate killing when reverse transcription and genome integration of the CAR gene was inhibited. 7 Using an anti-VSV-G antibody our study allowed a more accurate characterization of stCAR T cells with respect to residual vector particles.…”

Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models

“…Only after further cultivation and activation of the cells, more CAR is expressed on the cell surface, while vector particles disappear, demonstrating that transduction is not fully completed in stCAR T cells. Previous studies have not tracked the presence of vector particle components, whereas CAR transferred as a protein from packaging cells to transduced cells has been detected and studied (Ghassemi et al, 2022 ; Cordes et al, 2021 ; Jamali et al, 2019 ). However, CAR protein transfer alone could not mediate killing (Ghassemi et al, 2022 ).…”

Current updates on generations, approvals, and clinical trials of CAR T-cell therapy

“…The desired gene encoding CAR is genetically transduced into the T-cells’ genome using integrating viral vectors, transposons, or mRNA transfection. 99 , 100 T-cell-enriched apheresis products are cultured in a sterile bioreactor in the presence of CAR encoding vectors to deliver the desired genes and cell-based artificial antigen-presenting cells (aAPCs). Besides, genes can be transferred using non-viral transduction methods such as nanoparticles, liposomes, electroporation, or CRISPR/Cas9 technology.…”