Minimizing Tumor Antigen Escape by Triple CAR T Cells Simultaneously Targeting CD19 CD20 & CD22

Authors

• K Highfill
• L Chen
• M Igboko
• E Cherkasova
• RM Nadal Rios
• D Allan
• K Carney
• M Chakraborty
• R Reger
• W Murphy
• R Childs

Abstract

Chimeric antigen receptors (CARs) are composed of a single-chain variable fragment of a monoclonal antibody with the costimulatory domain and CD3 zeta. While CD19-CAR therapy has been successful in many patients, there remains a high relapse rate, with up to 25% of patients experiencing CD19 antigen-negative relapses. To address this, CARs targeting multiple antigens (CD19/CD20 and CD19/CD22) have been developed, showing improvements but still suboptimal results. We propose that triple CAR T cells simultaneously targeting CD19, CD20, and CD22 antigens will minimize tumor antigen escape.
We designed a triple CAR consisting of a tandem CAR chain targeting CD19 and CD20, linked to a mono-CD22 CAR chain. The NALM6 B cell line was chosen to generate various CD19, CD20, and CD22 (single, double and triple) knockout combinations, to illustrate the hierarchy and dynamics of tumor antigen escape to CD19, CD20, CD22 triple CAR T cell treatment. We developed a triple sgRNAs targeting strategy to disrupt target regions and validated this protocol by knocking out HLA-A2 in T2 cells. Three days following electroporation, we observed significantly reduced expression of HLA-A2. We plan to run additional optimization experiments with CD19, CD20 and CD22 in the NALM6 cell line.
We plan to transduce T cells with lentivirus coding for the CD19/CD20/CD22 triple CAR and a control CD19 CAR. The function of the CAR T cells will be evaluated in in-vitro co-culture experiments of NALM6 with CD19, CD20, CD22 single, double or triple knockout clones and subsequently in NSG xenograft mouse models.

Scientific Focus Area: Immunology

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