Abstract LB030: A novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19
Authors: Kim Ki-Hyun , Patel Ruchi P. , Lee Yong Gu , Kim Soohwan , Choi Ji-Hyeon , Kim Sung-Min , Kim Gae-Baik , Lee Jong-Ho , Lee Hyun-Jong , Park Ji-Ho , Lee Guewha , Cui LeiGuang , Yoon Min , Kim Ki Hyun , Kim Soohyun , Hwang In-Sik , Lee Youngha , Ko Bong-Kook , Lee Jong-Seo , Chung Junho , Ruella Marco 
Published: July 2021

Abstract
CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+ B cell malignancies. All the FDA-approved CART19, including tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel use anti-CD19 single-chain variable fragments (scFv) that bind to the same CD19 epitope. This epitope is located at the membrane-distal portion of CD19. We aimed to develop an anti-CD19 CAR that binds to a membrane-proximal domain. To this goal, we screened a chicken immune scFv library against the CD19 extracellular domain. We selected an scFv clone (1218) that was not competing with FMC63 for CD19 binding in competition enzyme immunoassay and real-time interaction analysis. Subsequent mutagenesis assay by replacing several residues with monkey residues showed that, as compared to FMC63, the epitope of the 1218 scFv is localized in a membrane-proximal location at three-dimensional modeling (Teplyakov A., Proteins, 2018). The chicken 1218 scFv was humanized by CDR-grafting to human germline genes and backmutations (h1218 scFv) and was confirmed to be specific for CD19 in cell microarray assays and flow cytometry analysis. We, therefore, designed a CAR construct using the h1218 scFv, 41-BB costimulatory domain in a lentiviral backbone. CAR-T cells were successfully generated using the Myltenyi Prodigy platform. In vitro cytotoxicity and interferon release assays showed potent effector functions of h1218 CAR-T cells that compared positively with FMC63-based CART19. Our group recently described the occurrence of a B-cell leukemia relapse characterized by the expression of the CAR19 itself on the cell surface due to accidental transduction during manufacturing (Ruella M., Nat Med, 2018). Interestingly, h1218 but not FMC63 CART19 were able to recognize and kill FMC63 CAR19-expressing tumor cells. In vivo, h1218 CART19 cells led to complete tumor regressions in both Raji (CD19+ B-cell lymphoma) and Nalm-6 (B-cell leukemia) xenograft models. Currently, h1218 CART19 is under advanced preclinical development and will be tested in a phase I-II clinical trial for relapsed and refractory B-cell lymphoma patients.
Citation Format: Ki-Hyun Kim, Ruchi P. Patel, Yong Gu Lee, Soohwan Kim, Ji-Hyeon Choi, Sung-Min Kim, Gae-Baik Kim, Jong-Ho Lee, Hyun-Jong Lee, Ji-Ho Park, Guewha Lee, LeiGuang Cui, Min Yoon, Ki Hyun Kim, Soohyun Kim, In-Sik Hwang, Youngha Lee, Bong-Kook Ko, Jong-Seo Lee, Junho Chung, Marco Ruella. A novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB030.