Author ORCID Identifier

https://orcid.org/

0000-0002-0115-711X

Date of Graduation

5-2022

Document Type

Thesis (MS)

Program Affiliation

Immunology

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Sattva S Neelapu

Committee Member

Richard Eric Davis

Committee Member

Laura Bover

Committee Member

Greg Lizee

Committee Member

Ryan Sun

Abstract

Despite high clinical responses, about 30%-60% of patients with B-cell malignancies relapse following CD19-targeting chimeric antigen receptor (CAR) T cell therapy. CD19 loss is a major cause of relapse and resistance after CD19 CAR T therapy. Dual antigen targeting could overcome resistance due to antigen escape and improve outcomes after CAR T cell therapy. We hypothesized that dual CD19-CD79b CAR T cells will be effective against B cell malignancies by targeting one or both antigens. I have engineered dual CAR targeting CD19 and CD79b antigens under the control of EF1α promoter in a 3rd generation lentiviral vector. The dual CD19-CD79b CAR was composed of CD19 CAR with 4-1BB co-stimulatory domain and CD79b CAR with OX-40 linked by T2A self-cleavable peptide. High transduction efficiency of dual CD19-CD79b CAR was achieved and stable dual CAR expression was observed in both CD4+ and CD8+ T cell subsets. My method of generation of dual CAR T cells attained the optimal CD4+:CD8+ ratio of 1:1 on day 9 after transduction. I demonstrated that both CD19 CAR and CD79b CAR are functional in the dual CAR construct by exhibiting degranulation, proliferation and robust cytotoxic activity against B cell lymphoma and leukemia cell lines expressing either CD19 or CD79b or both. The expression of two distinct CAR molecules in the same cell did not adversely affect T-cell differentiation during the CAR T generation as the phenotype and expression of various inhibitory receptors were similar to untransduced and CD19 CAR-transduced T cells. In summary, dual targeting of CD19 and CD79b could provide a feasible strategy to minimize antigen escape and improve v the efficacy of CAR T treatment for patients with relapsed or refractory B-cell non-Hodgkin lymphoma, hairy cell leukemia and chronic lymphocytic leukemia.

Keywords

dual CD19-CD79b CAR, bicistronic CAR, EF1α promoter, lentiviral transduction, CAR T cell degranulation, CAR T cell cytotoxicity, CAR T cell proliferation

Available for download on Thursday, April 20, 2023

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