Language
English
Publication Date
11-1-2023
Journal
Molecular Therapy
DOI
10.1016/j.ymthe.2023.09.008
PMID
37705245
PMCID
PMC10638030
PubMedCentral® Posted Date
9-13-2023
PubMedCentral® Full Text Version
Post-print
Abstract
Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.
Keywords
Humans, Receptors, Chimeric Antigen, Protein Domains, Receptors, Antigen, T-Cell, T-Lymphocytes, Neoplasms, Immunoglobulin G, Immunotherapy, Adoptive, Antigens, CD19, cancer immunotherapy, cell therapy, protein engineering, chimeric antigen receptors
Published Open-Access
yes
Recommended Citation
Rios, Xavier; Pardias, Osmay; Morales, Marc A; et al., "Refining Chimeric Antigen Receptors via Barcoded Protein Domain Combination Pooled Screening" (2023). Faculty, Staff and Students Publications. 8247.
https://digitalcommons.library.tmc.edu/baylor_docs/8247