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3264 Fully Human Tandem CD22-CD19 CAR-T Cells with Superior Sensitivity to Low Antigen Density Derived by Optimization of Co-Stimulation and CAR Architecture

Program: Oral and Poster Abstracts
Session: 703. Adoptive Immunotherapy: Mechanisms and New Approaches: Poster III
Hematology Disease Topics & Pathways:
ALL, Leukemia, Biological, apheresis, Adult, Diseases, bioengineering, Therapies, CAR-Ts, cellular interactions, Pediatric, Biological Processes, white blood cells, Technology and Procedures, cell expansion, immune cells, Young Adult, immunotherapy, Cell Lineage, Xenograft models, Lymphoid Malignancies, Study Population, Clinically relevant, imaging, immune mechanism, flow cytometry
Monday, December 7, 2020, 7:00 AM-3:30 PM

Peirong Hu, PhD*, Ying Xiong, PhD*, Darong Wu, MS*, Zhongyu Zhu, PhD*, Boro Dropulic, PhD, MBA* and Dina Schneider, PhD

Lentigen Technology, Inc., a Miltenyi Biotec Company, Gaithersburg, MD

In the treatment of B cell leukemia, relapse due to antigen to loss or downregulation remains a major challenge. Tumor antigen escape may be mitigated by multi-targeting CAR T cells redirected to CD19 and CD22, and possessing a superior sensitivity to low-density antigens. Using lentiviral transduction of primary T cells, flow cytometry, cell-based assays, and xenograft mouse models, we systematically optimized 22-19 CAR architecture and co-stimulatory domains for best functionality.

Fully-human tandem 22-19 CARs with co-stimulatory domains derived from 4-1BB, CD28, ICOS, OX40 or CD27, and hinge and transmembrane domains derived from CD8, CD28, or OX40 were evaluated. The tandem targeting ScFv domain orientation 22-19 was selected based on greater expression and cytotoxicity vs 19-22. All CARs achieved high T cell expression (mean 50-90%), and efficient dose-dependent killing of RajiCD19+CD22+, 293TCD19+, 293TCD22+, but not 293TCD19-CD22- target cells, and elaborated IL-2, IFN-γ, and TNF-a in antigen-dependent manner. CARs' potency in vitro varied by co-stimulatory domain: 4-1BB< OX40, ICOS, CD27<CD28, 4-1BB-CD28. The 22-19 tandem CAR with hinge and transmembrane domains (H/TM) derived from CD8 (CD8/CD8) performed as well as the CAR with H/TM CD28/CD28 configuration, whereas configuration OX40/OX40 was superior to CD8/OX40.

In RajiCD19+CD22+ mouse xenografts, the potency of tumor rejection by the 2nd generation tandem 22-19 CARs was also dependent on co-stimulatory domain, ranking 4-1 BB<CD27, ICOS<OX40<CD28. Tandem 3rd generation CAR 22-19, combining CD28 and 4-1BB co-stimulation, achieved the greatest anti-tumor effect. In antigen-heterogeneous Raji xenografts of a mixture of CD19-CD22+, CD19+CD22-, and CD19+CD22+ clones, the 22-19 tandem CARs with CD28, OX40 or ICOS co-stimulation mediated rapid and complete tumor rejection, whereas the conventional single-targeting CAR19 or CAR22 enabled tumor progression due to antigen escape.

Low antigen density Raji clones were generated by CRISPR-Cas9-mediated disruption of both CD19 and CD22 expression, followed by lentiviral transduction to express a limited number of antigen molecules on the cell surface. When challenged with Raji CD22 low clone in vitro, 2nd generation tandem CARs with CD28 and ICOS co-stimulation, and the 3rd generation tandem CAR combining CD28 and 4-1BB co-stimulatory domains were more effective than 4-1BB-, CD27-, or OX40-containing tandem CARs. Against RajiCD19 low clone, CARs with CD27 and OX40 domains were more effective than CARs with 4-1BB, and CARs with ICOS or CD28 co-stimulation were the most potent.

In summary, the fully-human tandem 22-19 CARs incorporating ICOS and CD28 co-stimulatory domains mitigate tumor antigen escape, exhibit robust anti-tumor function in pre-clinical models, enable superior lysis of CD22low and CD19low tumor clones, and may help improve clinical outcomes.

Disclosures: Hu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Xiong: Lentigen, a Miltenyi Biotec Company: Current Employment. Wu: Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

*signifies non-member of ASH