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2336 Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia

Program: Oral and Poster Abstracts
Session: 703. Adoptive Immunotherapy: Mechanisms and New Approaches: Poster II
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts
Sunday, December 6, 2020, 7:00 AM-3:30 PM

Shoji Saito, MD, PhD1,2, Aiko Hasegawa, Ph.D.1*, Mika Nagai1*, Yoichi Inada1,3*, Hirokazu Morokawa, M.D.1*, Ikumi Nakashima1*, Daisuke Morita, M.D., Ph.D.1,4*, Yuichiro Ide5*, Kazuyuki Matsuda, PhD6*, Haruko Tashiro, MD7*, Shigeki Yagyu, MD, PhD1,2,8*, Miyuki Tanaka, MD, PhD1* and Yozo Nakazawa, MD, PhD1*

1Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan
2Center for Advanced Research of Gene and Cell Therapy, Shinshu University, Matsumoto, Japan
3Department of Drug Discovery Science, Shinshu University, Matsumoto, Japan
4Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Japan
5Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, JPN
6Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan
7Department of Hematology/Oncology, Teikyo University School of Medicine, Tokyo, JPN
8Department of Pediatrics, Kyoto Prefectural Medical University, Kyoto, JPN

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%–80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells.

Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads.

Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal.

Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells.

Disclosures: Saito: Toshiba Corporation: Research Funding. Hasegawa: Toshiba Corporation: Research Funding. Inada: Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima: Toshiba Corporation: Research Funding. Yagyu: Toshiba Corporation: Research Funding. Nakazawa: Toshiba Corporation: Research Funding.

*signifies non-member of ASH