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4514 Chimeric Antigen-Receptor (CAR)-Engineered Natural Killer (NK) Cells Targeting Chronic Myeloid Leukemia (CML) Blast CrisisClinically Relevant Abstract

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
AML, Acute Myeloid Malignancies, Biological therapies, Research, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, CML, Chronic Myeloid Malignancies, Diseases, Therapies, Myeloid Malignancies, Natural Killer (NK) Cell Therapies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Jusuf Imeri1*, Paul Marcoux1*, Matthias Huyghe1*, Christophe Desterke, PhD2,3*, Daianne Fantacini4*, Frank Griscelli, PharmD, PhD1,5*, Dimas T. Covas, MD, PhD6,7,8, Lucas Edouardo Botelho de Souza9,10*, Annelise Bennaceur Griscelli, MD, PhD1,11,12* and Ali Turhan, MD, PhD13

1Inserm U1310, Villejuif, France
2Faculty of Medicine, University Paris Saclay, Le Kremlin Bicetre, France
3Inserm U1310, Villejuif, FRA
4Ribeirao Preto School of Medicine, Blood Center, RibeirãO Preto, BRA
5Center for iPSC Therapies, CITHERA, Evry Genopole, France
6Biotechnology Cluster of Ribeirao Preto, Butantan Institute, Ribeirao Preto, Brazil
7Faculdade De Medicina De Ribeirao Preto -USP, Ribeirao Preto, Brazil
8Department of Clinical Medicine, Division of Hematology, School of Medicine of Ribeirão Preto - University of São Paulo (FMRP-USP), Ribeirão Preto, Brazil
9Ribeirão Preto/Ribeirão Preto School of Medicine/University of São Paulo - Ribeirão Preto/SP, Brazil., Blood Center, Ribeirao Preto, Brazil
10Biotechnology Nucleus of Ribeirão Preto, Butantan Institute, Ribeirao Preto, Brazil
11Center for iPSC Therapies, Evry Genopole, France
12University Paris Saclay-Division of Hematology, 94805, Villejuif, FRA
13CITHERA, Center for iPSC Therapies, Evry, France


The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab.

Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.106 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .106/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200).

Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly, in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20.

Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb’s scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Disclosures: No relevant conflicts of interest to declare.

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