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3282 Establishing Immunocompetent Leukemia Models to Investigate the Impact of CAR T Cells on the Immune Microenvironment and Bone Marrow Niche

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Gene Therapy, Immunotherapy, immunology, Therapies, Biological Processes
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Sarah E. Moore, BS1,2, Jaquelyn T. Zoine, PhD2, Jeremy Chase Crawford, PhD3*, Deanna Langfitt, PhD2*, Juan M. Barajas, PhD4, Sherif Abdelhamed, PhD4, Ilaria Iacobucci, PhD4, Dalia Haydar, PhD2*, Giedre Krenciute, PhD2*, Charles G. Mullighan, MBBS, MD4, Jeffery M. Klco, MD, PhD4, Stephen Gottschalk, MD2, Paul G. Thomas, PhD3* and Mireya Paulina Velasquez, MD2

1St. Jude Graduate School of Biomedical Sciences, Memphis, TN
2Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN
3Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN
4Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN

Background

Chimeric antigen receptor (CAR) T cells need to overcome limited expansion and persistence within the highly immunosuppressive tumor microenvironment (TME) to successfully eliminate acute myeloid leukemia (AML) in the clinic. CD123 has been identified as a promising target for AML because of its high expression on leukemia stem cells and AML blasts, with a clear increased differential expression when compared to healthy hematopoietic progenitors (HPCs). To date, preclinical evaluation of CD123-CAR T cells have used human xenograft models, which lack a functional immune system. This cross-species evaluation of CAR T cells inadequately addresses interactions between CAR T cells and the immune microenvironment, a limitation of many CAR T cell therapy studies. We have generated immunocompetent murine leukemia models to evaluate murine CD123 (mCD123)-specific CAR T cells and thus investigate the impact of the AML TME on CAR T cell dysfunction.

Methods and Results

We used biopanning of a murine phage display library on recombinant mCD123 to select a panel of mCD123-specific single chain variable fragments (scFvs). 4 scFvs, encoding ‘top binders’, were subcloned into a MSGV1 retroviral vector encoding a CD28.z-CAR to generate 4 mCD123-CARs. Second-generation mCD123-CAR T cells were generated by retroviral transduction of activated splenocytes. Only one mCD123-CAR construct was expressed on the cell surface as judged by flow cytometric analysis using F(ab’)2 (55.8±11.5% N=7) or recombinant mCD123 (59.0±12.1% N=6) for detection, and T cells expressing this CAR were selected for further analysis.

To test the antigen specificity of mCD123-CAR T cells, we expressed full-length mCD123 on 2 murine leukemia cell lines: C1498, a spontaneous myelomonocytic AML cell line, and a Arf-null, BCR-ABL1 preB-ALL. In addition, we analyzed a panel of virally induced NUP98::KDM5A AML generated by retroviral transduction of murine HPCs and determined all samples naturally expressed mCD123 antigen at different densities (range 38-77% N=4). We co-cultured mCD123-CAR in the presence of mCD123+ leukemia cell lines, with mB7-H3-CAR, SP6-CAR, or non-transduced T cells serving as controls. mCD123-CAR T cells recognized CD123+ targets, as evidenced by significantly increased cytokine secretion and cytolytic activity in comparison to controls (N=6, p<0.01). Antitumor activity was maintained in repeat stimulation assays for up to 5 stimulations.

To determine in vivo toxicity of mCD123-CAR T cells, non-tumor bearing C57BL/6 mice were given 200 mg/kg cyclophosphamide (Cy) and one effector T cell infusion generated from transgenic luciferase mice (N=5) and monitored by bioluminescence imaging for 10 days. Bone marrow and spleen were harvested 10 days after injection for flow cytometry evaluation and colony formation unit (CFU) readouts. We detected no significant differences in toxicities between mCD123-CAR and control T cells. We next evaluated the anti-leukemia activity of mCD123-CAR T cells. C57BL/6 mice were injected with preB-ALLArf-/CD123+ cells on day 0 and received 100 mg/kg of Cy on day 2. On day 4, they were injected with 10x106 mCD123-CAR T cells. Mice receiving tumor only, tumor + Cy, or SP6-CAR served as controls. mCD123-CAR T cells had significant anti-leukemia activity resulting in a prolonged survival advantage in comparison to controls (N=5 per group, p<0.001). We focused on delineating the TME in our established models and preliminary analysis of comprehensive flow cytometry and single cell transcriptomic data on bone marrow. Our findings revealed that the TME of our established immune competent leukemia CAR T cell therapy models recapitulate human disease, having an increase in dying tumor cells within the bone marrow of mice treated with mCD123-CAR, with a number of other populations (e.g. dendritic cells, regulatory T cells) approximating the tumor-only microenvironment profile.

Conclusions

We have developed functional mCD123-CAR T cells targeting AML and ALL and have established immunocompetent models to test them. mCD123-CAR T cells demonstrated potent anti-leukemic activity in vitro and have anti-leukemia activity with minimal to no toxicity in vivo. These findings will provide insight into the impact of the AML TME on CAR T cell functionality, and inform future strategies to improve CAR T cells in the immunosuppressive TME.

Disclosures: Zoine: St. Jude Children's Research Hospital: Patents & Royalties. Crawford: St. Jude Children's Research Hospital: Patents & Royalties. Iacobucci: Mission Bio: Honoraria. Gottschalk: Data and Safety Monitoring Board of Immatics: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Sanofi: Honoraria; TESSA Therapeutics: Honoraria; St. Jude Children's Research Hospital: Patents & Royalties; Tidal: Honoraria; Catamaran Bio: Honoraria. Thomas: Cytoagents: Membership on an entity's Board of Directors or advisory committees; Immunoscape: Membership on an entity's Board of Directors or advisory committees; PACTBio: Honoraria; JNJ: Honoraria; Illumina: Honoraria; 10X: Honoraria; Elevate Bio: Honoraria.

*signifies non-member of ASH