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871 Development of a First-in-Class CAR-T Therapy Against Calreticulin-Mutant Neoplasms and Evaluation in the Relevant Human Tissue Environment

Program: Oral and Poster Abstracts
Type: Oral
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: New Insights into The Biology of Myeloproliferative Syndromes
Hematology Disease Topics & Pathways:
Research, Clinical trials, Translational Research, Clinical Research, Real-world evidence
Monday, December 9, 2024: 2:45 PM

Alexandros Rampotas, MBBS, MRCP1*, Zoë Wong, BSc2*, Isaac Gannon1*, Camelia Benlabiod, PhD2*, Yuqi Shen2*, Charlotte Brierley2*, Aude-Anais Olijnik3*, Saif Khan1*, Nawshad Hayder, MSc, BSc2*, Gordon Weng-Kit Cheung1*, Marina Mitsikakou, MRes4*, Manuel Rodriguez-Justo5*, Abdullah O Khan2*, Adam J Mead, PhD, MRCP, FRCPath6, Jonathan Lambert, PhD, BSc, BMBS, FRCP, FRCPath7*, Claire Roddie, MD8*, Bethan Psaila, MD, PhD9 and Martin A Pule, MD10*

1UCL Cancer Institute, University College London, London, United Kingdom
2MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
3MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
4Haematology Department, UCL Cancer Institute, London, United Kingdom
5Research Department of Pathology, UCL Cancer Institute, London, United Kingdom
6Haematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
7Department of Haematology, University College Hospital, University College of London Hospitals NHS Foundation Trust, London, United Kingdom
8University College London Cancer Institute, London, United Kingdom
9Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
10Research Department of Haematology, University College London Cancer Institute, London, United Kingdom

Introduction

Myeloproliferative neoplasms (MPNs) are characterized by a TGFb-rich, fibrotic tumor microenvironment (TME), posing a challenge for targeted immunotherapies. Current treatments are largely palliative and do not selectively target MPN clones. In 1/3 of patients, MPNs are driven by mutant calreticulin (mutCALR), that binds to the thrombopoietin receptor (TpoR) causing activation and prominent display of the cancer neoantigen on the surface of TpoR-expressing cells stem/progenitors (HSPCs) and megakaryocytes. Bispecific and inhibitory antibodies targeting mutCALR recently entered clinical trials. Here, we present pre-clinical validation of a pioneering chimeric antigen receptor T-cell (CAR-T) therapy targeting mutCALR, including efficacy in relevant clinical settings.

Methods

Killing efficacy and target cell TpoR expression was assessed by flow cytometry. To evaluate CAR-T in the relevant TME, we used scRNAseq to analyze iPSC-derived human bone marrow (BM) organoids co-engrafted with CAR-T, patient-derived CD34+ HSPCs +/- immunoregulatory factors abundant in the myelofibrotic niche (TGFβ and Galectin-1,Gal1).

Results:

The novel CAR-T demonstrated robust and highly selective eradication of low- and high- mutCALR expressing human cell lines in vitro, and CAR-T administration led to a dramatic reduction in leukemic burden and improved survival in NSG mice xenografted with mutCALR+ TpoR+ AML cells. Excellent BM infiltration of CAR-T, and near-complete ablation of leukaemia confirmed remarkable efficacy of CAR-T against mutCALR+ malignancies in vivo.

CAR-T efficacy was tested on CD34+ HSPCs from MPN patients (n=12), including myelofibrosis, ET, and accelerated/blast phase MPN (AP/BP-MPN) with both ins5 and del52 variants (n=8) in 2D and 3D cultures. Excellent depletion of HSPCs (40->90%) and extreme specificity was observed, with minimal killing of JAK2V617F+ samples (n=4).

Killing of AP/BP-MPN HSPCs was notably lower than chronic phase samples (myelofibrosis/ET), and correlated with lower surface TpoR expression. We hypothesized that treatment with TpoR agonists might increase TpoR expression on AP/BP-MPN HSPCs, by promoting megakaryocytic differentiation. Serial doses of rhTPO and TpoR agonists were tested. Pre-treatment of cells with Eltrombopag, but not with Romiplostim or rhTPO, increased surface TpoR expression by >50% and improved CAR-T killing, confirming that pharmacologically enhancing TpoR expression might boost vulnerability to anti-mutCALR immunotherapies.

As expected, CAR-T killing efficacy was substantially diminished by prolonged exposure to JAK inhibitors at clinically-relevant concentrations, with a substantial drop in cytotoxicity and CAR-T proliferation. However, 24hr pre-incubation of CAR-T with peak plasma equivalent doses of JAK inhibitors prior to exposure to target cells led only to a smaller reduction in killing, ranging from <10-40%. Less potent JAK1 inhibitors demonstrating only a minor drop in efficacy, suggesting that selected JAKi agents/dosing regiments or short washout periods may be possible for synergistic co-therapy with CAR-T treatment

Human organoids were used to evaluate CAR-T efficacy in the relevant TME. A scRNAseq dataset of 161,970 cells was generated from ‘healthy’ and ‘myelofibrotic’ (TGFb/Gal1 rich) organoids co-engrafted with CD34+ myelofibrosis HSPCs and CAR-Ts. CAR-Ts exposed to mutCALR+ primary cells had a 2-fold expansion of CD8+ effector memory cells. The proportion of immunosuppressive, regulatory T cells (Tregs) increased in a high TGFβ environment, but not with Gal1. GSEA showed significant enrichment in pathways associated with IFNγ, TNFa and IL-2 in CAR-Ts exposed to mutCALR+ samples. Ongoing analyses will evaluate vulnerability of specific patient HSPC/lineage subsets to CAR-T, the ability of healthy (non-mutant) haematopoiesis to reconstitute after treatment, and the impact of CAR-T killing on the BM niche.

Conclusions:

Here we present a novel CAR-T therapy showing potent and remarkably selective targeting of mutCALR-driven malignancies. This study also evaluates the impact of JAKi on CAR-T efficacy and presents the first use of human organoids to evaluate immunotherapies in the relevant human tissue environment, including features of the TME, validating this as a powerful platform for pre-clinical development of targeted therapies across blood cancers.

Disclosures: Mead: Incyte: Consultancy, Honoraria; Alethiomics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Research Funding; Galecto: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Medscape: Honoraria; Ionis: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Roche: Research Funding. Lambert: Novartis: Honoraria; Kite-Gilead: Consultancy, Honoraria; Blueprint: Consultancy; Takeda: Honoraria. Roddie: Autolus, BMS, Gilead, Janssen: Consultancy; Autolus, BMS, Gilead: Honoraria, Speakers Bureau. Psaila: University of Oxford: Patents & Royalties: 2203947.3; Alethiomics: Consultancy, Current equity holder in private company, Research Funding; Blueprint Medicines: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Research Funding. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company; Autolus: Other: Entitled to royalty payments from related intellectual property.

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