{
  "abstract": "Background CD19-targeted CAR T cell therapy has demonstrated high initial response rates (80-90%) for relapsed or refractory (R/R) pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, long-term event-free survival (EFS) remains limited (40-50%), and many patients require consolidative hematopoietic stem cell transplant following CAR T cell therapy to maintain durable responses. 1 We aim to improve long-term EFS for R/R pediatric B-ALL by employing CD19CAR T cell therapy in combination with maintenance therapy (MT) consisting of mercaptopurine (6-MP) and methotrexate (MTX). Evidence suggests promising clinical outcomes from the use of 6-MP/MTX MT following loss of CD19CAR T cell persistence,2 but administration of 6-MP/MTX MT in combination with CAR T cell therapy is currently hindered by the immunosuppressive nature of these agents.3 Our lab has previously developed MTX-resistant CAR T cells that have demonstrated safety in the clinic.4–6 Here, we report on the development of a novel molecular system to confer 6-MP resistance in CD19CAR T cells.Methods We designed multiple mutant gene constructs of the human purine biosynthesis enzyme phosphoribosyl pyrophosphate synthetase I (PRPS1) based on point mutations identified in cohorts of relapsed pediatric ALL patients. 7 Single, double, and triple variants were introduced at residues within the allosteric site or dimer interface of the enzyme. Mutant PRPS1 (mPRPS1) constructs were cloned into a self-inactivating lentiviral vector (LVV) containing an mCherry reporter for initial screening. Human CD4/CD8 T lymphocytes were transduced with wild-type or mPRPS1-encoded LVV, exposed to 6-MP, and evaluated for resistance. We subsequently selected several top-performing PRPS1 variants to incorporate into our clinical CD19CAR LVV (FMC63scFv-41BB) for further evaluation.Results Expression of engineered mPRPS1 constructs in human CD4/CD8 T lymphocytes conferred resistance to 6-MP at concentrations relevant to therapeutic plasma levels (up to 2µM). 8 9 Exposure to 6-MP resulted in efficient selection of mPRPS1+ T cells, achieving 85-95% purity after 3-5 drug treatments. Phenotypic analysis by flow cytometry demonstrated that mPRPS1 did not alter the intrinsic T cell phenotype with respect to CD4/CD8 ratio, differentiation state, or expression of activation/exhaustion markers CD69 and PD1. Importantly, mPRPS1 variants conferred some protection against 6-MP-induced phenotypic alterations.Conclusions We developed a mutant PRPS1-based system that confers 6-MP resistance in CD19CAR T cells without altering T cell phenotype, supporting its potential as a clinically translatable platform to enable a combination immunotherapy/chemotherapy approach in pediatric B-ALL. Functionality and safety assessments are ongoing.References Gardner RA, Finney O, Annesley C, et al. Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood. 2017;129(25):3322–3331.Gabelli M, Oporto-Espuelas M, Burridge S, et al. Maintenance therapy for early loss of B-cell aplasia after anti-CD19 CAR T-cell therapy. Blood Adv. 2024;8(8):1959–1963.Dervieux T, Brenner TL, Hon YY, et al. De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo. Blood. 2002; 100(4):1240–1247.Jonnalagadda M, Brown CE, Chang WC, et al. Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase. Gene Ther. 2013; 20(8):853-860.Vitanza NA, Ronsley R, Choe M, et al. Intracerebroventricular B7-H3-targeting CAR T cells for diffuse intrinsic pontine glioma: a phase 1 trial. Nat Med. 2015;31:861–868.Vitanza NA, Wilson AL, Huang W, et al. Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety. Cancer Discov. 2023;13(1):114–131.Li B, Li H, Bai Y, et al. Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL. Nat Med. 2015;21:563–571.Zimm S, Collins JM, Riccardi R, et al. Variable Bioavailability of Oral Mercaptopurine: Is Maintenance Chemotherapy in Acute Lymphoblastic Leukemia Being Optimally Delivered? N Engl J Med. 1983;308:1005–1009.Lennard L, Keen D, Lilleyman JS. Oral 6-mercaptopurine in childhood leukemia: parent drug pharmacokinetics and active metabolite concentrations. Clin Pharmacol Ther. 1986;40(3):287–292.",
  "authors": [
    {
      "affiliations": [
        "Seattle Children’s Therapeutics, Seattle, WA, USA"
      ],
      "name": "Tavie Parker"
    },
    {
      "affiliations": [
        "Seattle Children’s Therapeutics, Seattle, WA, USA"
      ],
      "name": "Finn Watt"
    },
    {
      "affiliations": [
        "Seattle Children’s Therapeutics, Seattle, WA, USA"
      ],
      "name": "Kamila Gwiazda"
    },
    {
      "affiliations": [
        "BrainChild Bio, Seattle, WA, USA"
      ],
      "name": "Michael Fitzgerald"
    },
    {
      "affiliations": [
        "BrainChild Bio, Seattle, WA, USA"
      ],
      "name": "Michael CV Jensen"
    },
    {
      "affiliations": [
        "Seattle Children’s Therapeutics, Seattle, WA, USA",
        "Seattle Children’s Research Institute, Seattle, WA, USA",
        "Seattle Children’s Hospital, Seattle, WA, USA",
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Corinne Summers"
    }
  ],
  "title": "263 Engineering mercaptopurine-resistant CD19CAR T cells with a mutant phosphoribosyl pyrophosphate synthetase I (PRPS1)",
  "uid": "937d67db-8327-5e2d-a670-14e74244c921"
}