Development of an Off-the-Shelf Chimeric Antigen Receptor (CAR)-T Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL) without Gene Editing

CAR-T cell therapy has revolutionized the treatment of B-lineage ALL and other B-cell malignancies, but the development of cell therapies for T-cell malignancies has lagged behind. For T-ALL, CD7 is an excellent CAR target because it is consistently and highly expressed in leukemic cells and is retained during chemotherapy and at relapse. However, CD7 is also expressed in normal T-cells and the expression of anti-CD7 CARs triggers T-cell fratricide, resulting in a marked decrease in viable and/or effective CAR-T cells. We developed an anti-CD7 CAR-T cell product (PCART7) by utilizing a CD7 protein expression blocker (PEBL) to downregulate CD7 surface expression and prevent fratricide. PEBLs consist of a target binding domain fused to an intracellular retention domain that enables relocalization of surface proteins intracellularly (Png et al. Blood Adv 2017; Kamiya et al. Blood Adv 2018; Kamiya et al., JCI 2019). Autologous PCART7 cells lacking surface CD7 and expressing anti-CD7 CARs can be effectively produced under cGMP conditions and the technology is being tested in clinical trials for patients with relapsed/refractory T-ALL or T-cell lymphoblastic lymphoma.

Generation of autologous CAR-T cells, however, may be hampered by the presence of a high percentage of leukemic cells in peripheral blood and/or low T cell numbers. An allogeneic anti-CD7 CAR-T cell product available "off-the shelf" would represent a useful tool to reduce leukemic burden and provide a bridge to hematopoietic cell transplant or to autologous CAR-T cell therapy. Because allogeneic T-cells can be activated upon recognition of host tissue antigens by their T-cell receptor (TCR)/CD3 complex, their infusion carries a risk of causing graft-versus-host disease (GvHD).

We optimized a CD3 PEBL that can effectively downregulate surface TCR/CD3. We then developed a lentiviral-based process that allows simultaneous expression of CD3 and CD7 PEBLs together with anti-CD7 CAR. The PEBLs retained surface CD7 and TCR/CD3 intracellularly, thereby minimizing fratricide and abrogating TCR-mediated antigen recognition. PCART7 cells lacking TCR/CD3 were manufactured from healthy donor T-cells by using a double transduction with a bicistronic lentiviral vector delivering CD7 PEBL and anti-CD7 CAR (PCART7 LVV), and a second lentiviral vector delivering CD3 PEBL. Robust expansion of PCART7-CD3 PEBL cells was observed and more than 90% of the cells were of the desired CAR+CD7-CD3- phenotype. Following depletion of residual CD3/TCRαβ+ cells, CD3- cell purity was >99% (Fig. 1A). These results were reproduced across multiple donors. The efficient downregulation of surface TCR/CD3 by CD3 PEBL was similar to that achieved by CRISPR knockout of the TCR alpha chain locus (TRAC) and completely abrogated activation in response to TCR stimulation. In vitro short- and long-term assays confirmed strong cytotoxic potency against CD7+ leukemic cells but not CD7- target cells. Importantly, PCART7 cells were equally cytotoxic regardless of whether they were co-transduced with CD3 PEBL. In a xenograft model using the CCRF-CEM T-ALL cell line, PCART7-CD3 PEBL cells, like PCART7 cells, effectively suppressed tumor growth and improved survival (Fig. 1B).

This study demonstrates the feasibility of using two PEBLs simultaneously for intracellular protein retention without perturbing critical CAR-T cell functions. The use of PEBL technology to generate allogeneic anti-CD7 CAR-T cells fits seamlessly in cGMP manufacturing protocols and avoids the risk of genotoxicity arising from the use of gene-editing techniques. Because the resulting allogeneic anti-CD7 CAR-T cells had high potency and were devoid of alloreactivity potential, they represent an attractive new tool for off-the-shelf CAR-T cell therapy of T-ALL and other CD7+ malignancies.

Wong:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Ng:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Zheng:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Ismail:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Qian:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campana:MediSix Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties; Nkarta Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Juno Therapeutics (Bristol-Myers-Squibb): Patents & Royalties; Smart Immune: Membership on an entity's Board of Directors or advisory committees. Tan:MediSix Therapeutics: Current Employment, Current holder of stock options in a privately-held company.