Background/Purpose: Autologous CD19 chimeric antigen receptor (CAR) T cell therapies have recently shown to be well tolerated and highly effective in patients with autoimmune diseases (AID) including systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis. However, autologous CAR T cell therapies have major limitations such as complex logistics and manufacturing constraints that may hinder widespread access in these large patient populations. These challenges may be overcome with off-the-shelf allogeneic CAR T cell products derived from healthy donor T cells. Although allogeneic CAR T cells can provide immediate availability to patients and scalable manufacturing, they are more susceptible to rejection by the host immune system and therefore may have reduced persistence, limiting clinical responses. To address this challenge, we developed an anti-rejection CD70 CAR capable of selectively depleting activated (CD70⁺) alloreactive lymphocytes. This CD70 CAR may render allogeneic CD19 CAR T cells resistant to allorejection while endowing dual targeting of CD19⁺ B cells and CD70⁺ T cells. A CD19/CD70 dual CAR T cell therapy may therefore reduce or eliminate the need for lymphodepleting conditioning regimens that are typically required prior to CAR T cell infusion. Furthermore, because CD70 itself has been shown to be upregulated on T cells in AID, this approach could provide additional benefit to patients through elimination of CD70⁺ autoreactive T cells.

Methods: CRISPR gene-editing technology (ABR-001) combined with adeno-associated virus (AAV) transduction was employed to knock-in a bicistronic, CD19/CD70 dual CAR construct into the T Cell Receptor Alpha Constant (TRAC) locus. Following expansion, dual CAR T cells were characterized by flow cytometry analysis of CAR expression as well as markers of T cell activation and differentiation. Cytotoxicity and expansion of the dual CAR T cells, referred to as ALLO-329, were assessed in vitro and in vivo using B cell lymphoma models and humanized mouse models. The susceptibility of ALLO-329 to rejection by HLA-mismatched T cells was assessed in mixed lymphocyte reaction (MLR) assays.

Results: ALLO-329 exhibited cytotoxic activity and target-mediated expansion in the presence of tumor cell lines expressing CD19 and/or CD70. Primary CD19⁺ B cells and CD70⁺ T cells from healthy donors and patients with SLE were rapidly eliminated by ALLO-329 in vitro and in vivo, both in mouse peripheral blood and spleen, resulting in a concomitant reduction of antibody production. In in vitro MLR assays, ALLO-329 eliminated CD70⁺ alloreactive T cells and demonstrated resistance to rejection and longer persistence relative to CD19 CAR T cells, which were rapidly depleted.

Conclusion: ALLO-329 can be successfully produced via ABR-001-mediated site-specific integration of a single bicistronic construct in the TRAC locus, successfully yielding high numbers of CAR⁺ cells that show specific cytotoxic activity, rejection avoidance, and the ability to eradicate B cells in mouse tissues. This novel off-the-shelf allogeneic CD70/CD19 CAR T cell product is a promising candidate for clinical evaluation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/preclinical-evaluation-of-allo-329-allogeneic-cd19-car-t-cells-expressing-an-anti-rejection-cd70-car-for-the-treatment-of-autoimmune-diseases/