Background/Purpose: CD19 single and BCMA-CD19 compound chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable activity against autoimmune disorders, although relapses with CD19 CAR are reported. However, such CARs rely on classical single-chain fragment variable (scFv) with light and heavy chains connected by a peptide linker. This linker may induce immunogenicity in humans. FDA approved CD19 CAR uses the murine derived scFv FMC63 clone which has demonstrated induction of neutralizing antibodies against the CAR after infusion, impairing the potential to re-dose the patient. Additionally, the scFv structure may exhibit instability when binding to the antigen, and result in T cell exhaustion. To address such limitations, this study aimed to produce a novel CAR construct utilizing camelid-derived nanobodies that target both B and plasma cells.

Nanobodies derived from the variable heavy-chain (VHH) of camelid antibodies, naturally lack the light chain structure, and thus, the use of immunogenic peptide linker is not required. Their smaller size and greater stability make them ideal for constructing more complex CARs. Furthermore, nanobodies exhibit a high homology with the human immunoglobulins. Unlike scFvs, nanobodies do not induce neutralizing antibodies. To date, this is the first investigation of the pre-clinical efficacy of BCMA-CD19 nanobody-based CAR T cells in vitro and in vivo.

Methods: Alpacas were immunized with human BCMA or CD19 protein. Peripheral blood mononuclear cell (PBMC) mRNA was isolated and processed into a VHH gene library, transformed into phage-competent bacteria to generate a bacteriophage library. This library was panned against BCMA or CD19 antigens to enrich for binding clones, identified by high-throughput ELISA and cellular binding by flow cytometry. VHH candidates with binding greater than two-fold above background were sequenced. Using Biolayer Interferometry (BLI), association and dissociation rates were determined. Selected nanobodies were used to create a novel construct, termed BCMA-CD19 nanobody compound CAR (ncCAR). Human T cells were transduced with ncCAR and evaluated for cytotoxic efficacy through co-culture with BCMA or CD19-positive cell line. ncCAR cell-mediated cytotoxicity was assessed via flow cytometry and cell viability assays. In vivo studies were conducted using NSG (NOD scid gamma) mouse models engrafted with target cells, followed by infusion of ncCAR.

Results: Single nano-CARs (nCAR) and ncCAR demonstrated potent and selective cytotoxicity against BCMA or CD19-positive cells in vitro. Co-culture assays revealed significant BCMA or CD19 positive cell lysis, indicating effective dual-antigen targeting. In mouse models, both nCAR and ncCAR showed substantial cytotoxic activity, with depletion of BCMA and CD19 positive cells and significant durable persistency.

Conclusion: nCAR and ncCAR represents a promising therapeutic strategy for patients with autoimmune diseases who relapse after CAR therapy. The novel construct may provide improved efficacy and potential to re-dose a CAR relapsed patient. Future clinical studies are needed to explore this possibility in treating autoimmune disorders.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/preclinical-evaluation-of-bcma-and-or-cd19-nanobody-based-single-or-compound-car-t-cells-targeting-b-and-plasma-cells-associated-with-autoimmune-disorders/