Targeting Heterogeneous B Cell Tumors with Tri-Specific VHH CAR-T (LCAR-AIO): Enhanced Expansion and Persistence in Preclinical Models

Anti-CD19 CAR-T therapy has shown significant success in treating B cell lymphoma, but its effectiveness is limited to approximately half of the patients with B cell malignancies due to the reliance on a single CD19 antigen target. The FDA-approved therapies using the murine FMC63 scFv binder have seen relapse rates between 39% and 88%, possibly due to antigen mutations or downregulation, and the potential immunogenicity of the murine binder. To address these challenges, a new tri-specific VHH CAR-T therapy was developed, targeting not only CD19 but also CD20 and CD22, for patients who have relapsed after previous CAR-T treatments.

The methodology involved the creation of mono-, bi-, or tri-specific VHH CAR constructs in a lentiviral vector, designed to target CD19, CD20, and/or CD22. These constructs were tested in vitro for cytotoxicity against tumor lines with varying antigen expression. The study also examined the impact of different CAR structures and the combination of scFv, VH, or VHH on the CAR design. The hypothesis was that the tri-specific VHH CAR-T, LCAR-AIO, would effectively combat tumors with diverse antigen expression and hinder antigen evasion.

In vitro assays showed that the tri-specific VHH CAR-T cells exhibited dose-dependent cytotoxicity against tumor lines. LCAR-AIO demonstrated cytolytic activity comparable to or better than mono- or bi-specific VHH or scFv CAR-T cells. It was able to lyse cells over-expressing single targets such as CD19, CD20, or CD22, indicating that all three VHHs in LCAR-AIO were functional. LCAR-AIO also displayed higher lytic activity and IFN-γ production compared to anti-CD19 FMC63 scFv CAR-T against tumor lines in vitro.

In vivo studies using a NCG murine model with Raji tumor line showed that LCAR-AIO resulted in better T cell expansion, longer persistence, and more effective tumor elimination compared to mono-scFv CAR-T therapies. The tri-specific VHH CAR-T, LCAR-AIO, was especially effective in maintaining lytic activity and IFN-γ production against CD19-deficient tumor cells, suggesting its potential to prevent tumor escape due to CD19 loss.

The conclusion drawn from the preclinical data is that the tri-specific CD19xCD20xCD22 VHH CAR-T has the potential to target tumors that have lost CD19 expression, offering a new option for treating patients who have relapsed after initial CD19 CAR-T therapy. The development of this tri-specific CAR-T approach could also be significant for extending the application of CAR-T technology to solid cancers, where tumor heterogeneity is a significant hurdle.