Tri-Specific CD19xCD20xCD22 VHH CAR-T Cells (LCAR-AIO) Eradicate Antigen-Heterogeneous B Cell Tumors, Enhance Expansion, and Prolong Persistence in Preclinical In Vivo Models
Authors: Zhou Zhe (joy) , Han Yue , Pan Hong-Bo , Sang Cai-Jun , Shi Dong-Lin , Feng Chong , Xiao Hui , Zhuang Qiu-Chuan , Wang Ping-Yan , Fan Xiao-Hu (Frank) 
Published: November 2021

Abstract
Introduction: Anti-CD19 CAR-T therapy has achieved remarkable treatment efficacy in B cell lymphoma. However, targeting CD19 antigen alone can only benefit about half of patients with B cell malignancies. The FDA-approved CD19 CAR-T therapies all use same binder, which is murine FMC63 scFv targeting CD19 and up to 39%-88% of patients have relapsed. Possible mechanisms of relapse include mutations or downregulation of the targeted antigen, CD19, however, the targetable expression of CD20 and CD22 is preserved. In addition, immunogenicity against murine FMC63 scFv could have a negative impact on possible re-dosing regimen. To overcome these limitations, we designed and developed a novel tri-specific VHH CAR-T, targeting three antigens that include CD19, CD20 and CD22, for treating patients who relapsed from prior CAR-T therapies.
Methods: We engineered mono-, bi-, or tri-specific VHH CAR constructs targeting CD19, CD20 and/or CD22 respectively in a lentiviral vector. The mono-, bi- or tri-specific CAR-T cells were tested against tumor lines expressing single, dual or triple antigens in an in vitro cytotoxicity assay. In addition, we evaluated the contribution of different CAR backbones, and possible combinations of scFv, VH or VHH to CAR design. We hypothesized that our lead tri-specific VHH CAR-T, LCAR-AIO, would potently inhibit tumors with heterogeneous Ag expression and prevent Ag escape. To validate this, we compared in vitro cytolytic activity and cytokine production of LCAR-AIO CAR-T to anti-CD19 FMC63 CAR-T against CD19 +CD20 +CD22 + Raji.Luc and CD19KOCD20 +CD22 +Raji.Luc cells . In vivo treatment efficacy and CAR-T persistence were also investigated in NCG murine model xenografted with Raji tumor line. 0.3x10 6 CAR +T cells or dose-matched untransduced T cells were given to NCG mice four days post i.v. implantation of Raji.Luc tumor cells. Tumor growth was monitored weekly by bioluminescence imaging until achieved endpoint (55 days), and CAR-T persistence was determined using genomic DNA level.
Results: Tri-specific VHH CAR-T cells can mediate dose-dependent cytotoxicity against Raji tumor lines. Compared to mono- or bi-specific VHH or scFv CAR-T, tri-specific VHH CAR-T demonstrated equal or better cytolytic activity. Our lead tri-VHH CAR-T, LCAR-AIO, was able to specifically lyse K652 over-expressing single target such as CD19, CD20 or CD22, at the similar level to mono-specific CD19, CD20 or CD22 VHH CAR-T. Since no blocking effect of recognition against these three antigens was observed, our result suggested that all three VHHs in LCAR-AIO are functional. In comparison to anti-CD19 FMC63 scFv CAR-T, LCAR-AIO exhibited higher lytic activity and IFN-γ production against Raji.Luc tumor lines in vitro. In addition, LCAR-AIO retained its robust lytic activity and IFN-γ production when co-cultured with CD19KO-Raji.Luc cells while anti-CD19 FMC63 scFv CAR-T could not, suggesting LCAR-AIO may prevent tumor escape due to loss of CD19. Furthermore, comparison of LCAR-AIO to mono-scFv CAR-T (anti-CD19 FMC63-BBz, anti-CD20 Leu16-BBz or anti-CD22 m971-BBz) was performed in NCG mice xenografted with Raji cell line, LCAR-AIO exhibited better T cell expansion, longer persistence, and superior efficacy in eliminating tumors.
Conclusions: Based on in vitro and in vivo preclinical data, tri-specific CD19xCD20xCD22 VHH CAR-T can be effective targeting tumors lack of CD19 expression, therefore, it has the potential of treating relapsed patients with prior CD19 CAR-T therapy. The feasibility of making tri-specific CAR-T would help to extend this technology to solid cancers where heterogeneity poses a major challenge at current stage.
Figure 1 Figure 1.

Disclosures
Zhou: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Han: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Pan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Sang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Shi: Legend Biotech: Current Employment. Feng: Legend Biotech: Current Employment. Xiao: Legend Biotech: Current Employment. Zhuang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Wang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Fan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company.