Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

Hongxia Wang,Dan Jiang,Yanting Zhang,Liyan Wang,Yanning Li,Guangqi Li,Yuankui Chu,Liyuan Liu,Xiaochun Zhang,Guangxian Xu

doi:10.1186/s12935-021-02151-z

Abstract

BackgroundTaking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells.MethodsCD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function.ResultsThe nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%.ConclusionsNanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Highlights

Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nbbased chimeric antigen receptor T cells (Nb Nb-based Chimeric antigen receptor T cells (CAR-T)) against lymphoma cells
Recent studies have shown that nanobody-based CAR-T cells exert obvious antitumour effects [21,22,23]. nanobodies, known as the variable domain of the heavy chain of heavy chain antibody (VHH), were first found in dromedaries by Hamers Castermans in 1993 and identified in Camelidae and sharks. nanobodies belong to the variable region of the heavy chain antibodies (HcAbs), which only contain variable regions of heavy chain and CH2, and CH3 but are devoid of light chain and CH1 [24, 25]
Surface plasmon resonance (SPR) revealed that the dissociation rate constant (KD) of CD19 was 0.29 μM and that of CD20 was 0.147 μM, indicating the high binding ability to CD19 and CD20 (Fig. 1H, I)

Summary

Introduction

Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nbbased chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. Cancer immunotherapy has shown excellent clinical therapeutic effects against many cancers, CAR-T cell therapy is one of the most promising immunotherapy approaches [1,2,3,4,5,6]. Most sequences with identity to the human VH gene family III result in weak immunogenicity, applying Nb as part of the antibody recognition of CAR-T cells may be safer than mAbs derived from mice [27]. Nbs have been applied in antibody-drug conjugates owing to their small molecular weight (15 kDa), stability and strong penetrating power [29,30,31]. Nanobodies show promising therapeutic applications due to their favourable characteristics [32,33,34,35,36]

Methods

Results

Discussion

Conclusion