Single VHH-directed BCMA CAR-T cells cause remission of relapsed/refractory multiple myeloma

Abstract

Chimeric antigen receptor T (CAR-T) cells have shown remarkable effects in treating hematological malignancies [1, 2]. Typically, the antigen recognition domain of CAR-T cells is a single-chain variable fragment (scFv) linked to a costimulatory domain and a cytoplasmic activation domain, such as CD28, 4-1BB, and CD3ζ [3, 4]. The scFvs are composed of a heavy-chain variable fragment connected to a light-chain variable fragment by a flexible linker optimized to preserve the pairing of heavyand light-chain variable regions. These are usually derived from a fulllength mouse immunoglobulin and can lead to human antimouse immune response. This immunogenicity can lead to adverse events and loss of efficacy during CAR therapy [5, 6]. In addition, scFvs do not always fold efficiently and can be prone to aggregation [7, 8]. As an alternative to scFvs, nanobody may serve as suitable antigen recognition domains in CAR-T cells. Nanobody (also called variable domain of heavy chain of heavy-chain antibody, VHH), is the variable fragment of heavy-chain antibodies of Camelidae. The heavy-chain antibodies are composed of only two heavy chains, with no light chain, but have the function of conventional antibodies. The VHH is the small, stable, single domain structure with high affinity and specificity comparable to those of scFvs [9, 10] and is easy to be humanized for therapeutic purposes [11]. In recent years, the CAR-T cells targeting B cell maturation antigen (BCMA) for treating multiple myeloma (MM) have shown dramatic effect in clinical trials [12–14]. Among these targeted therapies, the LCAR-B38M CAR-T is the most noteworthy. Unlike most CAR-T cells designed for targeting one epitope of antigen, the LCAR-B38M targets two epitopes of BCMA using two tandem VHH sequences. However, whether single VHH targeting one epitope has similar potential in CAR-T therapy further needs to be explored. We immunized an alpaca with the BCMA-Fc fusion protein. Following the protocol (Supplementary Fig. 1A), we got one VHH with a high-binding affinity of 1.1 nM for BCMA, as determined using the Octet RED system (data not shown). After further humanization, we constructed the humanized VHH-human IgG1 Fc fusion plasmid and expressed it in 293T cells. This protein recognized the BCMA overexpressed on K562 cells, and the expression level was comparable to the level detected using a commercial anti-BCMA antibody (55.0% vs. 44.2%, Supplementary Fig. 1B). We performed the membrane protein panel screening and found that the VHH-Fc specifically recognized BCMA (TNFRSF17) (Supplementary Fig. 1C). These results showed that the VHH we screened has a high affinity and specificity for BCMA targeting. We constructed the CAR gene using the humanized VHH sequence linked with CD8α extracellular, transmembrane These authors contributed equally: Lu Han, Ji-Shuai Zhang, Quan-Li Gao, Yong-Ping Song