T Cells Expressing Anti-B-Cell Maturation Antigen (BCMA) Chimeric Antigen Receptors with Antigen Recognition Domains Made up of Only Single Human Heavy Chain Variable Domains Specifically Recognize Bcma and Eradicate Tumors in Mice

Multiple myeloma (MM) is an almost-always incurable malignancy of plasma cells. Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T-cell signaling domains. CAR T cells have been shown to have substantial activity against leukemia and lymphoma, which has generated great interest in developing CAR T-cell therapies for MM. CAR T cells targeting B-cell maturation antigen (BCMA) have been shown to have substantial activity against MM (Ali et al. Blood. 2016. 128(13). 1688-1700). Improvements in CAR T-cell therapies for MM are needed. One possible limitation of CAR T-cell therapies is generation of recipient anti-CAR immune responses. Recipient anti-CAR immune responses have been reported in patients on clinical trials of anti-CD19 CARs (Turtle et al. Journal of Clinical Investigation. 2016. 126 (6). 2123-3138). Recipient anti-CAR immune responses could potentially develop against murine antibody variable region domains that are included in the single-chain variable fragments (scFvs) of most CARs. In addition, recipient anti-CAR immune responses could develop against the artificial linker domain in the scFv or against junctions between different CAR components.

We designed CARs with antigen-recognition domains of single fully-human heavy-chain-only variable domains (FHVH). The FHVH CARs do not contain a light chain variable region domain or a linker. The fully-human variable region, lack of a linker, and the minimized number of junctions should all lead to reduced immunogenicity for the FHVH antigen-recognition domain compared with a standard scFv.

We constructed CARs with 4 different FHVH domains designated FHVH32, FHVH33, FHVH74, and FHVH93. We designed CARs with each of these FHVH domains along with a CD8-alpha hinge and transmembrane domain, either a CD28 or a 4-1BB costimulatory domain, and a CD3-zeta T-cell activation domain. DNA encoding these CARs was ligated into a gamma-retroviral vector, and primary human T cells were transduced with gamma-retroviruses encoding each of the CARs. We tested the T cells for CAR expression and a variety of T-cell functions. We assessed T-cell surface CAR expression by staining with phycoerythrin-labeled BCMA protein. T cells from 3 MM patients were transduced with genes encoding each of the CD28-containing CARs. The following mean percentages of T cells expressed each CAR after transduction: FHVH32-CD828Z (47.3%), FHVH33-CD828Z (49.6%), FHVH74-CD828Z (51.3%), and FHVH93-CD828Z (46.6%). The mean T-cell surface CAR expression of the 11D5-3-CD828Z CAR containing a standard murine scFv with a heavy chain variable domain and a light chain variable domain connected by a linker was 60.4%. Importantly, we found that each of the FHVH CARs specifically recognized BCMA in functional assays. Specific interferon gamma production by the CARs is shown in the Table. We also demonstrated BCMA-specific tumor-necrosis factor-alpha release, cytotoxicity, and proliferation by T cells expressing each of the 4 FHVH CARs containing CD28. We have previously reported clinical anti-myeloma responses with infusions of T cells expressing the 11D5-3-CD828Z CAR (Ali et al. Blood. 2016. 128(13). 1688-1700). T cells expressing FHVH CARs with CD28 domains, especially FHVH33-CD828Z and FHVH74-CD828Z, had BCMA-specific functions including cytokine release that were equal to or superior to those of T cells expressing 11D5-3-CD828Z (Table). We have demonstrated that human T cells expressing either FHVH33-CD828Z or FHVH74-CD828Z can eliminate established tumors of BCMA-expressing human RPMI8226 cells from immune-deficient mice.

We also assessed FHVH CARs with 4-1BB costimulatory domains. Of the 4-1BB CARs, FHVH33-CD8BBZ CAR had the highest percentage of T cells with surface CAR expression (mean 52.1%, n=3). T cells expressing FHVH33-CD8BBZ exhibited BCMA-specific cytokine-release, degranulation, cytotoxicity, and proliferation.

In summary, we have designed, constructed, and tested a novel type of CAR with an antigen-recognition domain made up of only single fully-human heavy-chain variable region domains. These CARs specifically recognized BCMA in vitro, and they eradicated tumors from mice. The simple, fully-human antigen-recognition domains in these CARs offer a potential advantage of decreased immunogenicity compared to other CAR designs.

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