Background Chimeric antigen receptor T cells (CAR-T) therapy has been applied in the treatment of B cell lymphoma; however, CAR-T manufacturing requires virus or non-virus based genetic modification which causes high manufacturing cost and potential safety concerns. The novel antibody cell conjugation (ACC) technology has the advantage to direct innate immune cells, such as γδ T and NK cells, to find and erase cancer cells by linking cancer-targeting antibodies on cell surface proteins without genetic modification. In this study, ACC technology was applied to generate a novel off-the-shelf CD20-targeting γδ T cell therapy ACE1831, and the potential mechanism of ACC-mediated cytotoxicity was elucidated.
Methods PBMC-derived γδ T cells were expanded from healthy donors. DNA linker-1 and linker-2 were conjugated to γδ T cells and Rituximab, respectively. Linker-1 conjugated Rituximab was attached to linker-2 conjugated γδ T cells by DNA hybridization to generate off-the-shelf CD20-targeting γδ T cell product, ACE1831. In vitro and in vivo potency of ACE1831 against CD20-expressing cancer cells was evaluated in B cell lymphoma models. Mass spectrometry analysis was performed to identify Rituximab-linked surface proteins of γδ T cells. Jurkat T cells expressing a luciferase reporter driven by an NFAT-response element were used to examine the mechanism of ACC-mediated T cell activation.
Results ACE1831 exhibited CD20-specific binding activity and viability after recovery from cryopreservation. In vitro cytotoxicity analysis demonstrated the superior potency of ACE1831 against B cell lymphoma including Raji, Rituximab-resistant Raji and Daudi cells, while no significant off-target toxicity against CD20-negative cells or PBMCs was observed. The enhanced secretion of IFNγ and TNFα has been detected when ACE1831 encountered Raji cells, whereas IL-6 remained undetectable. Furthermore, ACE1831 showed excellent in vivo potency in B cell lymphoma xenograft model, and ACE1831-treated mice remained cancer-free to the end of the study. By mass spectrometry analysis, Rituximab-linked proteins were identified, and the protein functions were mainly related to cell activation and immune synapse. Additionally, NFAT signaling was significantly activated when Rituximab-linked Jurkat cells encountered CD20-expressing cancer cells. Blocking TCRγδ activation partially inhibited ACE1831-mediated cytotoxicity, indicating both TCRγδ and external ACC-linked Rituximab involved in ACE1831 potency against B cell lymphoma.
Conclusions This study provides the evidence for the efficacy and safety of ACE1831 to support the clinical application against CD20-expressing tumors and elucidates the potential mechanism of ACC-mediated activation and cytotoxicity. ACE1831 will be evaluated in clinical trials for relapsed/refractory B cell lymphomas.
Ethics Approval SCID-Beige mice were purchased and housed under the regulation of the Institutional Animal Care and Use Committee (IACUC) of the contract research organizations.
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