Bispecific CD19-CD20 and CD19-CD22 CAR-T Cells with Glycogen Synthase Kinase (GSK)-3β Inhibitor TWS119 Treatment Have Superior Therapeutic Effects on Mantle Cell Lymphoma
Authors: Jin Jingling , Li Yijing , Liu Yang , Jordan Alexa A , McIntosh Joseph , Vargas Jovanny , Che Yuxuan , Yao Yixin , Wang Michael 
Published: November 2021

Abstract
Introduction: Chimeric antigen receptor (CAR) - expressing T cells have shown breakthrough clinical successes to hematological malignancies such as diffuse large B cell lymphoma. Intensive development on CAR-T cell therapies are being developed in many common lymphomas. However, the development of CAR-T cell therapies in mantle cell lymphoma (MCL), a rare lymphoma has lagged behind. So far only CD19 CAR-T has been applied to MCL treatment, and MCL frequent relapse from prior CD19 CAR-T therapy with CD19 antigen loss. Bispecific CAR-T cells target two tumor antigens at once could reduce the risk of relapse. In addition, immune checkpoint blockade such as GSK3β and PD-1inhibition has been successfully used to enhance CAR-T cell therapies. Therefore, we attempt to develop a dual CAR for mantle cell lymphoma with checkpoint blockade.
Methods: CD19-CD20 and CD19-CD22 bispecific CAR-T constructs were generated by subcloning the fragments in-frame into the pSFG retroviral vector. The BiCAR-T cells were cultured with/without a small molecule GSK3β inhibitor TWS119. Then, we performed a serial of experiments using CARs. 1, We assayed the CAR expression and cell expansion rates in vitro. 2, We assessed the tumor-killing capacity in vitro using MCL cell lines and primary MCL patient samples. 3, We performed phenotypic analyses by determination of proteins expression profiles of exhaustion markers and stem cell markers by flow cytometry. 4, We examined in vivo antitumor efficacy using preclinical models of MCL cell line-derived xenograft (CDX) .
Results: Firstly, we found CD19-CD20 and CD19-CD22 bispecific CAR-T cells showed similar CAR expression levels (55%-72% CAR+ ) and similar expansion rate as single CD19 or CD20 CAR. Secondly, bispecific CAR- T cells potently and specifically lysed tumor cells in vitro, and showed the better killing of MCL tumor than CD19 or CD20 single CAR after challenge with MCL cell lines in vitro. Finally, we also confirmed these effects in CDX mice models derived from MCL cell line, Jeko-1 in vivo. CD19-CD20 and CD19-CD22 bispecific CAR successfully eliminated MCL tumor, showed better antitumor efficacy, and prolonged mouse survival than single CAR treated groups (P &lt; 0.001). We also found that CD19-CD20 and CD19-CD22 bispecific-CAR T cells treated with TWS119 express significantly fewer exhaustion markers, such as PD-1 (P &lt; 0.001), and LAG3 (P &lt; 0.001), and showed better antitumor efficacy than the nontreated control group and single CAR groups in vivo. Furthermore, CAR-T cells expressing bispecific CD19-CD20 and CD19-CD22 CAR with TWS119 treatment had superior anti-MCL activity in vitro. In total, significantly better anti-MCL effects were observed for CD19-CD20 and CD19-CD22 bispecific CARs than single CAR. Their anti-MCL functions were further improved by TWS119 treatment.
Conclusion: Altogether, these findings suggest that bispecific CD19-CD20 and CD19-CD22 CAR-T cells are potent for treating MCL relapse and drug resistant patients. In addition, bispecific CD19-CD20 and CD19-CD22 CAR-T cells with TWS119 treatment will further bring up the full power of CARs. These data support the therapeutic potential of bispecific CARs in patients with MCL.

Disclosures
Wang: Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; BGICS: Honoraria; Bayer Healthcare: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; Lilly: Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; Epizyme: Consultancy, Honoraria; Dava Oncology: Honoraria; Chinese Medical Association: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Molecular Templates: Research Funding; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; VelosBio: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; CStone: Consultancy; Scripps: Honoraria; OMI: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Oncternal: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding.