Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections

Abstract

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4 + and CD8 + T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CART cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CART cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CART cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34 + cells with long-term human immune reconstitution were used to model HCMV/ GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CART cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high ontarget effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CART cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

“…It would be interesting to evaluate if reactive T cells directed against lytic-or latency-associated antigens could be generated. In fact, using humanized mice infected with HCMV at 17-19 weeks after HCT, we recently showed therapeutic effects of adoptive T cells expressing an engineered chimeric antigen receptor targeting gb (gB-CAR-T) administered 8 weeks later [46].…”

“…Mutagenesis was performed to change the fusion loop residues and avoid protein aggregation by modifying the YAYIH 154-158 and GSTWLY 238-243 residues into EEEEE and EEEEEE, respectively. Generation of the S2 cell lines secreting the gB ectodomains and the protein production were performed as described 46 . The recombinant gB ectodomains were purified from the supernatant by affinity chromatography using StrepTactin resin (IBA Biosciences, Göttingen, Germany), dialyzed into PBS and used as such for the experiments.…”

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease

“…Expression of gp350CARs on T cells was compared with gBCAR, recognizing the glycoprotein gB of HCMV and generated with the same vector backbone and signaling domains. 29 The mean fluorescence intensities of the different CAR + T cells were calculated (see flow cytometry gating strategy in Figures S2 B and S2C). The reference gBCAR showed significantly higher expression on the surface of CD4 + and CD8 + T cells than 7A1-gp350CAR (p ≤ 0.01) and 6G4-gp350CAR (p ≤ 0.001) ( Figures 1 C and 1D; Table S1 ).…”

“…In a recent related study, we have shown that CB-derived CAR-T cells targeted against the HCMV gB envelope also showed efficacy in vitro and in vivo . 29 Therefore, similar to VST cells expanded with peptides and targeted against several viruses, polyfunctional CAR-T cells targeted simultaneously against EBV and HCMV can be developed in the future.…”

“… 23 , 24 Beyond the oncology field, CAR-T cells redirected against viral antigens expressed on the surface of infected cells are in development; for example, against human cytomegalovirus (HCMV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV). 25 , 26 , 27 , 28 , 29 …”