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

ABSTRACT: Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3? signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.

SUBMITTER: Slabik C

PROVIDER: S-EPMC7479496 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Slabik Constanze C Kalbarczyk Maja M Danisch Simon S Zeidler Reinhard R Klawonn Frank F Volk Valery V Krönke Nicole N Feuerhake Friedrich F Ferreira de Figueiredo Constanca C Blasczyk Rainer R Olbrich Henning H Theobald Sebastian J SJ Schneider Andreas A Ganser Arnold A von Kaisenberg Constantin C Lienenklaus Stefan S Bleich Andre A Hammerschmidt Wolfgang W Stripecke Renata R

Molecular therapy oncolytics 20200808

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The sc ...[more]

PMID: 32953984

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Project description:EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (?3C EBV) in a cord blood-humanized mouse model (CBH). We found that the ?3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of ?3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and ?3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, ?3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that ?3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in ?3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, ?3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.

Project description:This study compares cellular and viral gene expression in a cord blood-humanized (CBH) mouse model infected with wild-type Epstein-Barr virus (WT), versus a mutant EBV deleted in the latent viral EBNA3C gene (∆3C). EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently infected cells, is required for EBV transformation of B cells in vitro. However, many EBV+ lymphomas in humans do not express this protein, suggesting that it may be less important in vivo or that cellular mutations and/or signaling pathways may obviate the need for EBNA3C. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. We found that the ∆3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and ∆3C viruses induced monoclonal B-cell lymphomas (without somatic hyper-mutation) that were infiltrated by polyclonal T cells. In comparison to WT-infected tumors, ∆3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that ∆3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. Unexpectedly, ∆3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.

Dataset Information

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

Publications

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

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