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/CD3ζ 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.

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.

The Jackson Laboratory, which commercially distributes the nonobese diabetic Rag1 null IL2rg null mice.

Lentiviral vectors (LVs) developed in the past two decades for research and pre-clinical purposes have entered clinical trials with remarkable safety and efficacy performances. Development and clinical testing of LVs for improvement of human immunity showed major advantages in comparison to other viral vector systems. Robust and persisted transduction efficiency of blood cells with LVs, resulted into a broad range of target cells for immune therapeutic approaches: from hematopoietic stem cells and precursor cells for correction of immune deficiencies, up to effector lymphoid and myeloid cells. T cells engineered for expression of chimeric antigen receptors (CARs) or epitope-specific transgenic T cell receptors (TCRs) are in several cancer immune therapy clinical trials worldwide. Development of engineered dendritic cells is primed for clinical trials for cancer and chronic infections. Technological adaptations for ex vivo cell manipulations are here discussed and presented based on properties and uses of the target cell. For future development of off-shelf immune therapies, direct in vivo administration of lentiviral vectors is warranted and intended. Approaches for lentiviral in vivo targeting to maximize immune therapeutic success are discussed.