Generation of a neutralizing antibody against RD114-pseudotyped viral vectors.

The feline endogenous RD114 glycoprotein has proved to be an attractive envelope to pseudotype both retroviral and lentiviral vectors. As a surface protein, its detection on packaging cells as well as viral particles would be useful in different fields of its use. To address this, we generated a monoclonal antibody against RD114 by immunization of rats, termed 22F10. Once seroconversion was confirmed, purified 22F10 was cloned into murine Fc and characterized with a binding affinity of 10nM. The antibody was used to detect RD114 and its variant envelopes on different stable viral packaging cell lines (FLYRD18 and WinPac-RD). 22F10 was also shown to prevent the infections of different strains of RD-pseudotyped vectors but not related envelope glycoproteins by blocking cell surface receptor binding. We are the first to report the neutralization of viral particles by a monoclonal αRD114 antibody.

Rapid generation of EBV-specific cytotoxic T lymphocytes resistant to calcineurin inhibitors for adoptive immunotherapy.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (PTLD) remains a major cause of morbidity and mortality after hematopoietic stem cell (HSCT) or solid organ transplant (SOT). Strategies to reconstitute immunity by adoptive transfer of EBV-specific cytotoxic T lymphocyte (CTL) therapy while highly effective in the HSCT setting where immunosuppression can be withdrawn have been less successful in the SOT setting where continued immunosuppression therapy is necessary. Additionally, the complexity and time taken to generate EBV-CTLs for adoptive transfer limit the clinical applicability. We have developed a system for the rapid generation of EBV-CTLs resistant to immunosuppression based on selection of interferon-gamma (IFN-γ) secreting EBV-CTLs and retroviral transduction with a calcineurin B mutant. With this methodology, EBV-CTLs resistant to the calcineurin inhibitor Tacrolimus (TAC) can be produced in 14 days. These CTLs show high specificity for EBV with negligible alloreactivity in both proliferation and cytotoxicity assays and are able to proliferate and secrete IFN-γ in response to antigen stimulation in the presence of therapeutic doses of TAC. This strategy will substantially facilitate clinical application of this approach for the treatment of PTLD in SOT recipients.

Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences.

BACKGROUND: Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used. METHODS: We have developed and validated a simple but effective technique for generating libraries of IHJ, which we term flanking-sequence exponential anchored-polymerase chain reaction (FLEA-PCR). Flanking-sequence random anchoring is used as an alternative to restriction enzyme digestion and cassette ligation to allow consistent detection of IHJ and decrease bias. RESULTS: Individual clones from plasmid libraries can be sequenced and assembled using custom-written software, and FLEA-PCR smears can be analyzed by capillary electrophoresis after digestion with restriction enzymes. DISCUSSION: This approach can readily analyze complex mixtures of IHJ, allowing localization of these sequences to their genomic sites. This approach should simplify analysis of retroviral integration.

Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells.

T cells can be engineered to target tumor cells by transduction of tumor-specific chimeric receptors, consisting of an extracellular antigen-binding domain and an intracellular signaling domain. However, the peripheral blood of cancer patients frequently contains an increased number of T regulatory cells, which appear to inhibit immune reactivity. We have investigated the effects of T regulatory cells on chimeric T cells specific for the B-cell antigen CD19, as B-cell malignancies are attractive targets for chimeric T-cell therapy. When a CD19 single-chain Fv antibody was coupled to the CD3 zeta (zeta) chain, there was sharply reduced activity on exposure to T regulatory cells, measured by CD19+ target-induced proliferation and cytotoxicity. By contrast, expression in T cells of a chimeric receptor consisting of the intracellular portion of the CD28 molecule fused to the zeta-chain and CD19 single-chain Fv not only produced a higher proliferative response and an increased nuclear factor kappaB activation but also sustained these activities in the presence of T regulatory cells. These effects are seen whether the chimeric T cells are derived from normal donors or from patients with B-cell chronic lymphocytic leukemia, indicating the potential for clinical application in B cell malignancies.

Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

Artificial T-cell receptors.

Artificial T-cell receptors are generated by joining an Ag-recognizing domain (ectodomain) to the transmembrane and intracellular portion of a signaling molecule (endodomain). The ectodomain is most often derived from Ab variable chains, but may also be generated from T-cell receptor variable chains, as well as from other molecules. Various alternative ectodomain designs exist, with some comparative studies suggesting optimal forms. The endodomain most often used is the intracellular portion of CD-zeta. Although signaling by CD-zeta leads to IFN-n release and cell killing, it fails to transmit a full activation signal. Recently, unions of different signaling molecule segments have facilitated transmission of more potent signals, stimulating T-cell proliferation and overcoming this major limitation. Artificial T-cell receptors allow grafting of nearly any specificity to T cells. This allows generation of large numbers of specific T cells, without laborious selection and expansion procedures. Efficacy against tumors has been demonstrated in animal models. Phase I and II studies of T-cells transduced with artificial T-cell receptors as therapy for HIV infection have been performed. This rapidly advancing technology will make new strategies of adoptive immunotherapy possible.