Work

768. Redirected T Cells to Selectively Target Mature B Cell Derived Malignancies

Year: 2006

Type: article

Abstract: Adoptive transfer of CD19 or CD20 chimeric artificial receptors (CARs) to T-lymphocytes has been proposed to treat B-cell malignancies. If successful, however, the approach would likely severely impair humoral immunity. Since low grade lymphomas express surface monoclonal immunoglobulins (Igs) carrying kappa (|[kappa]|) or lambda (|[lambda]|) light chain, we explored whether chimeric T-cells targeting these immunoglobulin chains could be used instead. This approach would selectively eliminate |[kappa]|+ or |[lambda]|+ clonal tumor cells while sparing |[lambda]|+ and |[kappa]|+ normal B cells, respectively. We generated an anti-|[kappa]| scFv from the |[kappa]|-specific monoclonal hybridoma, using RT-PCR and phage display. The specific scFv was cloned in SFG-retrovirus vector in frame with the IgG1-CH2CH3 domain, CD28 endodomain and |[zeta]| chain of the TCR complex (CAR46/CD28/|[zeta]|). T-cells obtained from 6 normal donors were activated with OKT3/CD28 antibodies and then transduced. Seventy-one percent (range 68-76%) of T-cells were CAR46/28/|[zeta]|+. The cytotoxicity of transgenic T-cells was tested using the 51Cr release assay against several B-cell malignancies (Daudi, BJAB, JEKO-1 and CLL-120 as |[kappa]|+/|[lambda]||[minus]| targets; SP53: |[lambda]|+/|[kappa]||[minus]|target; RAJI, |[kappa]||[minus]|/|[lambda]||[minus]| target; K562). CAR46/28/z+ T-cells efficiently killed all k+ tumors (44%, range 48|[ndash]|80% at 20:1 ratio) with 2,000 pg/mL|[times]|106cells) and significantly expanded (11 fold, range 7|[ndash]|16) in response to autologous |[kappa]|+B-CLL cells. No significant expansion was observed in control T-cells or CAR46/|[zeta]|+ T-cells receptor lacking the CD28 domain. To discover whether free Ig-kappa in serum competed for the chimeric receptor we co-cultured CAR46/28/|[zeta]|+ T-cells with Daudi cells (ratio 10:1). Although the presence of plasma (1:1) reduced killing (residual activity 25%|[plusmn]|6%) in short term cytotoxicity assays, the chimeric T cells retained the ability to eliminate all viable tumor cells by day 5|[ndash]|7 of co-culture, while tumor overgrowth was observed in the presence of control T-cells. To explore the function of transduced cells in vivo we used a xenograft mouse model and in vivo bioluminescence. We injected i.p. Daudi cells (5|[times]|106) expressing renilla Luciferase (RL) and 5 days later CAR46/28/|[zeta]|+ T-cells (10|[times]|106) expressing firefly Luciferase (FL) or T-cells expressing only the FL. By day 16, FL signal was significantly enhanced in mice receiving CAR46/28/|[zeta]|+ cells (ROI=3.2|[times]|106counts) compared to mice receiving control cells (ROI=0.3|[times]|106), suggesting an in vivo expansion of CAR46/28/|[zeta]|+ T-cells. In parallel, RL signal was reduced in mice receiving CAR46/28/z+ T-cells (ROI=0.6x106) compared to mice receiving control T-cells (ROI=1.2|[times]|106), suggesting control of tumor growth. Adoptive transfer of T-cells targeting the appropriate Ig light chain could be a useful immunotherapy approach to treat clonal B cell malignancies.

Source: Molecular Therapy

DOI: 10.1016/j.ymthe.2006.08.853