T-Cells Redirected Against the kappa Light Chain of Human Immunoglobulins Target Mature B Cell Derived Malignancies In Vitro and In Vivo.
Authors: Vera J. , Savoldo B. , Vigouroux S. , Biagi E. , Pule M. , Rossig C. , Brenner M. K. , Dotti G. 
Published: April 2020

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 chronic lymphocytic leukemia (B-CLL), follicular lymphoma and mantle cell lymphoma, express surface monoclonal immunoglobulins (Igs) carrying kappa (κ) or lambda (λ) light chain, we explored whether chimeric T-cells targeting these immunoglobulin chains could be used instead. This approach would selectively eliminate κ+ or λ+ clonal tumor cells while sparing λ+ and κ+ normal B cells, respectively. We generated an anti-κ scFv from the κ-specific monoclonal antibody (hybridoma HP6053), 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 ζ chain of the TCR complex (CAR46/CD28/ζ). 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/ζ+. 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 κ+/λ− targets; SP53: λ+/κ−target; RAJI, κ−/λ− target; K562). CAR46/28/ζ+ T-cells efficiently killed all κ+ tumors (44%, range 48%–80% at 20:1 ratio) with &lt;15% killing of SP53, RAJI and K562 cells. Control T-cells (GFP+) did not show any significant activity (&lt;15%). In addition, T-cells isolated from 3 patients with κ+B-CLL were transduced with CAR46/28/ζ, they efficiently killed both autologous and allogeneic κ+B-CLL cells (55%, range 28%–71%), but not allogeneic λ+B-CLL cells. These transgenic T-cells produced IL-2 (&gt;2,000 pg/mLx106cells) and significantly expanded (11 fold, range 7–16) in response to autologous tumor cells (κ+B-CLL). No significant expansion was observed in control T-cells or T-cells transduced with CAR46/ζ receptor lacking the CD28 domain. To discover whether free Ig-kappa in serum competed for the chimeric receptor we co-cultured CAR46/28/ζ+ T-cells with Daudi cells (ratio 10:1). Although the presence of plasma (1:1) reduced killing (residual activity 25%±6%) in short term cytotoxicity assays, the chimeric T cells retained the ability to eliminate all viable tumor cells by day 5–7 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 (5x106) expressing renilla Luciferase (RL) and 5 days later T-cells (10x106) expressing both CAR46/28/ζ and firefly Luciferase (FL) or T-cells expressing only the FL. No exogenous cytokines were given. By day 16, FL signal was significantly enhanced in mice receiving CAR46/28/ζ+ cells (ROI=3.2x106counts) compared to mice receiving control cells (ROI=0.3x106) suggesting an in vivo expansion of CAR46/28/ζ+ T-cells. In parallel, RL signal was reduced in mice receiving CAR46/28/ζ+ T-cells (ROI=0.6x106) compared to mice receiving control T-cells (ROI=1.2x106) 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.