268. CD19-Targeted Normal and CLL Patient T Cells Expanded with Beads Can Eradicate Systemic Tumors In Vivo
Authors: J. Stefanski, R. Brentjens, D. Hollyman, M. Bonyhadi, M. Sadelain, I. Rivière
Journal: Molecular Therapy (2006)

In order to conduct a clinical trial of adoptive therapy with genetically modified T cells in patients with CLL, we are investigating the feasibility of ex vivo T cell transduction and expansion using Xcyte™ Dynabeads® to generate sufficient numbers of biologically active tumor-reactive T cells. T cells are genetically modified to express 19-28z chimeric antigen receptor (CAR). The 19-28z CAR is derived from a single chain fragment length murine antibody specific to the human CD19 antigen expressed on CLL tumor cells, fused to the transmembrane and cytoplasmic domains of the human CD28 co-stimulatory receptor and the cytoplasmic domain of the CD3ζ chain of the human T cell receptor.In four experiments, we find that transduction efficiencies of healthy donor-derived T cells initially activated with Xcyte™ Dynabeads® range from 50% to 88% 19-28z+ T cells as measured by flow cytometry analysis. The degree of 19-28z+ T cell expansion ranges from 2 to 3 log over 14 days of culture. Significantly, we find that T cells activated with Xcyte™ Dynabeads® efficiently lyse CD19+ tumor cells in vitro (70 to 90% killing at 25:1 E:T ratio) similarly to T cells activated with PHA and subsequently restimulated on artificial antigen presenting cells (PHA/AAPCs) as previously demonstrated (Brentjens et al. Nature Medicine 2002). To determine whether 19-28z+ T cells activated with Xcyte™ Dynabeads® have cytotoxic activity in vivo, SCID-Beige mice bearing disseminated Raji cell lymphoma were treated with a single intravenous injection of either 2 × 107 19-28z transduced T cells (n=22) or 2 × 107 control Pz1 transduced T cells (n=15) or 2 × 107 1928z T cells activated on PHA/AAPC (n=8). The latter group was used as a positive control as we have previously shown that PHA/AAPC-stimulated T cells could eradicate established CD19+ tumor cells in vivo (Brentjens et al. Nature Medicine 2002). After treatment, mice were routinely checked for tumor progression as determined by hind-limb paralysis. Mice with hind-limb paralysis were sacrificed. As expected, all control mice treated with Pz1 transduced T cells all developed hind-limb paralysis 4-5 weeks after tumor cell injection. In contrast, mice treated with 19-28z+ T cells activated with Xcyte™ Dynabeads® had either delayed progression of disease (n=2) or remain disease free (n=20) at >180 days. Mice treated with 19-28z activated with PHA/AAPC showed either delayed progression of disease (n=1) or remain disease free (n=7) at >180 days. These results demonstrate that 19-28z+ T cells activated with Xcyte™ Dynabeads® are as potent as 19-28z+ T cells activated with PHA/AAPCs. Subsequently, we have demonstrated that T cells derived from patients with CLL can also be transduced and expanded with Xcyte™ Dynabeads® and that mice (n=14) treated with these cells have delayed progression of disease (n=7) or remain disease free for > 120 days. These results suggest that a Xcyte™ Dynabeads® -based approach should be suitable to transduce and expand T cells for our planned clinical trial. In order to conduct a clinical trial of adoptive therapy with genetically modified T cells in patients with CLL, we are investigating the feasibility of ex vivo T cell transduction and expansion using Xcyte™ Dynabeads® to generate sufficient numbers of biologically active tumor-reactive T cells. T cells are genetically modified to express 19-28z chimeric antigen receptor (CAR). The 19-28z CAR is derived from a single chain fragment length murine antibody specific to the human CD19 antigen expressed on CLL tumor cells, fused to the transmembrane and cytoplasmic domains of the human CD28 co-stimulatory receptor and the cytoplasmic domain of the CD3ζ chain of the human T cell receptor. In four experiments, we find that transduction efficiencies of healthy donor-derived T cells initially activated with Xcyte™ Dynabeads® range from 50% to 88% 19-28z+ T cells as measured by flow cytometry analysis. The degree of 19-28z+ T cell expansion ranges from 2 to 3 log over 14 days of culture. Significantly, we find that T cells activated with Xcyte™ Dynabeads® efficiently lyse CD19+ tumor cells in vitro (70 to 90% killing at 25:1 E:T ratio) similarly to T cells activated with PHA and subsequently restimulated on artificial antigen presenting cells (PHA/AAPCs) as previously demonstrated (Brentjens et al. Nature Medicine 2002). To determine whether 19-28z+ T cells activated with Xcyte™ Dynabeads® have cytotoxic activity in vivo, SCID-Beige mice bearing disseminated Raji cell lymphoma were treated with a single intravenous injection of either 2 × 107 19-28z transduced T cells (n=22) or 2 × 107 control Pz1 transduced T cells (n=15) or 2 × 107 1928z T cells activated on PHA/AAPC (n=8). The latter group was used as a positive control as we have previously shown that PHA/AAPC-stimulated T cells could eradicate established CD19+ tumor cells in vivo (Brentjens et al. Nature Medicine 2002). After treatment, mice were routinely checked for tumor progression as determined by hind-limb paralysis. Mice with hind-limb paralysis were sacrificed. As expected, all control mice treated with Pz1 transduced T cells all developed hind-limb paralysis 4-5 weeks after tumor cell injection. In contrast, mice treated with 19-28z+ T cells activated with Xcyte™ Dynabeads® had either delayed progression of disease (n=2) or remain disease free (n=20) at >180 days. Mice treated with 19-28z activated with PHA/AAPC showed either delayed progression of disease (n=1) or remain disease free (n=7) at >180 days. These results demonstrate that 19-28z+ T cells activated with Xcyte™ Dynabeads® are as potent as 19-28z+ T cells activated with PHA/AAPCs. Subsequently, we have demonstrated that T cells derived from patients with CLL can also be transduced and expanded with Xcyte™ Dynabeads® and that mice (n=14) treated with these cells have delayed progression of disease (n=7) or remain disease free for > 120 days. These results suggest that a Xcyte™ Dynabeads® -based approach should be suitable to transduce and expand T cells for our planned clinical trial.