Selective elimination of a chemoresistant side population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous hCD40L/IL-2 tumor vaccine

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5+ CD19+ SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5+ CD19+ SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8+ RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T-cell-mediated effector activity after immunization of human subjects

Selective depletion of a minor subpopulation of B-chronic lymphocytic leukemia cells is followed by a delayed but progressive loss of bulk tumor cells and disease regression

Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their it in vitro it properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal

Comparison of two CD40-ligand/interleukin-2 vaccines in patients with chronic lymphocytic leukemia

Background aims. Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies. Methods. We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity. Results. We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine. Conclusions. CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients

In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specific CAR-T Cells in B Cell Non-Hodgkin's Lymphomas

Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy. CD19.CAR-T cells are highly active against B cell malignancies, but the optimal CAR structure is controversial. Ramos et al. show that combining 4-1BB and CD28 endodomains produces superior CD19-CAR-T cell expansion and persistence compared to CD28 alone and that these cells are clinically effective and safe in aggressive lymphomas