Endpoints for clinical trials testing treatment of acute graft-versus-host disease: a joint statement.

Currently, no agents are approved by the United States Food and Drug Administration (FDA) for either prevention or treatment of acute graft-versus-host disease (aGVHD). Formal precedents establishing a comparative basis for assessing the efficacy and safety of new investigational agents are still lacking. As a step toward addressing this problem, a panel of experts met on 2 occasions to reach consensus on recommendations for terminology describing a clinically meaningful primary endpoint in studies assessing treatment for aGVHD. The panel recommended terminology for "very good partial response" (VGPR) that includes both diagnostic and functional criteria. The central hypothesis leading to this proposal is that the potential harm of giving more treatment than needed to produce or maintain complete response exceeds the harm of slight undertreatment that may be associated with less than complete response. VGPR clearly cannot be used as the sole outcome measure in GVHD treatment trials, and must be considered in the context of survival and safety. The proposed use of VGPR as the primary endpoint in GVHD treatment trials will remain provisional until its use has been validated through experience.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction.

The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origins without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with a retroviral vector we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the tumor-associated ErbB2 (HER2/neu) antigen, which is overexpressed by many tumors of epithelial origin. The chimeric antigen receptor consists of the ErbB2-specific scFv(FRP5) antibody fragment, a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta cells express high levels of the fusion protein on the cell surface as determined by fluorescence-activated cell-scanning (FACS) analysis. In europium release assays, no difference in cytotoxic activity of NK-92 and NK-92-scFv(FRP5)-zeta cells toward ErbB2-negative targets was found. However, even at low effector-to-target ratios, NK-92-scFv(FRP5)-zeta cells specifically and efficiently lysed established and primary ErbB2-expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2-expressing malignancies.

In vivo purging with rituximab prior to collection of stem cells for autologous transplantation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells express the CD20 antigen, and monoclonal antibodies against CD20 have resulted in remissions. We hypothezised that the anti-CD20 antibody rituximab (Rituxan) may be useful in reducing the number of contaminating CLL cells in stem cell collections for use in autologous transplantation. A pilot study in 5 patients was designed using rituximab 375 mg/m(2) as an in vivo purging step following cyclophosphamide 4 gm/m(2) and granulocyte colony-stimulating factor/granulocyte-macrophage colony-stimulating factor (G-CSF/GM-CSF) mobilization therapy for patients with advanced-stage CLL undergoing autologous stem cell transplantation. Eligible patients had 0-30% marrow involvement prior to mobilization. A single pre-rituximab leukapheresis product was obtained after the white blood cells (WBC) reached 800/mm(3) to serve as a control but was not reinfused. Rituximab was administered the following day and subsequent leukaphereses were commenced 48 h later to reach a total of >2 x 10(6) CD34(+) cells/kg. Dual-color flow cytometry CD5/CD19 and consensus PCR using primers to the joining region and FR3 of the variable region of the immunoglobulin heavy chain (IgH) were used to evaluate the degree of contaminating CLL cells in the leukapheresis product and to monitor disease status post transplant. All 5 patients were informative for the consensus PCR assay. Four of 5 patients mobilized >2 x 10(6) CD34(+) cells/kg and proceeded to cyclophosphamide 120 mg/kg and total body irradiation (6 x 200 cGy) with stem cell rescue. All leukaphereses products were positive by PCR for the IgH rearrangement and 4/5 contained CD5/CD19 dual-positive cells. Comparing the pre- and post-rituximab leukapheresis products, a reduction in the percentage of CD5(+)/CD19(+) cells was seen in 4/5 patients. All patients engrafted at a median of 13.5 days to ANC > 500/mm(3) and 11 days to platelets >20,000/mm(3). No regimen-related mortality was seen. Although 2 patients tested positive on PCR for the IgH rearrangement early after transplant, all patients had absence of the IgH gene rearrangement at 1 year and no CD5/CD19 dual-positive cells were could be detected in the bone marrow. This includes 1 heavily pretreated patient who received stem cells containing up to 30% CD5(+)/CD19(+) cells. We conclude that purging with Rituximab 48 h prior to stem cell collection was able to reduce significantly (but not eliminate) the percentage of CLL cells in the leukaphereses. However, despite the infusion of CD5(+)/CD19(+) cells in the stem cell coions, patients were able to obtain durable complete molecular remissions, implying that the PCR-positive cells in the leukaphereses may not have long-term clonogenic potential. The results also support the recommendation to test if rituximab should be part of a maintenance regimen after transplant to prevent disease recurrence in high-risk patients.