ABSTRACT
The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.
Subject(s)
Antigens, Neoplasm/immunology , CD5 Antigens/immunology , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Lymphoma, T-Cell, Peripheral/therapy , Molecular Targeted Therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Recombinant Fusion Proteins/immunology , 4-1BB Ligand/genetics , 4-1BB Ligand/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/genetics , CD3 Complex/immunology , CD8 Antigens/immunology , Cell Line, Tumor , Coculture Techniques , Cytotoxicity, Immunologic , Humans , Killer Cells, Natural/transplantation , Lymphoma, T-Cell, Peripheral/pathology , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Salvage Therapy , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Transduction, Genetic , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Xenograft Model Antitumor AssaysABSTRACT
Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.
Subject(s)
Adoptive Transfer , CD8-Positive T-Lymphocytes/immunology , Lymphoma, T-Cell, Peripheral/therapy , Mutant Chimeric Proteins/genetics , Receptors, Artificial/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/transplantation , Cell Engineering , Cell Line, Tumor , Coculture Techniques , Cytotoxicity, Immunologic , Gene Expression , Humans , Leukemia/genetics , Leukemia/immunology , Leukemia/pathology , Lymphoma, T-Cell, Peripheral/genetics , Lymphoma, T-Cell, Peripheral/immunology , Lymphoma, T-Cell, Peripheral/mortality , Male , Mice , Mice, Inbred NOD , Mutant Chimeric Proteins/immunology , Neoplasm Transplantation , Primary Cell Culture , Receptors, Artificial/immunology , Survival AnalysisABSTRACT
A xenon-chlorine excimer laser was used to irradiate normal endocardium of fresh sheep and pig hearts as well as unfixed human endocardial scar. Forty pulses of 370 J and 35 ns each resulted in penetration of up to 12 mm in normal tissue and only 3.5 mm in scarred endocardium. Dosimetry indicated that the volume of vaporized scarred tissue was 1/10th that of normal endocardium (0.19 to 0.40 versus 1.35 to 3.22 mm3/J). Ultrastructurally, there was a sharp demarcation of only 10 mu between the region of injury and normal myocardium, with little evidence of heat injury. The high power and short duration of these lasers coupled with the lack of a boundary zone of injury suggest that excimers may be an ideal tool for arrhythmia ablation.
Subject(s)
Endocardium/surgery , Laser Therapy , Animals , Arrhythmias, Cardiac/surgery , Chlorine , Cicatrix/surgery , Coronary Disease/pathology , Coronary Disease/surgery , Endocardium/injuries , Endocardium/ultrastructure , Humans , Sheep , Swine , XenonABSTRACT
A new in vitro model has been developed for studying the changes in the ultrasound Doppler spectrum that occur in the region of a stenosis. Pulsatile flow in rigid acrylic tubes was produced by means of a modified hemodialysis pump. The Doppler spectral waveforms were measured using a continuous wave Doppler system, a probe of a known field pattern, a real-time high resolution frequency analyzer, and a video display and recording system. The flow velocity waveforms were found to be nearly identical to those seen in the human carotid. Measurements were made to determine the critical stenosis and the results are similar to those reported from in vivo studies. In a preliminary study, the extent of spectral broadening was found to be dependent on the recording site in relation to the stenosis, the severity of the stenosis, and the flow rate. Using qualitative methods it was not possible to determine either the influence of the shape of the stenosis or the phase of the cardiac cycle on spectral broadening.
