ABSTRACT
CD1A is a cell surface protein expressed on Langerhans cells and cortical thymocytes that could potentially be used as an immunotherapeutic target in Langerhans Cell Histiocytosis (LCH), the cortical subtype of T-cell acute lymphocytic leukaemia (T-ALL) and other CD1A-positive tumours. The monoclonal antibody (mAb) CR2113 was selected from a panel of six fully human mAbs isolated from a semi-synthetic phage display library, based on specificity and avidity against cells expressing CD1 antigen variants. CR2113 recognized CD1A in T-ALL cell lines and patient samples. Confocal microscopy revealed that the CR2113-CD1A complex was internalized at 37°C. Furthermore, while CR2113 induced moderate complement-dependent cytotoxicity (CDC), potent antibody-dependent cell cytotoxicity (ADCC) activity was observed against CD1A expressing cell lines as well as T-ALL cell lines and T-ALL patient samples. In vivo experiments showed that CR2113 as a naked antibody has modest but specific anti-tumour activity against CD1A-expressing tumours. CR2113 is a high-affinity human anti-CD1A mAb with significant ADCC activity. These properties make CR2113 a candidate for clinical diagnostic imaging and therapeutic targeting of LCH as well as potential use in other clinical applications.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD1/immunology , Cell Surface Display Techniques , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/isolation & purification , Antibody Affinity/immunology , Antibody Specificity/immunology , Antigens, CD1/metabolism , Cell Line, Tumor , Humans , Immunoglobulin G/immunology , Kinetics , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Peptide Library , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Single-Chain Antibodies/immunology , Single-Chain Antibodies/isolation & purificationABSTRACT
Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8(+) T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8(+) T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8(+) T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8(+) T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-gamma mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8(+) T cells in vaccine trials. The technology should be transferable to analysis of CD8(+) T-cell responses to other antigens.
