Cytokine induction of VCAM-1 but not IL13Rα2 on glioma cells: a tale of two antibodies.
The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Vaidehi
V
Mahadev
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Renate
R
Starr
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Sarah L
SL
Wright
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Catalina
C
Martinez
Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, United States of America.
Michael C
MC
Jensen
Department of Neurosciences, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Michael E
ME
Barish
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Stephen J
SJ
Forman
Departments of Hematology and Hematopoietic Cell Transplantation, Cancer Immunotherapy & Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, California, United States of America.
Christine E
CE
Brown
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis / Q000378:metabolism*; D018450:Disease Progression; D000939:Epitopes / Q000276:immunology; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology; D005910:Glioma / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000473:pathology*; D006801:Humans; D053663:Interleukin-13 Receptor alpha2 Subunit / Q000235:genetics / Q000276:immunology*; D058990:Molecular Targeted Therapy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D016207:Cytokines; D000939:Epitopes; D053663:Interleukin-13 Receptor alpha2 Subunit; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1