In situ isolation of immunoglobulin sequences expressed by single tumor-infiltrating B cells using laser-assisted microdissection.
The isolation of fully human monoclonal antibodies (MAb) against tumor targets has to date relied largely on combinatorial library-based antibody display techniques, which generally require lengthy antigen selection procedures due to a low frequency of clones expressing compatible heavy (VH) and light chain (VL) variable genes. Here we describe a method to directly isolate immunoglobulin sequences in situ from antibody-producing cells infiltrating human tumor tissue. Single B cells and plasma cells infiltrating cervical cancer were microdissected from tissue sections using laser-assisted microscopy, and VH and VL expressed by each individual cell amplified using nested reverse transcriptase- polymerase chain reaction (RT-PCR), thus retaining the native VH and VL pairing. Sequencing analysis determined that the isolated cells expressed functional immunoglobulin variable genes, consistent with an antitumor antibody response. The immunoglobulin sequences can be reassembled as Fab or scFv fragments using conventional recombinant antibody expression plasmids. This method will allow a more direct assessment of the humoral immune response to cancer, and the potential identification of novel human therapeutic cancer antibodies.
O'Brien
Philippa M
PM
Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow G61 1QH, UK.
Millan
David W M
DW
Davis
Jonathon A
JA
Campo
M Saveria
MS
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007834:Lasers; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000473:pathology*; D042282:Microdissection / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000276:immunology* / Q000473:pathology*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments