Unknown

Dataset Information

0

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.


ABSTRACT: The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.

SUBMITTER: Cai X 

PROVIDER: S-EPMC39013 | BioStudies | 1996-01-01T00:00:00Z

SECONDARY ACCESSION(S): U58144

REPOSITORIES: biostudies

Similar Datasets

1997-01-01 | S-EPMC23147 | BioStudies
1997-01-01 | S-EPMC23241 | BioStudies
2017-01-01 | S-EPMC5742356 | BioStudies
2010-01-01 | S-EPMC2998965 | BioStudies
2015-01-01 | S-EPMC4966335 | BioStudies
1999-01-01 | S-EPMC96443 | BioStudies
2020-01-01 | S-EPMC7075871 | BioStudies
2015-01-01 | S-EPMC4517675 | BioStudies
2017-01-01 | S-EPMC6296615 | BioStudies
2019-01-01 | S-EPMC6726835 | BioStudies