A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes

H. Zhang, Douglas Lake, J. A M Barbuto, R. M. Bernstein, W. J. Grimes, E. M. Hersh

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

With the development of recombinant DNA technology, it has become feasible to clone, construct, and express fully human immunoglobulin molecules. Here we report a novel methodology to make human antitumor single-chain Fv (scFv) antibodies from tumor-infiltrating B lymphocytes. We isolated and expanded tumor-infiltrating B lymphocytes from melanomas in the presence of Epstein- Barr virus. The transformed B cells secreting tumor-specific antibodies were identified and cloned by limiting dilution. From one B cell clone with specific melanoma reactivity, we captured the immunoglobulin variable region genes V(H) and V(k) by PCR, sequenced the genes, and linked them together by PCR assembly with the use of a (Gly4Ser)3 linker. The scFv gene was then cloned into the pET21d vector and expressed. The obtained scFv protein with a M(r) of 29,000 was purified and biotinylated for further characterization. The scFv demonstrated specific tumor reactivity to 21 of 24 different melanoma cell lines and not to 14 nonmelanoma tumor cell lines, such as breast, ovarian, and colon cancer cells lines; normal human melanocytes as well as normal human leukocytes. These results were obtained in (a) a tumor cell ELISA, (b) fixed cell immunofluorescence, and (c) live cell flow cytometry. The immunoprecipitation results indicated that a protein antigen of M(r) 45,000 was recognized by the scFv. Since we reported previously that about 70% of human tumors of different histological types contain tumor- infiltrating B lymphocytes producing specific antitumor antibodies, this approach offers a rapid, effective method by combining in vitro B-cell expansion and PCR gene cloning to elucidate the repertoire of the human antitumor immune response and to make human monoclonal antitumor antibody molecules.

Original languageEnglish (US)
Pages (from-to)3584-3591
Number of pages8
JournalCancer Research
Volume55
Issue number16
StatePublished - 1995
Externally publishedYes

Fingerprint

Tumor-Infiltrating Lymphocytes
Single-Chain Antibodies
B-Lymphocytes
Melanoma
Polymerase Chain Reaction
Genes
Clone Cells
Immunoglobulin Variable Region
Neoplasm Antibodies
Cell Line
Neoplasms
Recombinant DNA
Melanocytes
Tumor Cell Line
Human Herpesvirus 4
Immunoprecipitation
Ovarian Neoplasms
Colonic Neoplasms
Fluorescent Antibody Technique
Immunoglobulins

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Zhang, H., Lake, D., Barbuto, J. A. M., Bernstein, R. M., Grimes, W. J., & Hersh, E. M. (1995). A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes. Cancer Research, 55(16), 3584-3591.

A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes. / Zhang, H.; Lake, Douglas; Barbuto, J. A M; Bernstein, R. M.; Grimes, W. J.; Hersh, E. M.

In: Cancer Research, Vol. 55, No. 16, 1995, p. 3584-3591.

Research output: Contribution to journalArticle

Zhang, H, Lake, D, Barbuto, JAM, Bernstein, RM, Grimes, WJ & Hersh, EM 1995, 'A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes', Cancer Research, vol. 55, no. 16, pp. 3584-3591.
Zhang H, Lake D, Barbuto JAM, Bernstein RM, Grimes WJ, Hersh EM. A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes. Cancer Research. 1995;55(16):3584-3591.
Zhang, H. ; Lake, Douglas ; Barbuto, J. A M ; Bernstein, R. M. ; Grimes, W. J. ; Hersh, E. M. / A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes. In: Cancer Research. 1995 ; Vol. 55, No. 16. pp. 3584-3591.
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