Isolation of monoclonal antibodies with predetermined conformational epitope specificity.
Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Anton M
AM
Sholukh
Muhammad M
MM
Mukhtar
Michael
M
Humbert
Sosthène S
SS
Essono
Jennifer D
JD
Watkins
Hemant K
HK
Vyas
Vivekanandan
V
Shanmuganathan
Girish
G
Hemashettar
Maria
M
Kahn
Shiu-Lok
SL
Hu
David C
DC
Montefiori
Victoria R
VR
Polonis
Peter H
PH
Schur
Ruth M
RM
Ruprecht
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D001324:Autoantigens / Q000737:chemistry; D001402:B-Lymphocytes / Q000276:immunology; D001435:Bacteriophages / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry*; D005434:Flow Cytometry; D006678:HIV / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000821:virology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D054299:env Gene Products, Human Immunodeficiency Virus