An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells.
A procedure was developed to generate recombinant single chain Fv (scFv) antibody fragments reacting with the extracellular domain of human cell surface antigen CD13 (hCD13; aminopeptidase N) on intact cells. Membrane fractions prepared from a stably transfected hCD13-positive murine NIH/3T3 cell line were used to immunize BALB/c mice, with the intention that hCD13 would be the major immunogenic molecule recognized by the immune system. Spleen RNA from the immunized mice served to generate a combinatorial scFv phage display library. The library was adsorbed against non-transfected NIH/3T3 or Sf21 insect cells to eliminate nonrelevant binders. The supernatant was then used for panning with either hCD13-transfected Sf21 insect cells or a hCD13-expressing human leukemia-derived cell line. Therefore, the key concepts of the procedure were the presentation of hCD13 as the sole human antigen on murine NIH/3T3 cells and a screening strategy where hCD13 was the major common antigen of the material used for immunization and panning. Two different hCD13-reactive phages were isolated and the soluble scFvs were expressed in E. coli and purified. The two scFvs, anti-hCD13-1 and anti-hCD13-3, differed at four amino acid positions in their V(H) regions and both had high affinities for hCD13 as determined by surface plasmon resonance (K(D)=7 and 33x10(-10) M, respectively). Both efficiently recognized hCD13 on intact cells. Therefore, the procedure allowed the production of high affinity scFvs reacting with a desired antigen in its native conformation without requiring extensive purification of the antigen and should be useful for the preparation of scFvs against other conformation-sensitive cell-surface antigens.
Peipp
M
M
University of Erlangen-Nürnberg, D 91058, Erlangen, Germany.
Simon
N
N
Loichinger
A
A
Baum
W
W
Mahr
K
K
Zunino
S J
SJ
Fey
G H
GH
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018411:Spodoptera; D014162:Transfection
D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D018826:CD13 Antigens