Generation of human Fab monoclonal antibodies against preS1 of hepatitis B virus using repertoire cloning.
Human monoclonal antibodies (MAbs) have considerable potential in the prevention and treatment of many viral diseases. A combinatorial antibody library of heavy (Fd)- and light-chain genes derived from peripheral blood lymphocytes of a volunteer with high antibody titer to preS1 of HBV was constructed and expressed on the surface of filamentous phages. The library contained 7 x 10(9) independent clones. A phage antibody population from the third panning against preS1 was converted to one expressing soluble Fabs by removal of the g3 sequences from the pComb3 phagemid vector and subsequent transformation into E. coli TG1 cells. Screening of the library led to the identification of two clones, 3DW and 8GW, showing high reactivity toward preS1. The authenticity of the Fabs was confirmed by immunoblot analysis which yielded approximately 60 and approximately 30 kDa bands under nonreducing and reducing conditions, respectively. The soluble Fabs of 3DW and 8GW exhibited relative affinities of 6 x 10(5) and 8 x 10(6) M(-1), respectively. The sequencing results demonstrate that all Fd sequences belong to subgroup II and all light chain sequences belong to subgroup I. There are differences in CDR length and composition, especially in the FW3 and CDR3 regions of the heavy- and light-chain genes. These human Fab MAbs specific to preS1, generated from a combinatorial library, represent prototypes of passive immunotherapy candidates for viral hepatitis B.
Choi
I H
IH
Department of Microbiology, College of Medicine, Inje University, Pusan, Korea.
Park
S G
SG
Chung
J H
JH
Kim
I J
IJ
Hong
H J
HJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D015723:Gene Library; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000276:immunology*; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018076:DNA, Complementary; D006514:Hepatitis B Surface Antigens; D007140:Immunoglobulin Fab Fragments; D011498:Protein Precursors; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen