A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers.
Heavy chain antibodies are naturally occurring in camelidae (camels and llamas). Their variable domain (VHH) can be efficiently produced as a recombinant protein in E. coli with a large range of applications in the fields of diagnostics and immunotherapy. Standard cloning approach involves resolution of VHH from the heavy chain variable domain of conventional antibodies (VH) by a nested PCR amplification followed by a phage display based selection. Present work illustrates that in contrast to usual finding, specific, good affinity and efficiently expressed VH domain of conventional antibodies can be selected from the co-amplification products of VH and VHH cDNAs. Sequence analysis illustrated that following the two first rounds of selection against cancer markers, similar number of VH and VHH binders were observed. However, after a third round, the more specific binders directed against p53, VEGF, BCL-2 proteins surprisingly contain only VH specific hallmarks. Characterisation of the specificity, affinity and productivity of selected VH binders is described. Because llama VHs show higher sequence and structural homology with the human VH III group than llama VHHs (Vu et al., 1997), they constitute very interesting agents in therapeutic applications, especially in human immunotherapy and cancer treatment.
Kastelic
Damjana
D
CNRS, Centre de Génétique Moléculaire FRE3144, Unité associée à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
Frković-Grazio
Snjezana
S
Baty
Daniel
D
Truan
Gilles
G
Komel
Radovan
R
Pompon
Denis
D
D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000276:immunology; D002161:Camelids, New World; D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry* / Q000276:immunology
D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019253:Proto-Oncogene Proteins c-bcl-2; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A