[Construction and secretory expression of single-chain Fv fragment M97 with therapeutic potential against tumor invasion and metastasis].
OBJECTIVE
To manufacture single chain Fv antibody fragment for cancer targeting therapy.


METHODS
Based on recombinant phage display techniques, the construction, screening and expression of functional single-chain Fv fragment (scFv) from murine hybridoma C2H5 which secretes antibody specifically binding to (92 kD) type IV collagenase were performed. By RT-PCR for VH and VL genes, assembly of scFv genes and cloning into phagemid pCANTAB5E, transformation of TG1 cells and M13KO7 helper phage rescue, a recombinant phage scFv library with titre of 5 x 10(9) pfu/ml was established. Panning against immobilized type IV collagenase (92 kD) was performed by one round. Finally, 30 antigen-positive recombinant phage clones were isolated and identified by ELISA.


RESULTS
DNA sequencing result showed that the entire gene of scFv against type IV collagenase (92 kD) designated as scFv-M97 is 732 bp, consisting of the 351 bp VH gene encoding 117 amino acids, the 336 bp VL gene encoding 112 amino acids and a 45 bp linker encoding (Gly4Ser)3. The antigen-positive phage with titre of 10(10) pfu/ml was used to infect HB2151 cells in which soluble scFv was expressed. After induction of Lac promotor by 1 mM IPTG for 20 h, the scFv-M97 yield of HB2151 in SBA medium is about 2 micrograms/ml. Immuno-blot indicated that the 27 kD scFv-M97 retained the affinity and specificity of the original intact antibody to type IV collagenase (92 kD).


CONCLUSIONS
Single chain antibody scFv-M97 expressed by secretion was successfuly produced. As type IV collagenase plays an important role in tumor invasion and metastasis by the mechanism of proteolytic degradation of type IV collagen in the basement membrane, scFv-M97 may have therapeutic potential against tumor invasion and metastasis.
Zhou
C
C
Institute of Medicinal Biotechnology, CAMS and PUMC, Beijing 100050.
Jiang
M
M
Xu
L
L
Zhen
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006825:Hybridomas / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D020780:Matrix Metalloproteinase 9 / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; C429184:single chain Fv fragment M97; D020780:Matrix Metalloproteinase 9