[The mutation of anti-CD3 antibody (HIT3a) gene and its expression].
OBJECTIVE
To improve the expression of anti-CD(3) single chain Fv (scFv) by site mutation and identify its biological activity.


METHODS
Anti-CD(3) scFv gene was mutated by PCR, the target clones were screened by both the fingerprints of DNA restriction endonuclease digestion and Western blot, the antigen-binding activity of scFv was examined by FACS, competitive inhibition was performed with (125)I-labeled HIT3a and the cytotoxic effect mediated by the anti-CD(3) scFv-activated T lymphocytes was analyzed by (51)Cr-released assays.


RESULTS
The DNA sequencing showed that the 6th amino acid of the anti-CD(3) antibody (HIT3a) heavy chain gene was mutated from E (GAG) to Q (CAG). The expression of mutated anti-CD(3) scFv (m2) was increased by 100 times higher than that of the parent scFv, and there was no difference in the Jurkat cell (CD(3)(+))-binding activity between the (m2) and parent scFv. The preliminary results of competitive assays showed that m2 could partially block the sites of CD(3)(+) Jurkat cells where the parent antibody bound to. Cytotoxicity assays demonstrated that CD(3)AK cells induced by IL-2 and m2 showed stronger cytotoxic effect than that of LAK cells induced by IL-2 alone in vitro.


CONCLUSIONS
By site mutation, a high expression fragment m2 of anti-CD(3) scFv antibody was obtained. The results of some experiments indicated that m2 could bind to CD(3)(+) Jurkat cells, furthermore, by co-stimulated with IL-2, it could activate peripheral T lymphocytes and induce CD(3)AK cytotoxic effect.
Xu
Y
Y
State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS&PUMC, Tianjin 300020, China.
Xiong
D
D
Yang
C
C
Lai
Z
Z
Liu
H
H
He
X
X
Peng
H
H
Shao
X
X
Xu
C
C
Liao
X
X
Xie
Y
Y
Zhu
Z
Z
D017252:CD3 Complex / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D006801:Humans; D008213:Lymphocyte Activation; D009154:Mutation; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
D017252:CD3 Complex; D057127:Single-Chain Antibodies