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[The mutation of anti-CD3 antibody (HIT3a) gene and its expression]
1State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS&PUMC, Tianjin 300020, China.
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.
Conclusion:
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.