Study On The ScFv Of Mouse Anti-human CD14 New Clone ZCH-7-2F9

Acute monocytic leukemia (AML-M5) is the common type of acute myeloid leukemias. Many studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells. It plays a great role on the diagnosis of M5 since it can be recognized by anti-human CD14 monoclonal antibodies (McAb), furthermore, the antigen-antibody complex will be internalized when CD 14 is recognized and bound by specific antibodies, which provides advantage for the antibody targeting therapy for M5 leukemia through CD14 molecules. Up till now, anti-tumor drugs' combined chemotherapy and radiation therapy are still the main modality of treatment for leukemias. Though they have worked effectively to most leukemic patients, the severe side effects and long-term poor prognosis are two major obstacles for the successful treatment of this leathal disease. In contrast to conventional cytotoxic chemotherapy and radiation therapy, McAb targerting therapy is more effective with less side effects owing to its perfect selectivity and specificity. ZCH (Zhejiang Children's Hospital)-7-2F9(2F9) gemerated in this laboratory is an anti-human McAb belonging to murine IgG1k subtype and has been designated as a new clone of CD14 by the 6th International Workshop and Conference on Human Leukocyte DifferentiationAntigens (HLDA6). According to the data obtained through side by side comparison studies with another standard anti-human CD14 McAb LeuM3, the 2F9 antibody holds higher specificity and sensitivity on the diagnosis of M5 which may provide a perfect target for the treatment of this particular type of leukemia. However, the clinical application of this mouse antibody in patients has been greatly restricted due to the occurrence of severe serum disease and human anti-mouse antiboby (HAMA) in patients during the targeting therapy. So it is necessary to reduce the immunogenicity of the mouse antibody maximally in order to administer large doses of antibody repeatedly in the patients. As we all know, the immunogenicity of a given antibody mainly exists in its constant (Fc) region while the antigen binding site is located at its varable (Fv) region. If the Fc fragment was removed properly while Fv was remained, the immunogenicity could be reduced greatly while the antigen recognizing and binding capacity could be kept. One of the practical methods currently being used is to reconstruct single chain Fv (ScFv) gene and expression in certain host cells. In this reseach, variable region (V) genes of both heavy (H) (VH_2F9) and light chain (L) (VL_2F9) of ZCH-7-2F9, a new clone of anti-human CD14 antibody were cloned and sequenced successfully. Reconstruction, purification, refolding and characterization of the genetic antibody ScFv_2f9 were carried out.1. Cloning and sequencing of the VL_2F9 and VH_2F9 genes from 2F9 antibodyFrom the mouse hybridoma cell line 93A10 subcloned from 2F9 and its fusion partner murine myeloma cell line NS-1, total RNA was prepared by using the Trizole kit. The VL and VH genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) with family specific primer pairs, respectively. The PCR products were cloned into pGEM?-T easy vectors, then transfected into DH5a and positive recombinants were identified and purified. After sequencing with automatic DNA sequencer, the sequenceswere analyzed online. The results showed that VL_2F9 consists of 321 bps encoding a peptide of 107 amino acid residues, and VH _2f9 contains of 360 bps encoding a peptide of 120 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/ V-QUEST, the VL_2F9 and VH_2F9 genes belong to mouse IGkV and mouse IGHV subgroups, respectively. On the position 23/88 of the light chain and 22/96 of the heavy chain genes, there are cysteine pairs, which play the key role in forming disulfo-bond between the two chains. Both VL and VH chain have 4 definite frame regions (FR) and 3 complementary determinant regions (CDR). In this part, the VH_2F9 and VL_2F9 genes were clon...