Study Of Gene Engineered Antibody Against Humanized NH₂ Terminal Lipopolysaccharide Binding Protein And Its Biological Activity

Background:Endotoxemia induced by lipopolysacchride(LPS) is one of the complications in traumatic and burn patients. LPS is a prominent cell wall component of gram-negative bacteria and being released into blood when the bacteria are killed. Lipopolysacchride binding protein(LBP) secreted by hepatocytes may bind with LPS and transfer it to the membrane-bound isoform of CD14(mCD14) or soluble CD14(sCD14), after that the forming tri-complex(LPS/LBP/CD14) activates the effectors cells by the combined actions of the transmembrane signaling unit of Toll-like receptor 4 (TLR-4) and the accessory protein MD-2. As the result, the nuclear transcription factor Kappa B which locates in the cytoplasm interchanges into nucleolus, and then various of transcript factors known as pro-inflammatory mediators and cytokines are activated, by which the systematic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) is induced. Normal concentration of LBP in human serum is about 5 to 10 fig/ml and increases intensively follow the increasing of LPS in blood circulation. It has been documented that the lower concentration of LBP enhances the sensitivity of LPS by catalyzing the binding of LPS with CD14. In addition, it has been verified that the site of full-length LBP binding with LPS is at its N-terminal 91^st to 100^th amino acids. It is supposed that the endotoxemia can be inhibited if the function of LBP is blocked off, moreover the antibody which against the NH-LBP may also antagonize the full-length LBP.There are a few antagonistic reagents towards the LBP such as monoclonal antibody from animals, small molecular polypeptide et al. The antibodies from animals have some limitations: to produce these antibodies in large-scale and keep the hybridoma-cells stabilization is difficult; these antiboies may induce human anti-mouse antibody easily andhave short half-life in vivo; furthermore cannot be used to prepare protein fusion toxin. Otherwise the size of full-length antibody is too large to penetrate into target tissue and lack of site specificity. With the development of molecular biology and molecular immunology, the McAb from animals can be remodeled to form human-mouse chimerical antibody or gene engineered antibody, such as scFv, dsFv, bispecificdiabodies, Fab, F(ab')2> miniantibody, et al. Gene engineered antibodies can be constructed not only from hybridoma cells, but also from spleen tissue or peripheral blood mononuclear cells with/without being immunized, and the full-length antibody, small molecular antibody or antibody fusion toxin, et al can also be constructed.The scFv is a kind of linear polypeptide with a Mr of 26 kDa translated from a single gene. It is composed of the variable domains from matching heavy and light chains which have been connected by a short linker sequence. The scFv is the smallest functional molecular of antibodies against the specific antigen. Due to its relatively small size, the scFv penetrates into the target tissue easily and being cleared quickly from blood circulation as compared with the larger size antibodies, such as Fab', Fab'2 and IgG The preparation of gene engineered antibodies includes: construction of antibody repertoire; screening antibody against a specific antigen from repertoire; identification the screened antibody. In the course of the preparation for antibodies, the size of antibody repertoire is the key factor for screening the high affinity antibody, the repertoire size of being transformed into bacteria(109-1010) is too small to screen high affinity antibody, otherwise this limitation is solved with the introducing of expression technology in vitro (ribosome display). Ribosome display has some potential advantages, such as construction of large size repertoire easily, reduction of discrepancy during expression in bacteria and screening the high affinity antibody rapidly.Objective:To construct humanized scFv antibody repertoire and screen the high affinity scFv against NH-LBP by ribosome display technology. To express the scFv antibody against NH-LBP in P. methanolica and study the biological activity of the antibody in vitro and in vivo.Methods:1. High titer recombinant virus was obtained by infection sf21 insect cells with shuttle plasmid pBacmidtLBP which contained the binding-site of LBP with LPS over 3 cycles. Andthen the sf21 insect cells were infected by recombinant virus to express NH-LBP, the expressive supernatant was purified by affinity chromatography and the production was identified by SDS-PAGE and Western-blot.2. The VH and VL chains of antibody were amplified by RT-PCR from the peripheral blood mononuclear cells of healthy donor respectively and linked by a soft polypeptide (Gly4Seri) x3 through SOE-PCR to form a scFv repertoire.3. The scFv antibody repertoire was expressed in vitro by using the ribosome display technology and screened with micro-plate coated with NH-LBP, meanwhile the obtained ARM complex was reverse-transcript into cDNA which coded scFv antibody against NH-LBP.4. According to the gene homologous recombinant principle, the recombinant expression vector pPMETaA/scFv was constructed and transformed into P. Methanolica, after being induced by methanol, the lyses of cells was purified by using metal affinity resin and the scFv antibody was identified by protein electrophoresis and immunoblotting.5. The inhibition role of the scFv antibody on the LPS binding with U937 cells was observed by FCM; the morphologic changes of liver, lung and kidney of the burned mice combined with endotoxemia being intervened by scFv antibody were observed, at the same time the concentration of TNF-a and IL-6 in serum was detected.Results:1. The result display that the 2.624mg NH-LBP with Mr 30kDa was obtained after being expressed with baculovirus expression vector system.2. The humanized scFv repertoire was constructed with PCR method and expressed, screened in vitro by ribosome display technology and the production with Mr 27kDa after being assayed by immunoblotting. After being selected by transcription/translation system in vitro over five cycles, the cDNA of coding scFv antibody against NH-LBP was recovered by RT-PCR.3. The cDNA of coding scFv was sequenced and blasted in Gene Bank, the results revealed that the 1st to 811th bp of sequence coded VH chain of antibody, 812th to 856th coded soft linker polypeptide and 857th to 1200th bp coded VL chain of antibody. And then the amino acids sequence of cDNA coding scFv was also blasted and got the analogic results.4. The cDNA of coding scFv against NH-LBP was transformed into PMAD11 yeast cells,and the lyses of yeast cells was purified and identified, it was obtained about 5.4062mg scFv with the Mr 35kDa.5. The result of FCM indicated that the absorption apex dropped down obviously after the U937 cells were treated with FITC-LPS and the scFv antibody simultaneously.6. After being treated with scFv, the pathological changes of the organs in mice burned combined with endotoxemia alleviated and the concentration of TNF-a in serum decreased significantly within 3hrs (p<0.05). The content of IL-6 in scFv-treated groups was indiscriminate as compared with the model controls (p>0.05) except the 6hrs group (p<0.01).Conclusions:1. The NH-LBP was obtained through baculovirus expression vector system. The scFv antibody repertoire was constructed successfully from peripheral blood mononuclear cells of healthy donor, and then the cDNA which coded the scFv against NH-LBP was gotten by ribosome display and verified by the utilization of blast in Gene Bank.2. The scFv with Mr 35kDa was obtained after being expressed in P. methanolica and purified by affinity chromatography. The results of experiments in vivo and in vitro indicated that the scFv against NH-LBP inhibited the endotoxemia to some certain degree.3. In this study, we firstly introduced humanized antibody technology in prevention and treatment of endotoxemia, and the scFv antibody was expressed in yeast cells. Therefore, these results conduce to understand the role of LBP in endotoxemia and found a basis for the prevention and treatment of infection in traumatic (burn) patients.