Construction, Expression And Identification Of Anti-CD45 Mouse/Human Chimeric Antibody

BackgroundAML kills 60% to 70% of affected adults despite improvements in induction chemotherapy and HCT. High-dose chemoradiotherapy followed by stem cell transplantation offers the only chance of cure for many patients with treatment-resistant leukemia. Several groups have documented the feasibility, safety, and efficacy of treating AML. Therefore, targeted radiation to leukemic progenitor and stem cells residing in hematopoietic bone marrow was the safest resolution to use radiolabeled antibodies directed to epitopes expressed on leukemic cells or normal hematopoietic bone marrow cells.For treatment of acute myeloid leukemia, monoclonal antibodies have been directed to CD33,CD66, and CD45, and studies using anti-CD45 have shown the most promising results.CD45 antigen is stably expressed at a high density on the surface of all leukocytes, their precursors and more than 70% of nucleated cells in normal bone marrow, with an average copy number of approximately 200,000 molecules per cell. CD45 (also called GP180, T200, or leukocyte common antigen) is a～200-kDa tyrosine phosphatase that is stably expressed at a high density on the surface of virtually all hematopoietic cells except mature erythrocytes and platelets. At least 90% of myeloid leukemias express CD45 and the antigen is not found on tissues of nonhematopoietic origin, making CD45 an attractive target for treatment of leukemia.The abundant expression of CD45 on virtually all leukocytes, including myeloid precursors in bone marrow and mature lymphocytes in lymph nodes as well as more than 90% of AML samples, provides a high number of Ab binding sites in these tissues for patients in remission or relapse. It is expressed by both normal and malignant cells, and therefore anti-CD45 antibodies can deliver to marrow, spleen, and lymph nodes in patients with acute leukemia.In radioimmunotherapy, a specific antigen is targeted to selectively deliver radioactivity to cancer cells. Radioimmunotherapy of blood-borne cancers has been the most successful because of high accessibility of the target antigen expressed on inherently radiosensitive cells. The CD45 antigen acts as an attractive alternative target for radioimmunotherapy of AML and MDS.Radiolabeled anti-CD45 Ab can delivery radiation to antigen-positive or surrounding antigen-negative cell in myeloid tissues. Therefore, the radioimmunoconjugate does not need to bind to every leukemia cell at a high density or penetrate homogeneously into bone marrow or tumor cell masses to induce lethal DNA damage. For patients in remission, even blasts that do not express CD45 may be killed if they are surrounded predominantly by nonmalignant hematopoietic cells, because of the bystander effect. The characteristics of the CD45 antigen have prompted investigations of radiolabeled antibodies against CD45 with transplantation therapy. With the CD45 molecular isomer research, the development of flow cytometry immunophenotyping and anti-CD45 monoclonal antibody application,CD45 molecules in the application of immunology and hematology research would get further attention. We can expecte, CD45 could play greater role in clinical diagnosis, treatment, prognosis. Our group has documented the promise of anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high. So we have been committed to continuous improvement.In this article, we report in the first time the construction of anti-CD45 mouse/human chimeric antibody, and describe its merits on the blood system diseases. To lay solid foundation for further study on this chimera label with 131I on the pretargeted radioimmunotherapy (PRIT) to use autologous and allogeneic haematopoietic stem cell transplantation.Humanized or chimeric monoclonal antibodies (MoAbs) have been developed that react with antigens expressed by hematopoietic cells. They have been used either unlabeled('native' or 'naked'antibodies) or conjugated with toxins, radioisotopes, or antitumor drugs. According to whether they carry the material into other connections not bound monoclonal antibody, anti-cancer drug conjugated monoclonal antibody and isotope conjugated monoclonal antibody. So far, five drugs by the U.S.FDA approval for clinical, which are no-binding human-mouse chimeric antibody or humanized antibody. But use no-binding CD45 monoclonal antibody had little effect in the human body and with the limitation of murine application, the current research on the CD45 monoclonal antibodies mainly on antibodies connect with radionuclides 131I and 213Bi as part of hematopoietic stem cell transplantation conditioning to reduce the dose of TBI and its side effects. Animal testing andⅠ/Ⅱclinical trials have confirmed, CD45 monoclonal antibody can be selectively deliver radionuclides 213Bi and 131I into bone marrow, liver, spleen and lymph nodes. Vallera et al. used 90Y labeled anti-CD45 monoclonal antibodies treat for experimental animal models of lymphoma, the results showed that give appropriate doses of 90Y-Anti-CD45 to mice, tumors disappeared after 6 days, and without one case happen to recurrence until 135 days. Liver, kidney, small intestine biopsy found no apparent damage. We have used anti-CD45 monoclonal antibody labeled with 90Y for the treatment of acute leukemia, preliminary experiments to select CDTPA to chelate 90Y and anti-CD45 monoclonal antibody. CDTPA is a classic metal ion chelating agent, as early as 1985,Hnatowich took CDTPA chelae IgG and 90Y, we obtained specific activity was 1.7μCi/μg, the radiochemical purity more than 90%,24 hours dissociation was 13%.The products 90Y-CDTPA-CD45 monoclonal antibody under the conditions of 20:1 CDTPA/IgG, the labeling efficiency was 95%, the radiochemical purity was more than 99%; good stability,24-hour dissociation rate was 8.32%. These results in accordance with Zhang Jinming et al.reported that the dissociation rate was 11.9%. Indirect immunofluorescence assay also proved that the immunological activity of the chelates have not been obviously affected by the combination with the high rate of nuclides. The binging rate with AML cells was good. It is a better targeted therapeutic agents and laying the basis for the further study. Antibody therapy is ideally suited to the treatment of acute myeloid leukemia because of the ready accessibility of neoplastic cells in the circulation.But mouse-derived whole-molecule antibodies have been demonstrated to have many limitations for therapeutic applications due to high immunogenicity and large molecular weight, and human anti-mouse antibody (HAMA) responses that can cause allergic reaction and damage in the human body.In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effect or mechanism in vivo, we herein developed its chimera. Genetically engineered antibody could have more important practical significance. To remove these difficulties, humanized antibody with a human antibody constant region was genetically engineered. Humanized human-mouse chimeric antibody has employed the constant region of human antibody to replac C region of murine monoclonal antibody. Human-mouse chimeric antibody not only retains the specificity of mouse monoclonal antibody but also decreases the immunogenicity. This genetically engineered antibody has more advantage than the mouse antibody in the function of complement mediated cell killing of target antigens and phagocytic effect. In addition, with construction of chimeric antibodies, we purposely chose the type of antibody, making it more effective to play the effectiveness of antibodies. Therefore, the variable region genes of this MoAb were cloned and ligated into chimeric antibody expression vector pFUSE-CHIg-hGl and pFUSE2-CLIg-hk, generating chimeric anti-human CD45 MAb (chi-mAb CD45)expression vectors,transfected CHO cells,expressed and purified chimeric antibody.Now we first generated and report a novel anti-CD45 mouse/human chimeric antibody (Chi-CD45).Untill now, it is no reported about reconstruction of humanized anti-CD45 monoclonal antibody at home and abroad. In vitro assay, it revealed that pro-competitive inhibition with the monoclonal antibody showed the change by gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit Jurkat/human PBMC proliferation were enhanced with the increase of the concentrations of chimeric antibody.Aim To construct the eukaryotic expression vector of chimeric anti-CD45 mouse/human chimeric antibody and realize its expression.Methods VH gene and VL gene of anti-CD45 monoclonal antibody were amplified by PCR. Use DNAtools, IMGT/QUEST and EBI TOOLS:ClustalW2 analysis software to compare the homology of the light chain and heavy chain gene respectively.After identification by DNA sequencing and PCR, the recombinant expression vectors transfected into CHO cells, and then Western blotting was used to identify the expression of chimeric antibody. Molecule modelling simulated protein secondary and tertiary structure of chimeric antibody, and fatherly verified its structural integrity and functional effectivity.The supernatants of transfected CHO cells were harvested and purified by a HiTrap Protein A HP.The purified antibody samples were identified by SDS-PAGE. Furthermore, the assays of the ability of V region of chimeric antibody binding to surface CD45 on CD45-expressing tumor cells and human PBMC cells, pro-competitive inhibition with murine-derived monoclonal antibody, C region of chimeric antibody mediating CDC to CD45-expressing tumor cells and inhibit the activity of target cells were valuated by FACS.Results The PCR amplified gene fragments of VL and VH were identified by 1% agarose gel electrophoresis and DNA sequencing, and the length and sequence of amplified fragments were identical to the theoretical values. Competitive V-gene PCR is a convenient and rapid method to determine the sequence of functional antibody V-genes in hybridoma that express the endogenous aberrant myeloma mRNA. The primer VL5'5 hybridized the variable VL genes belong to IGKV1-117*01 family by IMGT/QUEST system analyze, the identity was 99.32%.VJ genes showed characteristic according to IGKJ1*01 family, the homology was 100.00%.Sequence analysis revealed these functional VL gene products anti-CD45 variable region 333bp long,before it has a 57 bp signal peptide.The recombinant expression vectors were respectively amplified by the specific primers, and both the lengths of amplified fragments and the reading frames were identical to the theoretical values. The expression of chimeric antibody screening revealed that took the way of co-transfected CHO cells the yield was very low, follow-up results were not satisfactory. The low yield of recombinant antibody genetically engineered antibody preparation biggest obstacle, high expression cell line transfected cells relative to the whole group is very rare,cultured in a stable output can form gradient descent, excessive growth of non-expressing cells stable transfected cells can yield a whole was more obvious, and even the formation of non-expression of cell groups. During cell lines produced IgG most of them can produce more light chain genes. In the research of human-mouse hybrid cell line study, we found the correlation of the ratio of light chain secretion and intracellular content of light-chain. The speed of produce is faster than the synthesis proportion of heavy chain, and the concentration has the same result. Some literature reports step by step transfection can increase the functional cells expression to 5～30%. At first we used high concentration antibiotics to transfect light chain gene to CHO cells stably, and then transfected heavy chain gene. Reduction the amount of medium volume in the follow-up culture process, extend the cultured time of the transfected cells for 7 to 10 days or use etc can increase the relative concentration of antibodies. DMSO/sodium can inhibit cell growth,promote antibody secretion on the CHO cells (promote the yield of recombinant proteins approximately 2-fold).We studied on the relevant literature and used the step by step transfection method. We designed an alternate inducing methods, which used medium with or withour NaBu, ELISA results showed that production of chimeric antibodies have greatly improved. The detection index of molecular modeling shows the anti-CD45 antibody was according to the functional chimeric antibody. InsightⅡsoftware simulated Fab segment domain of anti-CD45 antibody, the model showed that the antibody has antigen-binding groove completely can exert the antibody-related functions.The detection of transfected cells expression confirmed successfully. We freezing the part of the transfected cells into the liquid nitrogen, after 5 months recovered them. After conventional limiting dilution cloning and ELISA, we identified that after frozen in liquid nitrogen and passaged several times,the cells still able to maintain good growth conditions in vitro.Western blotting demonstrated that the expression products were mainly in the supernatants of culture for 3d. The supernatants of tranfected cells were purified by a HiTrap Protein A HP. SDS-PAGE showed that there were only two bands of heavy chain and light chain in the purified products, and the molecular weight of purified antibody was identical to the theoretical values. FACS indicated that the purified antibodies were able to specially bind to surface target antigen on CD45-expressing tumor cells and human PBMC cells, and the capability of binding was similar to that of murine-derived monoclonal antibody at the same concentrations. The assay of pro-competitive inhibition with the monoclonal antibody showed the change of gradient. The effects of CDC mediated by chimeric antibody and ability to inhibit tumor cell proliferation were enhanced with the increase of the concentrations of chimeric antibody.Conclusion:We successfully constructed the recombinant eukaryotic expression vector of anti-CD45 mouse/human chimeric antibody, and the complete chimeric antibodies having biologic activity were obtained.To lay solid foundation for further study on on the blood system diseases.