Construction And Characterization Of A High-affinity Humanized CD34 Monoclonal Antibody

The CD34 antigen,a transmembrane glycoprotein of 110 kDa,is expressed on all measurable hematopoietic stem and progenitor cells.In the clinical hematopoietic stem/progenitor cell transplantation,CD34 antibodies were widely used to purify human hematopoietic stem/progenitor cells.But all the current anti-human CD34 monoclonal antibodies(mAbs) are murine,which have the potential to elicit human antimouse antibody(HAMA) immune response.In the present study,we have developed novel humanized CD34 antibodies to reduce the immunogenicity and improve the safety of hematopoietic stem cell transplantation.Three hybridomas,which secreted mAbs reacting with the human CD34 molecule, were established and named 5B12,4C8 and 2E10.The isotypes of mAbs 5B12,4C8 and 2E10 were determined to be(IgG2a,κ),(IgG1,κ) and(IgG3,κ),respectively.All the three mAbs 5B12,4C8 and 2E10 were shown to be able to effectively bind to CD34 positive KG-1a cells similar to the commercial anti-CD34 mouse mAb 581.Western blotting analysis showed that the mAbs 5B12,4C8 and 2E10 reacted with one specific band with a molecular weight of about 110 kDa,further indicating that 5B12,4C8 and 2E10 specifically recognized the CD34 molecule.Epitope identification results indicated that the epitopes detected by 5B12,4C8 and 2E10 belonged to classâ… ,classâ…¡and classâ…¢epitopes,respectively.The variable regions cDNAs for the light and heavy chains of 5B12,4C8 and 2E10 were cloned from the hybridoma cells by 5'RACE.The obtained variable region genes were sequenced and analyzed.To construct the chimeric antibody light and heavy chain expression vectors,the cloned anti-CD34 antibodies light and heavy chain variable region cDNAs(VH) were respectively fused to the human antibody light and heavy chain constant region genes (CH).The chimeric light and heavy chain genes were respectively inserted into the expression vectors,resulting in the chimeric light and heavy chain expression vectors.Then appropriate light and heavy chain expression vectors were co-transfected into CHO-K1 cells and the clones producing the highest amount of chimeric antibodies were selected.Finally,the three chimeric antibodies were purified by Protein A affinity chromatography from the serum-free culture supernatants of the selected transfectant and designated as c5B12,c4C8 and c2E10,respectively. Antigen-binding activity assay demonstrated that all of the three mAbs were shown to bind well to KG-1a cells,indicating that the three chimeric antibodies were correctly constructed and produced.In the competitive binding assay,c5B12,c4C8 and c2E10 could effectively compete with their respective parental mouse antibodies for binding to KG-1a cells.The avidity(mean IC_{50±SD} of c5B12,c4C8 and c2E10 was equal to that of 5B12,4C8 and 2E10,respectively,which indicated that the chimeric antibodies possessed affinity and specificity similar to that of the original murine antibodies.To further reduce the immunogenicity of chimeric antibodies,we developed a humanized antibody of 4C8 by complementarity-determining region(CDR) grafting based on computer-assisted molecular modeling.The three complementarity-determining regions from 4C8 light chain or heavy chain were grafted into human antibody light chain or heavy chain frameworks to generate humanized antibody genes.Then the humanized light chain and heavy chain genes were inserted into expression vectors respectively and were co-expressed transiently in COS cells. FCM was performed to determine the binding of humanized antibody to KG-1a cells. However,it was demonstrated that this antibody almost lost all the binding activity. Basing on the theory that some changes in the human FRs are essential to reconstitute full binding activity of the humanized antibody,important residues that may have influences on binding activity in the human FRs were analysis by computer-assisted designing and backmutation assay was carried out.A number of light and heavy chain versions were produced to evaluate the contribution of FR residues to antigen binding activity.It was demonstrated that residues 2,46,68,69,82,91 on the heavy chain FRs or residues 3,4,46 on the light chain FRs were important to the binding activity of heavy chain or light chain.Backmutation of all these residues generated humanized antibody h4C8.This humanized antibody completely restores the binding activity of chimeric antibody c4C8.In order to meet the need of further experimental studies and clinical applications,we expressed the humanized antibody in CHO-K1 cells and obtained the CHO cell transfectants that stably produced antibody by a positive selection procedure.Finally,h4C8 was purified from the CHO cell serum-free culture supernatant by Protein A affinity chromatography.In the competitive binding assay, h4C8 could effectively compete with 4C8 for binding to KG-1a cells.The avidity (mean IC₅₀±SD) of h4C8 was about equal to that of 4C8,suggesting that this humanized antibody possessed affinity and specificity similar to that of the original murine antibody,indicating that h4C8 was a successful humanized antibody.In summary,due to the expected better safety profiles,the humanized antibody h4C8 has the potential to be an alternative to mouse anti-CD34 antibodies routinely used for hematopoietic stem cell selection.