Study On The Preparation And Characteristics Of CD19 Human-mouse Chimeric Antibody Hm2E8b

Immunotherapy using monoclonal antibodies is an effective and safe method for the treatment of human lymphoid maglignancies. CD19 is a 95kDa transmembrane glycoprotein, expressed on most of B-lineage acute lymphoblastic leukemia, chronic lymphocytic leukemia, and lymphomas. Several CD19-recoginizing antibodies have been evaluated for the treatment of B-lineage malignancies in vitro, in mouse models, and in clinical trials, including unconjugated antibodies, antibody-drug conjugated, and bispecific antibody targeting CD19 and CD3.2E8, a murine IgM-type anti-CD19 antibdoy, was generated in our laboratory previously. We have demonstrated that the 2E8 and antibody-norcantharidin conjugated immunotoxin (2E8-NCTD) could specifically target the CD19 expressing B-lineage leukemia cells. However, as 2E8 is a murine MAb, it is immunogenic and does not mediate effector function in human due to the murine origin of its constant region. In the present study, we have connected the VH and VL domain by a short peptide linker to form a sigle-chain Fv (scFv) antibody fragment, then fused to the Fc (hinge,CH2,CH3) domains of human IgG1. The results revealed that the scFv-Fc fusion protein retained the specific antigen-binding affinity of the parental antibody.Methods:1. Confirm the sequence of the ZCH-4-2E8 signal peptides and variable domains: The sequence of the ZCH-4-2E8 signal peptides and variable domains was amplified by 5'RACE; the gene was cloned to pGEM?-T-Easy vector to establish the 2E8-cDNA clone and sequenced. The sequences of the signal peptides were predicted by Signal 1P3.0 software.2. Construction of recombinant expression vector pHMCH3-Hm2E8:1) The Fc fragment of human IgGl was amplified from a baculovirus expression vector pAc-?-CH3 by PCR and subcloned into pGEM?-T-Easy vector to generate TA-Fc. After sequencing, the Fc domain was subcloned into backbone vector pcDNA3.1(+)to generate the vector pHMCH3.2) The genes encoding the VH and VL of 2E8 were constructed in to scFv through splicing overlap extension and subcloned into pGEM?-T-Easy vector to generate TA-scFv2E8 After sequencing, the scFv2E8 domain was subcloned into pHMCH3 to generate the expression vector pHMCH3-Hm2E8b.3. Stable transfection of CHO cells by recombinant expression vector and expression of the chimeric antibody Hm2E8b:1) The recombinant vector was transfected to CHO cells by using lipofectamineTM 2000. Transfected cells were selected in the presence of G418 (600?g/ml).2) Two weeks after selection, the total RNA from the Hm2E8b gene transfected CHO cells (Hm2E8b-CHO) was extracted and analysed by RT PCR.3) Hm2E8b-CHO was incubated with fluorescein isothiocyanante-conjugated goat anti-human IgG Fc antibody (GAH-Fc-FITC) and observed under a muticolor fluorescence microscope.4) The supernatant of Hm2E8b-CHO was detected by flow cytometric analysis.5) The positive clone was isolated by limiting dilution, the highest-producing clone was chosen.6) The supernatant was harvested, and then purified by SPA Sepharose columns. The purified chimeric antibody Hm2E8b was identified by SDS-PAGE and Western-Blot.7) Study on the function of Hm2E8b antibody:The ability of Hm2E8b killing target cells was analyzed by complement dependent cytotoxicity (CDC). Results:1. Confirm the sequence of the ZCH-4-2E8 signal peptides and variable domains:The sequences of the signal peptides were predicted by Signa11P3.0 software. The signal peptides of light and heavy chains were 60bp and 57bp, encoding 20 and 19 amino acid residues, respectively. Compared with the sequences of the variable region genes of the 2E8 antibody obtained before, there were 3 (at 3bp(T-C),18bp(G-A) and 19bp(A-T)) and 5 (at lbp(G-C),4bp(G-A),6bp(G-C),7bp(A-C) and 13bp(G-C)) differences in positions exsisted in VL2E8 and VH2E8, respectively.2. Construction of recombinant expression vector pCDNA3.1 (+)-L2E8H-scFv2E8-Fc:The Fc domain and scFv2E8 were amplified successfully and subcloned into pCDNA3.1 (+)to generate eukaryogenic expression vector pHMCH3, then confirmed by DNA sequencing.3. Stable transfection of CHO cells by reeombinant expression vector and expression of the chimeric antibody Hm2E8b:The recombinant expression vector was transinfected into CHO cell and selected by G418 for two weeks. The band of the Hm2E8b gene was amplified from the CHO-Hm2E8b cells by RT-PCR. Under multicolor fluorescence microscope, the fluorescence could be clearly observed in the cytoplasm of CHO-Hm2E8b cells. The supernatant of Hm2E8b-CHO was detected by flow cytometric analysis. The positive rate of Hm2E8b on CD19+Nalm6 cells was 93.32%with mean fluorescence intensity (MFI) of 20.14%. Hm2E8b could block its parental murine antibody 2E8-FITC on Nalm-6 cells indicating that the binding activity and specificity of the engineered antibody Hm2E8b were not significantly changed. The supernatant was purified, and then identified by SDS-PAGE and Western-Blot analysis. The molecular weights of Hm2E8b monomer and dimmer were approximately 50KDa and 130KDa, and a 200KDa multiner antibody was identified simultaneously. Hm2E8b could kill the target cells through the effects of CDC Conclusions:The human-mouse chimeric antibody Hm2E8b was successfully expressed in CHO cells with excellent binding activities and the ability of killing the target cells, comparable with its parental antibody 2E8 of murine origin.