| OBJECTIVESAcute leukemia is the most common type of malignancy in children. There are at least 15000 new cases each year in China. About 70% of these patients are acute lymphoblastic leukemia (ALL), about 25% of them are acute myeloid leukemia (AML). Leukemia has the highest mortality in children and adolescent malignancy. In recent years, the prognosis of childhood ALL has greatly improved by combined chemotherapy. But about 20%-30% of high risk patients lack effective therapy. The treatment of AML is even less satisfied than the ALL. The chemotherapy is not only of poor selectivity, but also has serious side effect. The chemoresistance and relapse of leukemia cells lead to the failure of therapy. So it is necessary to search for more new treatment methods with better treatment selectivity and fewer side effects. Targeting therapy has been becoming a promising alternative for cancer treatment in the recent decades. Rituximab (Mabthera) is a human mouse chimeric anti-CD20 monoclonal antibody, which is the most successful case of targeting therapy. Mabthera is the first monoclonal antibody to enter the clinical use. This monoclonal antibody has great therapy effect in CD20+B cell lymphoma.CD45 is leukocyte common antigen (LCA) located on the surface of leukocyte and is expressed on all the hematopoietic cells except the mature erythrocyte and platelets. 85%-90% of leukemic cells also express CD45 antigen on their surface. But the non-hematopoietic cells do not express CD45 so that CD45 can be used as an optimal target for hematopoietic malignancy. Anti-CD45 monoclonal antibodies provide a new way of leukemia targeting therapy.Human CD45 protein is structurally heterogenous, consisting of 5 isoforms ranging in size from 180 to 220kDa (CD45RO, CD45RB, CD45RAB, CD45RBC and CD45RABC). The heterogeneity of the protein results from differential RNA splicing of exon 4 (or A),5 (or B) and 6 (or C). CD45RAB and CD45RABC are named as CD45RA.ZCH-6-3A4 (3A4), a new clone of CD45 isoform monoclonal antibody (mAb) generated in our lab was assigned into CD45RA category by the 7th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA7) in 2000. According to our previous study,3A4 can not block the reaction of CD45 antibody (clone name 2D1, IgGl, recognizes all the members of the human CD45 family with molecular masses of 180 to 220 kDa) to the KGla cells, indicating that 3A4 and CD45 recognize the different epitope.3A4 can not block the reaction of CD45RO antibody (clone name UCHL-1, reacts with the 180 kDa isoform of CD45 antigen not encoded by exon A, B, or C) to the CD45RO positive T lymphocytes, indicating that 3A4 does not recognizes CD45RO antigen. However,3A4 can block the reaction of CD45RA antibody (clone name L48, recognizes a MW of 220 kDa isoform of the CD45, CD45RABC) to the KG1a cells. Since 3A4 can block the the reaction of CD45RA antibody (clone name L48) to the KGla cells, it should recognize the same antigen epitope with the regular CD45RA. But our previous studies showed that 3A4 and the regular CD45RA had different reactive profiles to the leukemic cells. So it is necessary to clarify the antigen epitope recognized by the antibody of 3A4.METHODS(1) Preparation and identification of 3A4 FITC.The ascites containing 3A4 antibody (Ab) derived from Balb/c mice was purified by affinity chromatography using SPA Sepharose column.3A4 FITC was prepared according to Marshall's method and evaluated by flow cytometry (FCM).(2) The antigen recognized by the 3A4.The alternative splicing of exon 4,5 and 6 generates variants of CD45 protein. We designed a pair of PCR primers to amplify the sequence of CD45 from the exon 2 through the exon 8 (CD45-1) and to detect the distribution of the CD45 variants on the gene level. The reactivity of 3A4 with normal hematopoietic cells, PHA activated T lymphocytes, leukemic blasts and leukemic cell lines were identified by FCM. Western Blot was used to detect the reactivity of 3A4 with leukemic cell lines.(3) Construction of recombinant expression vector pcDNA3.1+/CD45RBC.According to the CD45 gene sequence (NM002838) from the gene bank, the entire sequence of CD45 contains 33 exons with 5330 bp. In order to facilitate the PCR amplification of the products, we arbitrarily divided CD45 into 4 parts, i.e. CD45-1, CD45-2, CD45-3 and CD45-4. The PCR product of CD45-1 was the sequence from exon 2 through exon 8;'CD45-2 from exon 8 through exon 15; CD45-3 from exon 15 through exon 24 and CD45-4 from exon 24 through exon 33. Then splicing by overlap extension PCR (SOE-PCR) was used to connect up the 4 parts. The restriction sites for Hind?and Xho?were added by PCR. The cDNAs of human CD45 were obtained by SOE-PCR and cloned to pCR(?)-2.1 vector to establish the TA clones. The clone pCR(?)-2.1/CD45RBC was first confirmed by the sequencing, so the expression vector of CD45RBC was constructed first. The pCR(?)-2.1/CD45RBC and expression vector pcDNA3.1+were both cleaved by endonucleases Hind?and Xho?and followed by the T4 DNA ligase connection. After transformation to E.coli DH5?, the recombinants were selected, amplified, confirmed by restriction endonuclease and sequencing. The inserted sequence was then compared with the CD45RBC gene sequence to confirm the correct insertion.(4) Construction of recombinant expression vectors pcDNA3.1+/CD45RA, pcDNA3.1+/CD45RAB and pcDNA3.1+/CD45RABC.Because of the alternative splicing of the exon 4,5,6 in the geng part of CD45-1, we named the CD45-1 isoforms as CD45RO-1, CD45RA-1, CD45RB-1, CD45RC-1, CD45RBC-1, CD45RAB-1 and CD45RABC-1, the TA clones were constructed respectively. The restriction site for Afe I is located in the CD45-1 sequence of each variant of CD45 gene. The pCR(?)-2.1/CD45RA-1, pCR(?)-2.1/CD45RAB-1, pCR(?)-2.1/CD45RABC-1 and expression vector pcDNA3.1+were cleaved by endonucleases Hind III and Xho I and followed by the T4 DNA ligase connection. After transformation to E.coli DH5?, the recombinants were selected, amplified, confirmed by restriction endonuclease and sequencing. The inserted sequences were then compared with the CD45RA, CD45RAB and CD45RABC gene sequences respectively to confirm the correct insertions.(5) Stable tranfection of CHO cells by recombinant expression vectors of CD45 gene and identification of 3A4 antigen epitope.The empty vector and the recombinant expression vectors pcDNA3.1+/CD45RA, pcDNA3.1+/CD45RBC, pcDNA3.1+/CD45RAB and pcDNA3.1+/CD45RABC were amplified and highly purified plasmid DNA were isolated by QIAGEN plasmid purification mini kit. Then the vectors were transfected into CHO cells by using LipofectaminTM 2000 kit. G418 with certain dilution were put into cell culture meidum to establish stable clones. Total cell RNAs were extracted and were reverse transcripted to cDNA. The transfected genes were detected by PCR. The expression of CD45 proteins were detected by the FCM. The antigen epitope recognized by the 3A4 was identified by the FCM and Western blot. (6) Construction of recombinant expression vector of CD45 truncked gene (CD45-B17).The recombinant expression vectors of CD45 gene were almost 10000bp, it was very difficult to establish stable transfected cell line. In order to improve the efficiency of transfection, we also constructed the recombinant expression vectors of CD45 truncked genes.The exon 16 is encoded the transmembrane domain of CD45 protein. We inserted a stop codon TAG in the exon 17. The restriction site for BsrG?is located in the exon 10. The stop codon TAG and the restriction sites for Xho?were added by PCR. The cDNAs of human CD45 from the restriction sites for BsrG?to the restriction sites for Xho?was obtained by PCR and cloned to pCR(?)-2.1 vector to establish the TA clone pCR(?)-2.1/B17. It was confirmed by the sequencing. The pCR(?)-2.1/B17 and pcDNA3.1+/CD45RA, pcDNA3.1+/CD45RBC, pcDNA3.1+/CD45RAB, pcDNA3.1+/CD45RABC were cleaved by endonucleases BsrG?and Xho?and followed by the T4 DNA ligase connection. After transformation to E.coli DH5?, the recombinants were selected, amplified, confirmed by restriction endonuclease and sequencing.The pCR(?)-2.1/CD45RC-1 and pcDNA3.1+/CD45RBC-B17 were both cleaved by endonucleases Hind?and Afe?and followed by the T4 DNA ligase connection. After transformation to E.coli DH5?, the recombinants were selected, amplified, confirmed by restriction endonuclease and sequencing.(7) Stable tranfection of CHO cells by recombinant expression vectors of CD45 truncked gene and identification of 3A4 antigen epitope further.The empty vector and the recombinant expression vectors pcDNA3.1+/CD45RA-B17, pcDNA3.1+/CD45RC-B17, pcDNA3.1+/CD45RBC-B17, pcDNA3.1+/CD45RAB-B17 and pcDNA3.1+/CD45RABC-B17were amplified and highly purified plasmid DNAs were isolated by QIAGEN plasmid purification mini kit. Then the vectors were transfected into CHO cells by using LipofectamineTM 2000 kit. G418 with certain dilution were put into cell culture medium to establish the stable cell lines. Total cell RNAs were extracted and reverse transcripted to cDNAs. The transfected genes were detected by PCR. The expression of CD45 truncked proteins were detected by the FCM. The antigen epitope recognized by the 3A4 was further identified by the FCM and Western blot.RESULTS(1) The 3A4 turned out to be two bands of approximately 58kDa and 30kDa when identified by SDS-PAGE. The positive rates of 3A4 FITC on KG1a cells exceeded 98% with a narrow peak.(2) The 3A4 antigen was highly expressed on the human T lymphocytes, B lymphocytes and NK lymphocytes (78.16±8.77%?99.31±1.01%?88.58±10.81%), partially on the monocytes (39.39±12.28%), and nearly negative on the granulocytes (2.07±0.35%). The longer the T lymphocytes activated by PHA, the less the 3A4 antigen expressed on the lymphocytes. The positive rates of the regular CD45RA (L48) were significantly lower those of the 3A4 in 30 cases of leukemic blasts (14 B lineage ALL,5 T lineage ALL,11 AML) demonstrated by the FCM.(3) The 3A4 antigen was highly expressed on the KG1a, Raji and U937 cells, lowly on the Molt-3 cells and negatively on the Nalm-6, K562 and HL60 cells. PCR was used to detect the CD45-1 gene in the cell lines U937, KG1a, Raji, Molt-3, Nalm-6, K562 and HL60. There were different subtypes of CD45 variations expressed on human leukemic cell lines. U937 and KGla cells expressed CD45RABC-1 and CD45RBC-1. Raji cells expressed CD45RABC-1, CD45RBC-1 and CD45RB-1 (tracely). Nalm-6 cells tracely expressed CD45RABC-1, CD45RBC-1, CD45RB-1 and CD45RO-1 in gene level. Molt-3 cells expressed CD45RAB-1, CD45RBC-1, CD45RB-1 and CD45RO-1. K562 cells and HL60 cells (tracely) expressed CD45RB-1 and CD45RO-1. According to the result of Western Blot, the antigen recognized by 3A4 was about 200kDa and found much on the KGla and Raji cells, negatively on the Nalm-6 and K562 cells.(4) The recombinant expression vectors pcDNA3.1+/CD45RA, pcDNA3.1+ /CD45RBC, pcDNA3.1+/CD45RAB and pcDNA3.1+/CD45RABC were proliferated and then the DNAs were extracted from each individual vector, digested with endonuclease and sequenced. The results indicated that correct sequences were inserted with CD45RA (3714bp), CD45RBC (3801bp), CD45RAB (3855bp) and CD45RABC (3999bp).(5) The empty vector and the recombinant expression vectors pcDNA3.1+/CD45RA, pcDNA3.1+/CD45RBC, pcDNA3.1+/CD45RAB and pcDNA3.1+/CD45RABC were transfected into CHO cells.600?g/ml G418 was used to select resisitant cells according to sensitive toxicity test. The CHO cells transfected were named as CD45RA-CHO, CD45RBC-CHO, CD45RAB-CHO and CD45RABC-CHO respectively. After 2-3 weeks of culture, the expressions of target genes and proteins were detected. RT-PCR revealed that the correct genes were integrated into the genome of CHO cells. CD45 antigen can be detected on the CD45-CHO cells by FCM.(6) According to the results of FCM, the antigen that 3A4 can recognize was on the CD45RBC-CHO and CD45RABC-CHO, but not on the CD45RA-CHO and CD45RAB-CHO cells. The Western Blot showed that the antigen recognized by 3A4 was about 200kDa and found in the CD45RBC-CHO and CD45RABC-CHO cell lysates.(7) Because the recombinant expression vector of CD45 gene was almost 10000bp, it was very difficult to establish stable transfected cell line. The positive cells were lower than 10% after 5-6 passages. In order to improve the efficiency of transfection. we also constructed 5 CD45 truncked gene recombinant expression vectors. The recombinant expression vectors pcDNA3.1+/CD45RA-B17, pcDNA3.1+/CD45RC-B17, pcDNA3.1+/CD45RBC-B17, pcDNA3.1+/CD45RAB-B17 and pcDNA3.1+/CD45RABC-B17 were proliferated and then the total DNA was extracted, digested with endonuclease and sequenced. The results indicated that the vector sequences were correctly inserted with CD45RA-B17 (1583bp), CD45RC-B17 (1529bp), CD45RBC-B17 (1670bp), CD45RAB-B17 (1724bp) and CD45RABC-B17 (1868bp) individually.(8) The empty vector and the recombinant expression vectors pcDNA3.1+/CD45RA-B17, pcDNA3.1+/CD45RC-B17, pcDNA3.1+/CD45RBC-B17, pcDNA3.1+/CD45RAB-B17 and pcDNA3.1+/CD45RABC-B17 were transfected into CHO cells.600?g/ml G418 was used to select resisitant cells according to sensitive toxicity test. The CHO cells transfected were named as CD45RA-B17-CHO, CD45RC-B17-CHO, CD45RBC-B17-CHO, CD45RAB-B17-CHO and CD45RABC-B17-CHO respectively. After 2-3 weeks of culure. the expression of the target genes and proteins were detected. RT-PCR amplification revealed that the correct genes were integrated into the genome of CHO cells. CD45 antigen could be detected on the CD45-CHO cells by FCM.(9) According to the results of FCM and cell immunofluorescence, the antigen that 3A4 can recognize was on the CD45RC-B17-CHO, CD45RBC-B17-CHO and CD45RABC-B17-CHO, but not on the CD45RA-B17-CHO and CD45RAB-B17-CHO cells. The Western Blot showed that the trunckted protein recognized by 3A4 was about 70kDa and found in the CD45RBC-CHO and CD45RABC-CHO cell lysates.(10) Two cell lines separately transfected with either CD45RC-B17 or CD45RBC-B17 recombinant expression vector were obtained.CONCLUSIONS(1) The antigen recognized by 3A4 is present highly on the human T lymphocytes, B lymphocytes and NK lymphocytes, partially on the monocytes. and nearly negative on the granulocytes.(2) We have successfully constructed four eukaryotic transfection vectors of CD45 isoform genes (pcDNA3.1+/CD45RA, pcDNA3.1+/CD45RAB, pcDNA3.1+/CD45RBC and pcDNA3.1+/CD45RABC) and five eukaryotic transfection vectors of CD45 truncked genes which were from the start codon through the 17 exon including the sequence of transmembrane domain (pcDNA3.1+/CD45RA-B17, pcDNA3.1+/CD45RC-B17, pcDNA3.1+/CD45RAB-B17, pcDNA3.1+/CD45RBC-B17 and pcDNA3.1+/CD45RABC-B17). The CD45 genes were integrated into the genome of CHO cells successfully. The various recombinated proteins of CD45 successfully expressed on the CHO cell membrane. The CHO cells transfected with the CD45 truncked genes CD45RBC-B17 and CD45RC-B17 have been successfully established as stable cell lines.(3) 3A4 recognizes the transfected cells of CD45RBC-CHO and CD45RABC-CHO. We also find out that 3A4 can recognize the transfected cells of CD45RC-B17-CHO, CD45RBC-B17-CHO and CD45RABC-B17-CHO.(4) 3A4 can only recognize the C exon containing isoforms, i.e. CD45RBC and CD45RABC among the 5 isofoms of human CD45. |
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