| Background and aims:Acute myeloid leukemia is a malignant disease originating from myeloid hematopoietic stem/progenitor cells.The immature and dysplasia myeloid cells in bone marrow and peripheral blood is the main characteristics.The morbidity of AML is about 25%amoug childhood acute leukemia in children.However,compared with acute lymphoblastic leukemia,the cure rate is much lower and the relapse rate is higher.According to reports in the literature,the 5-year survival rate of children with AML is about 65%,and 20%-60%will experience relapse.Therefore,exploration of a new therapy for relapsd/refiractory AML is an urgent issue.At present,the initial remission rate of AML in children can reach more than 85%under the current chemotherapy.However,the relapse rate is still over 30%.For children with adverse prognostic factors,allogeneic hematopoietic stem cell transplantation may be considered after the second remission.The advance of chimeric antigen receptor modified T cells,bispecific antibodies,and immune checkpoint inhibitor has opened a new chapter in immunotherapy.The treatment of AML has come to a new field.Blinatumomab,an anti-CD 19/CD3 BiTE,was approved by FDA in July,2017 for the treatment of relapse or refractory B-cell acute lymphoblastic leukemia with great clinical success.A study of relapse/refractory ALL after transplantation was conducted in children and included nine patients.Four of them reached CR after the first course of treatment.In another study,out of 70 patients receiving recommended doses,27 patients reached CR within the first two courses of treatment,14 patients’ MRD turned negative,suggesting that bispecific antibodies have great therapeutie potential.However,no highly effective bispecifie antibodies targeting AML has been made in the world.The main reason why many patients with AML can not be completely cured is that the leukemia stem cells(LSCs)or leukemia initiating cells(LICs)existing in their bodies can not be completely eliminated by current therapies.For AML,the current main target antigens of interest include CD33,CD 123 and CLL-1.Several bispecific antibodies targeting CD3 3,CD 123 and CLL-1 have been reported.For example,AMG330 is a bispecific antibody against CD33/CD3.AMG330,when given the dose as low as 0.002 mg/kg per day,can significantly prolong the survival of immunodeficient mice that received human MOLM-13 AML cells and human T cells.However,none of the above antibodies can completely eradicate the myeloid leukemia cells.The gene expression and imnunophenotype of bone marrow leukemic cells from 74 patients with CD34+ AML were studied by Goardon et al.It was found that 87.8%of the leukemic stem cells were CD45RA positive.Kersten et al analyzed bone marrow cells from 57 patients with CD34+ AML,and found that CD45RA was expressed on the membrane of CD34+CD38-bone marrow stem cells in 80.7%of the patients.These studies suggest that most of AML-LSC expresses CD45RA.Therefore,CD45RA may be a potential target for the treatment of AML.At present,there is no report of CD45RA and CD3 combined bispecific antibody.3A4 is an anti-human CD45 isotype monoclonal antibody generated in our laboratory.It belongs to murine IgG1K subclass and has been named by the 7th International Conference and Workshop on the Human Leukocyte Differentiation Antigen(HLDA7).The antibody can recognize myeloid leukemia and its stem cells,normal and malignant B lineage cells,CD45RA positive T cells,partial monocytes,but not normal memory T cells,neutrophils,erythrocytes,platelets and primitive hematopoietic stem cells.More importantly,the antibody did not react with non-hematopoietic tissues,organs and cells,which minimized the toxicity and side effects on unrecognized tissue cells.In this study,we intend to construct CD3-3A4 BiTE and to study its biological activity in vitro to pave the way for the further development of this potentially therapeutic agent for the treatment of relapsed or refractory AML.Methods:1.Construction and expression of CD3-3A4 BiTE.1)Design CD3-3A4 BiTE.According to the data from genebank,and the results of our laboratory,we have confirmed the sequences of CD3 and 3A4,the hinge region and its size,and the order of each single chain variable region(ScFv).The gene fragments of SP-CD3VL-3A4VH and 3A4VL-CD3VH-6xhis were synthesized and returned with PUC5 7 plasmid vector.2)Two target gene fragments were amplified.The returned plasmids were amplified by PCR,TA cloning.As a result,we obtain plenty of SP-CD3VL-3A4VH and 3A4VL-CD3VH-6×his DNAs.3)Construction of pcDNA3.1-CD3-3A4 vector.SP-CD3VL-3A4VH(Hind Ⅲ,BamH I),3A4VL-CD3VH-6xhis(BamH I,EcoR I)and pcDNA3.1(+)(Hind Ⅲ,EcoR I),)were digested by the related restricted endonucleases,respectively.After connection fo the DNA fragments and vector via T4 ligase,the constructed vector of pcDNA3.1-CD3-3A4 needs to be transfected into DH5α by smearing bacteria,choose clonies of interest,proliferation of bacteria by shaking,extract DNAs and have them sequenced.A software(DNAman)was used to double check the sequence of interest expected.4)Transfection of CHO cells by pcDNA3.1-CD3-3A4 vector.Extract the the PcDNA3.1-CD3-3A4 plasmid,and then transfect the CHO cells with liposome method(Roche reagent).After 24 hours of transfection,G418 was added to the optimal concentration to obtain CD3-3A4 BiTE gene containing clone and purified the CD3-3A4 BiTE protein from the supermatants.2.The biological function testing of CD3-3A4 BiTE1.Construction and expression of CD3-3A4 BiTE.1)Cell lines:In order to facilitate checking of the recognizing ability of the molecules of interest,two cell lines were selected.One cell line is T lymphoblastic leukemia cell line Jurkat,another cell line is acute myeloid leukemia cell line KG la.In order to get those two cell lines valid for the experiment,flow cytometry was used to detect PE labelled CD3 and CD45RA on the targeting cell lines.2)Gene expression,protein expression and bifunctional identification of CD3-3A4 BiTE.The expression of pcDNA3.1-CD3-3A4 in transfected CHO cells was checked by RT-PCR.The expression of CD3-3A4 BiTE protein in the txansfected CHO cells was deternined by inummofluorescence assay,and the bispecific binding function of CD3-3A4 BiTE was analyzed by multi-color flow cytometry.Antibody block assay was used to detect the bispecific binding activity of CD3-3A4 BiTE protein.3)Purification and concentration determination of CD3-3A4 BiTE.The purification of CD3-3A4 BiTE protein was carried out by using protein high performance liquid chromatography.The molecular weight of the purified protein was checked by westem-blot method and the concentration of the purified protein were measured by BCA assay.The optimal titration concentration of the purified protein was analyzed by flow cytometry.4)The evaluation of the target killing ant its T cell activation actlvites of the CD3-3A4 BiTE protein.CCK-8 assay was used to detect the targeting ability of CD3-3A4 BiTE and to find the best effector:target ratio and the best time course of killing KG la cells.The cytometric bead array(CBA)technology was used to measure the cytokines secreted by the activated T cells during the targeting kill of KG1a cells mediated by CD3-3A4 BiTE.Results:1.Construction and expression of CD3-3A4 BiTE.1)The gene sequence of CD3,3A4 was determined by consulting data and existing results in our laboratory.We chose(G4S)3 as the linker to link the DNA fragments and CD3VL-3A4VH-3A4VL-CD3VH as the final arrangement order.2)SP-CD3VL-3A4VH and 3A4VL-CD3VH-6xhis were largely obtained after PCR and TA cloning.3)The digestion and purification of SP-CD3VL-3A4VH,3A4VL-CD3VH-6xhis and pcDNA3.1 was performed by related endonucleases,electrophoresed,gel cutting and DNA extraction.T4 ligase reaction was carried out overnight,then transfection to DH5 a bacteria,picking the colonies of interest and have them sequenced.The results showed that the sequence of CD3-3A4 BITE gene was confirmed as expected,indicating that the CD3-3A4 BITE plasmid was successfully constructed.4)Transfection of CHO cells with pcDNA3.1-CD3-3A4 plasmid.After 24 hours of transfection,the CHO cells were cultured under the appropriate concentration of G418(600 μg/ml).2.The biological function analyses of CD3-3A4 BiTE1)Cell lines:Flow cytometry analysis showed that Jurkat cells were wtrongly bound by CD3 with a positivity and mean flurescence intensity(MFI)of 92.37%and 60.5,but only very weakly bound by CD45RA(7.71%,MFI=5.37)antibodies,respectively,while KG1z cells were strongly bound by CD45RA antibodies with a positivity and MFI of 90.69%and 36.3,but not bound by CD3 antibody(0.29%,MFI=4.59),indicating that those two cell lines were the appropriate cells for further experiments.2)Confirmation of successful transfection.RT-PCR showed that the bands of CD3VL,CD3VH,3A4VL and 3A4VH were in accordance with the expected sizes,suggesting that the pcDNA3.1-CD3-3A4 plasmid had been successfully transfected into CHO cells.The culture supernatants of transfected CHO cells were harvested afer 10-14 days of culture.The CD3-3A4 BiTE protein was successfully purified by chromatography.The results of immunofluorescence assay showed that green fluorescent protein was observed in the green field of the experimental group,but not in the no-load group,suggesting that CD3-3A4 BiTE protein had been expressed in the transfected CHO cells.Flow cytometry showed that CD3 positive Jurkat cells reacted with CD3-3A4 BiTE and GAM FITC,CD45RA positive KGla cells reacted with CD3-3A4 BiTE and GAM FITC,suggesting that the CD3-3A4 BiTE had bispecific binding activity.The results of antibody block test showed that the positive rate of CD3-3A4 BiTE on Jurkat cells was significantly lower than that after blockage with CD3 monoclonal antibody,while the positive rate of the BiTE protein on KGla cells was also significantly reduced after blockage with 3A4 monoclonal antibody,indicating that CD3-3A4 BiTE was able to recognize the 3A4 target on KGla cells.This results eleary demonstrated that the BiTE protein had bispecific binding function.3)The molecular weight analysis of the purified CD3-3A4 BiTE protein.Western-blot study showed that the molecular weight of the purified CD3-3A4 BiTE protein was about 55 KDa as expected.BCA test showed the concentration of purified protein was 50ng/μl after concentration of the supernatants.The best titration concentration of the purified protein on KG1a was 15μg/ml or 106 KGla cells.4)Targeting killing activity of the CD3-3A4 BiTE protein.Human prephearal blood T cells were collected by drawing blood from volunteers and separated by Ficoll-Hypaque centrifugation and the mononuelared cell suspension were prepared as effector cells with RPMI1640 supplemented with 10%fetal buvine serum.AML cell line KG la cells were selected as the target cells.The CCK-8 assay was used and the result showed that T cells could significantly kill the 3A4 positive target KGla cells but not 3A4 negative 293T cells after co-cultere with CD3-3A4 BiTE protein,suggesting that CD3-3A4 BiTE had targeted killing activity.The effector:target(E:T)ratios of CD3-3A4 BiTE killing KGla cells were set as 1:1,5:1,10:1,25:1 and 50:1.However,there was no significant difference between any groups.This result indicated that even 1:1 E:T ratio is enough to be used for the testing.The time course of killing time of CD3-3A4 BiTE to kill KGla cells showed tha 4.8%±2.28%,23%±6.71%,50.4%±8.42%,68.1%±7.27%,84.3%± 10.68%and 79.8%± 9.03%(n=5)of cell kill were obtained when co-culture time was set at 6h,12h,18h,24h,48h and 72h,respectively.Statitically testing results showed that all but 72h co-culture time points were significantly different while no statistical significance was found between 48h and 72h,indicating that the appropriate co-culture time was 48h_The results of cytokine measurement test showed that when T cells were engaged to KGla cells mediated by CD3-3A4 BiTE,IL-2,IL-4,IL-6 would increase with statistical significance in paired T test,while no significant difference was found between IL-10,TNF-α and IFN-γ groups.Conclusions:1)pcDNA3.1-CD3-3A4 plasmid was successfully constructed and transfected into CHO cells.2)The gene and protein of CD3-3A4 BiTE were successfully expressed in the transfected CHO cells with excellent bispecific targeting activity via activating the allogeneic prepheral T cells.3)CD3-3A4 BiTE may hold the potential targeting therapy for patient with AML. |
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