| ObjectivesPrevious studies have showed that a small subpopulation of leukemia cells with self-renewal ability can initiate and maintain leukemic disease. These cells are insensitive to chemotherapy and often can escape from drug killing. It is believed that these cells are partially responsible for drug resistance and relapse. These cells are defined as LSCs (Leukemia stem cells, LSCs).Therefore, it is necessary to eliminate LSCs for curing leukemia. Consequently, a novel antigen specifically expressed on LSCs while sparing normal stem cells are of great significance on the study of LSCs biology and leukemia treatment.3A4antibody as a novel monoclonal antibody was generated in our laboratory. Our preliminary data have showed that3A4antibody had a strong reactivity to CD34+CD38-CD123+LSCs. Therefore, in order to discover the potential application of3A4antigen as AML LSCs targets in targeted therapy for human leukemia, we conducted the study to explored the biological characteristics of3A4antigen including the following four parts:the first part is the study on3A4antigen expression; the second part is the study on the epitope of3A4antigen; the third part is the study on the stem cell biology of3A4antigen and the fourth part is preparation and detection of3A4-HHT conjugates.Methods 1Study on3A4antigen expression1.1Expression of3A4and other antigens on leukemia stem cells and other cell subsets:(1)37ALL patients (B-ALL31, T-ALL6),54patients with AML (MO-M14, M214, M35, M4-M529, M62) and6patients with CML were newly diagnosed and examined for antigen (3A4, CD45RA, CD45, CD45RO, CD33, CD123, CD133, CD44, HLA-DR and CD19) expression on LSCs, hematopoietic stem cells(HSCs) and other cell subsets by flow cytometry. Normal control bone marrow (NBM) was obtained from11patients with immune thrombocytopenia (ITP).(2) Patients with B-ALL(n=10) and AML(n=10) at relapse were examined for antigen (3A4, CD45RA, CD33, CD44, CD123, CD133, CD44, HLA-DR and CD19) expression on bone marrow leukemia cells by flow cytometry.(3) Cell definition:CD34+CD38-Lin-cells were seen in the stem cell population both for HSCs and LSCs. CD34-, CD34+CD38+, CD34+CD38-and CD34+CD38-Lin-cells represented different cell population based on different diffentiation stages.1.2The expression of3A4antigen on multiple organ normal tissues and tumor tissues:The expression of3A4antigen in99cases of multiple organ normal tissues and96cases of tumor tissues were determined by tissue microarray combined with immunohistochemistry.2Study on the epitope of3A4antigen2.1Construction and expression of the truncked recombinant expression vectors CD45RC:Four eukaryotic transfection vectors were constructed according to the truncked sequence of CD45excon6gene. They were pcDNA3.1+/CD45RC-1(99bp in front of exon6), pcDNA3.1+/CD45RC-2(99bp in back of exon6), pcDNA3.1+/CD45RC-3(48bp in front of exon6) and pcDNA3.1+/CD45RC-4(48bp in back of exon6), respectively. The empty vector pcDNA3.1+, the untruncked recombinant expression vector pcDNA3.1+/CD45RC and the four truncked recombinant expression vectors were isolated by QIAGEN plasmid purification minikit and then were transfected into CHO cells by using LipofectamineTM2000kit. The transfected genes were detected by RT-PCR. Then the expression of truncked CD45RC proteins on CHO cells were detected by FCM and Western blot using3A4antibody.2.2The epitope analysis of3A4antigen by peptide scanning technique: Peptides were synthesized according to the sequence of amino acids (DVPGERSTAS TFPTDPVSPL TTTLSLAHHS SAALPARTSN TTITANTSD) coded by the excon6of CD45gene using solid-phase synthesis and were detected by mass spectrometry (MS) and high performance liquid chromatography (HPLC).The reactivity of the peptide to3A4antibody was detected by indirect enzyme-linked immunosorbent analysis (iELISA).3Study on the stem cell biology of3A4antigen3.1Study on the stem cell biology of3A4antigen in vitro:Kasumi cells and HL60cells were sorted based on3A4antigen through indirect magnetic-activated cell sorting (MACS). BrdU (Bromodeoxyuridine) was used to label3A4-positive leukemia cells and3A4-negative leukemia cells to detect the cell proliferation ability.3A4-positive leukemia cells and3A4-negative leukemia cells were incubated with IC50and IC70of THP, HHT and Ara-C. The growth of leukemia cells were observed by CCK-8colorimetric assay and apoptosis rates were detected by flow cytometry using Annexin V-FITC/PI staining.3.2Study on the stem cell biology of3A4antigen in vivo:The NOD/SCID mice were inoculated with leukemia cells and human bone marrow mononuclear cells blocking with3A4antibody and mouse IgG antibody, respectively.3A4-positive AML mononuclear cells and3A4-negative AML mononuclear cells were sorted based on3A4antigen through indirect MACS and then were injected into NOD/SCID mice. The ability of engraftment to NOD/SCID mice with the different subpopulation of cells was observed.4Preparation and detection of3A4-HHT conjugates:3A4antibody was modified by bifunctional crosslinking agent SPDP and SMCC as the intermediate drug linker. The hydroxyl of homoharringtonine(HHT) were produced to sulphur using thiourea.Then the modified3A4antibody and HHT-SH were reacting overnight at 4℃.The concentrations of3A4-HHT conjugates were measured by BCA and the antigen binding activities of3A4-HHT conjugates were detected by flow cytometry. KG1a cells and Nalm-6cells were incubated with different concentrations (0,1,2,4,8,16and32μg/ml) of3A4-HHT conjugates at different hours (24h,48h and72h).Then the growth of KG1a cells and Nalm-6cells was observed by CCK-8colorimetric assay.Results1Study on3A4antigen expression1.13A4and other antigens expressed on leukemia stem cells and other cell subsets:(1) The median expression level of3A4antigen on LSCs in AML was significantly higher than those on HSCs (median94.8%vs.21.8%, P<0.0001) and54AML samples were all positive for3A4antigen expression on LSCs.(2)3A4antigen had higher expression level on leukemia cells with relapse B-ALL (mean (90.9±4.9)%) and with relapse AML (mean (81.7±9.1)%).(3) The median expression of3A4antigen on LSCs in different subtypes of AML were (92.3±3.3)%(M0-1),(95.1±1.5)%(M2),(46.4±13.8)%(M3),(85.2±4.3)%(M4-5) and (61.2±28.9)%(M6). The median expression level of3A4antigen on LSCs was higher in M0-1and M2.(4)3A4antigen was preferentially expressed on CD34+CD38+cells with a higher level than on CD34+CD38-cells and CD34+CD38-Lin-cells in control group and showed no significantly reactive preference on CD34+CD38+, CD34+CD38-and CD34+CD38-Lin-cell components in AML.(5) The preferential expression for3A4antigen on AML LSCs but not on HSCs was more significant as compared with other antigens, such as CD33, CD123, CD133, CD44and HLA-DR.1.2The expression of3A4antigen on multiple organ normal tissues and tumor tissues:(1) The expression of3A4antigen was restricted to lymphoid tissues such as thymus, tonsil and spleen, while it was negative in central nervous system and other important organs such as heart, liver, lung, kidney and reproductive organs. Also, some3A4-positive cells were found in peripheral tissues such as lymphatic plexus of gastrointestinal tract and parathyroid gland.(2)3A4antigen was mainly expressed on B cell lymphoma, Hodgkin’s lymphoma and non-Hodgkin’s lymphoma that arise in lymphoid tissue such as the intestine, spleen, thyroid and lymph nodes, but not on T cell lymphoma.2Study on the epitope of3A4antigen2.1Construction and expression of the truncked recombinant expression vectors CD45RC:(1) The recombinant expression vectors pcDNA3.1+/CD45RC-1, pcDNA3.1+/CD45RC-2, pcDNA3.1+/CD45RC-3and pcDNA3.1+/CD45RC-4were amplified. The DNAs were extracted from each individual vector, digested with endonuclease and sequenced. The results indicated that sequences were correctly inserted into the recombinant expression vectors.(2)RT-PCR revealed that the correct genes were integrated into the genome of CHO cells. The CHO cells transfected were named as CD45RC-1CHO, CD45RC-2CHO, CD45RC-3CHO and CD45RC-4CHO, respectively.(3)FCM showed that3A4antigen was all expressed on CD45RC-1CHO,CD45RC-2CHO,CD45RC-3CHO and CD45RC-4CHO cells with the mean expression level of (36.7±6.9)%,(35.2±13.3)%,(43±7.9)%and (41.4±14.2)%, respectively. No significant difference was found within those cells. According to the results of Western blot,3A4antibody could not recognize CD45RC-1CHO, CD45RC-2CHO, CD45RC-3CHO and CD45RC-4CHO cells.2.2The epitope analysis of3A4antigen by peptide scanning techniques: MS showed that the molecular weight (MW) of the synthetic peptide was4996.5which was similar to the theoretical MW4996.35, and thus indicated that the peptide was synthesized correctly. HPLC showed that the purity of the synthetic peptide was about70%. Indirect ELISA showed that3A4antibody could not recognize the peptide coded by extron6of CD45gene. Maybe the epitope of3A4antigen was conformational epitope.3Study on the stem cell biology of3A4antigen3.1Study on the stem cell biology of3A4antigen in vitro:The cell proliferation ability of3A4-positive Kasumi cells and HL60cells were both lower than the corresponding3A4-negative Kasumi cells and HL60cells. The mean expression level of BrdU was ((4.6±1.9)%vs.(35.1±1)%, P<0.05) for Kasumi cells and ((44.9±1.3)%vs.(74.4±2)%, P<0.05) for HL60cells, respectively. The inhibition rate of3A4-positive Kasumi cells and HL60cells were lower than the corresponding3A4-negative Kasumi cells and HL60cells after treated by THP and Ara-C for48h with IC50and IC70concentration (P<0.05). No significant difference was found on the inhibition rate between3A4-positive leukemia cells and3A4-negative leukemia cells both for Kasumi cells and HL60cells after treated by HHT (P>0.05). The apoptosis rate of3A4-positive Kasumi cells and HL60cells were also lower than the corresponding3A4-negative Kasumi cells and HL60cells after treated by THP and Ara-C with IC50concentration, while no significant difference was found by HHT.3.2Study on the stem cell biology of3A4antigen in vivo:The mice became paralyzed rapidly15days after inoculation with Nalm-6cells blocking with3A4antibody or mouse IgG antibody followed by weight loss with bent spines, hogback and cachexia until death.The median survival time was18days and22days, respectively. All (3/3) mice developed with overt leukemia demonstrated by FCM and pathological characteristics after inoculation with KG la cells blocking with mouse IgG antibody. The median survival time was58days, while the mice after inoculation with KG la cells blocking with3A4antibody remain alive with no sign of disease.4Preparation and detection of3A4-HHT conjugates:The concentrations of3A4-SPDP-HHT and3A4-SMCC-HHT conjugates were0.2mg/ml and0.3mg/ml, respectively. The reactivity of3A4-SPDP-HHT and3A4-SMCC-HHT to KG1a cells analyzed by FCM was98.89%and94.68%, respectively. The inhibition rate of Nalm-6cells were significantly lower than KG1a cells after treatment by3A4-SPDP-HHT (P<0.05) and3A4-SMCC-HHT (P<0.05), suggesting that3A4-SPDP-HHT and3A4-SMCC-HHT had a recognizing ability to KGla cells but not to Nalm-6cells. With increasing concentrations of3A4-SPDP-HHT and3A4-SMCC-HHT, the inhibition of cells also gradually increased, but not in a time-dependent manner. Conclusions1.3A4antigen was preferentially expressed on AML LSCs with high level, especially in MO-1and M2subtypes, while lower on normal HSCs.The preferential expression for3A4antigen on AML LSCs but not HSCs was more significant when compared with other antigens such as CD33, CD123, CD133and CD44.3A4antigen also had higher expression level on leukemia cells with relapsed B-ALL and relapsed AML.2. The expression of3A4antigen was restricted to lymphoid tissues such as thymus, tonsil and spleen, while it was negative in central nervous system and other important organs such as heart, liver, lung, kidney and reproductive organs.3. The epitope of3A4antigen was observed as the site of amino acids coded by excon6of CD45gene and was possibly conformational epitope.4.3A4-positive Kasumi cells and HL60represented more resistant to chemotherapy than the corresponding3A4-negative Kasumi cells and HL60cells.5.3A4antibody can effectively inhibit the engraftment of KG1a cells in NOD/SCID mice.6.3A4-SMCC-HHT and3A4-SPDP-HHT conjugates were successfully prepared and showed targeting effect in vitro. |
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