Development And Preliminary Characterization Of MCSP-specific Monoclonal Antibody

1Purpose and significanceBreast cancer is the most common neoplasm for women. The incidence ofbreast cancer has an upward tendency all over the world since20th century. There’shighest incidence of breast cancer for European women while North America womenwith breast cancer ranked second. In China, there were20million breast cancerpatients in the early90s during which the number of patients went up by5millionsevery year. Triple negative breast cancer (TNBC) that estrogen receptors (ER),progesterone receptor (PR), and human epidermal growth factor receptor2(HER2)expression are all negative, accounted approximately for20%～25%of all breastcancer patients, and characterized with a high degree of malignancy, invasiveness.Currently there is no standard treatment options for TNBC, although TNBC issensitive to chemotherapy, but it is easy to relapse and metastasize, easy to producedrug resistance, and the5-year survival rate is less than15%.Cancer cells derived from the normal cells and there were minor differencebetween cancer cell and normal cell.Some proteins are unique to cancer cell,butabsent or low-expression and difficult to be found in normal cells. MCSP(melanoma-associated chondroitin sulfate proteoglycan), also known as CSPG4(chondroitin sulfate proteoglycan4) or MW-MAA (high molecular weight-melanoma associated antigen), is a kind of melanoma-associated antigens expressed on cellsurfaces in more than80%of melanoma, and little or no expression in the normaltissue cells; MCSP is very conserved in evolution, linked by the N-terminal of280KDglycoprotein and450KD chondroitin sulfate proteoglycan, and has strongimmunogenicity,therefore has already become an important target ofimmunotherapy for malignant melanomas. Recent studies have shown that MCSPexpressed in breast cancers of some people, especially in triple negative breast cancer(TNBC) was probably a potential marker and therapeutic target of TNBC.MCSP-specific antibodies in mice experiments showed good retardation of triplenegative breast cancer metastasis and recurrence, however the studies of MCSP inhuman breast cancer have been reported rarely, according to the literatures searched.Also no research on development of MCSP-specific antibody is seen in china.This project attempts to immunize mice with purified MCSP,then to make thehybridoma cell lines producing MCSP-specific monoclonal antibody by conventionalmethods, and then to characterize the antibody using SDS-PAGE.ELISA,flowcytometry and immunofluorescence staining etc. lastly to explore preliminarily theMCSP expression in human breast cancer cell lines. The main purpose will provideantibody tool for understanding of biological functions of MCSP in breast cancer andtarget therapy for breast cancer.2Main contents, methods and results of the research(1) Identification of the purified MCSP by SDS-PAGE.The sample had good purityand was as immunogen for mice immunization.(2) Immunization of BALB/C mice. In tramuscular injection with GM-CSF plasmidand then intraperitoneal injection with mixture of MCSP and saponin. The sera titer ofMCSP-specific antibody was1:6400(MCSP-coated ELISA) or1:1600(Cell-ELISA).(3) Establishment of screening methods for hybridoma cells producingMCSP-specific monoclonal antibody. MCSP-coated ELISA wsa for freeMCSP-specific hybridoma establishment and Cell-ELISA for cell boundMCSP-specific establishment. The two methods worked well. (4) Cell fusion and screening of hybridoma cell lines.Splenic cells were fused withmurine myeloma cells Ag8.653under the PEG-mediation.21wells showed strongpositive to free MCSP. Of them,4wells (P1C6, P2G2, P4E4, P5F5) also showedpositive to cell-bound MCSP. Well P4E4cells showed the strongest and cloned bylimited dilution method.100%of the single-clone wells showed positive to cell-boundMCSP.The well P1D2cells were used to generate monoclonal antibody,named asCSPG4-0711.(5) Generation and purification of ascite antibody. CSPG4-0711hybridoma cells wereinjected to BALB/C mice intraperitoneally for preparation of ascite antibody.Totally8ml ascites fluid was harvested.The antibody titer was1:32000,measured byMCSP-coated ELISA. The ascite antibody was sequentially purified with ammoniumsulfate precipitation method and Protein G affinity chromatography. The final yieldwas5.2mg,with concentration of1.6mg/ml.(6) Identification of hybridoma cells by colchicines blocking method. CSPG4-0711hybridoma cell has average chromosome number of66.(7) Physical and Chemical properties of the antibody.SDS-PAGE confirmed the IgGof the antibody with high purity.(8) Immunological characterization of the antibody.①Inhibition test of antigen. Following pretreated with different concentrations ofMCSP, the mAb CSPG4-0711has a dose-dependent decreasing binding tocell-bound MCSP tested by Cell-ELISA,indicating the specificity of the antibody toMCSP.②Flow cytometric analysis.mAb CSPG4-0711could bind to MV3cells(MCSPpositive) in dose-denpendent manner,with range of MFI of45.73-1191.32.③Immunofluorescence stain(IF). IF also showed the binding of mAb CSPG4-0711toMV3cells.(9) RT-PCR for MCSP mRNA expression. As to human breast cancer cell lines,MCSP mRNA expressions were positive in MDA-MB-231, MDA-MB-435s celllines,which are classified to triple negative breast cancer cells,and negative in MCF-7and T47D cells. As to human melanoma cell lines, MCSP mRNA expressions were also positive in M21and MV3cell lines,but negative in1520and M14cell Lines,which was consistant with aforementioned ELISA, flow cytometric analysis,immunofluorescence staining results.（10）Specific binding of CSPG4-0711antibody to TNCB cell lines by flowcytometric analysis. CSPG4-0711antibody could also specifically bind to the TNCBcell lines MBA-MD-231, MBA-MD-435s,but not to other type of breast cancer celllines MCF-7, T47D,which was consistent with the RT-PCR results.3Summarization and conclusionWe used mouse hybridoma technology to develope a set of monoclonal antibodiesagainst MCSP and MCSP positive human melanoma cell lines.mAb CSPG4-0711was sceened out with high affinity and specificity to MCSP and MCSP positivehuman melanoma cell lines. Preliminary studies have shown that mAb CSPG4-0711could also bind to human breast cancer cell lines MDA-MB-231, MDA-MB-435s,two kinds of triple negative breast cancer cell lines, which indicated mAbCSPG4-0711could be used to investigate expressions and biological functions inhuman breast cancer, moreover to immunotherapy for breast cancer. Further studiesare in progress.