The Preliminary Study On Cloning, Expressing, Monoclonal Antibodies Preparation And Radioimmunoimaging Of Human Renal Cell Carcinoma Associated Antigen G250

Renal cell carcinoma (RCC), whose morbidity ranks the second in urologic tumor, is the most common malignant diseases of the adult kidney and was showing an ascending tendency in recent years.As the early symptom of RCC is untypical, the usual diagnosis for patients with RCC mainly depends on imageology methods, such as ultrasound, CT scans and MRI et al. However, the sensitivity and specificity of these methods are limited, and the interpretation of abnormal findings is sometimes difficult. In particular, the distinction between benign and malignant tumor, enlarged regional lymph nodes and lymph-node metastases is often cumbersome. Fine needle puncture may result in needle track implantation and is seldom used.As RCC is resistant to conventional therapeutic methods, such as radiotherapy and chemotherapy, surgery is the major treatment for RCC at present. Immunotherapy seems to be the most promising therapeutic approach to patients with the advanced RCC accompanied with metastasis and with other situation not fit for surgery. However, until now, cytokine-based immunotherapy with IL-2 and IFN-αhave shown a maximum cure rate of only 10～20%. Renal carcinoma patients at advanced stage, especially those who are diagnosed diffusible metastasis, are not suitable for surgery and can not get efficient treatment. Therefore, the method of targeted therapy becomes a hot issue in the research of kidney cancer, and finding a specific RCC tumor-associated antigen is the key element for targeted therapy as well as for early diagnosis.G250, a transmembrane glucoprotein, is considered as a RCC-associated antigen which expresses on the surface of almost all RCCs except for normal renal tissues. It is one of isomerases of the carbonic anhydrase family. Expression of G250 is very tightly associated with RCC, while it is generally absent from the normal kidney tissues and its expression in normal organs is restricted to the gastric mucosal cell and the large bile ducts. Studies showed that G250 was expressed in 98% RCCs and 88% metastasis lesions. The specific expression in RCC of humans makes the G250 antigen a tumor marker for the diagnosis and prognosis, even for a potential vaccine of RCC.Studies, which use G250 as a RCC tumor antigen to develop tumor-cell vaccines and dendritic cell vaccines, have been developed and tested in vitro. Satisfied results were obtained by using G250 monoclonal antibody to the research of radioimmunoimaging and radioimmunotherapy. However, there is no purified G250 protein in China and the McAb against G250 antigen are not available as well, which limits the related studies.This study aimed at the expressing and purifying of G250 fusion protein in prokaryotic system. Based G250 fusion protein, monoclonal antibodies against G250 were prepared through hybridoma technology. Based monoclonal antibodies, nuclide ^99mTc was labeled and tested in nude mice bearing with RCC xenograft.The research can be divided into three parts as follows:1. Cloning, expressing and purifying of the human renal cell carcinoma tumor-associated antigen G250/MN/CAâ…¨in Prokaryotic expression system.The preparing immunogenic G250 protein would provide a mean of function of G250 protein, and production of McAbs against G250 protein for diagnosis and treatment of RCC. This part of the study was aimed to clone G250 gene and insert it into the prokaryotic expression vector. The recombinant plasmid was transducted into E. coli. and provided an excellent immnogen for the production of the McAbs against G250 protein. Firstly, the G250 gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pET-32a(+). the recombinant plasmid pET-32a(+)/G250 was digested by restriction endonucleases respectively and sequence analysis confirmed its coincidence with the base sequence of G250 registered in GenBank. The recombinant plasmid was transducted into Rosseta strains of Escherichia coli. The expression product, fusion G250 protein, was purified successfully with Ni²⁺ affinity chromatography. The immunogenicity of the fusion protein was analyzed by Western Blot using M75 antibody. The fusion protein could react to specific antibodies. These results suggested that G250 protein possesses excellent immunogenicity.2. Preparation and characterization of monoclonal antibodies against G250 protein.Satisfied results were obtained by usihg G250 monoclonal antibody to the research of radioimmunoimaging and radioimmunotherapy. However, there are no purified G250 protein in China and the McAb of G250 are not available as well. It limits the related studies. Based on the immunogenic G250 fusion protein obtained in the first part of this study, this part aimed to prepare the McAbs against G250 protein so as to provide essental basis for the establishment of a radioimmunoimaging method to detected RCC in next part. Firstly, the G250 fusion protein was used to immunize the BALB/c mice for the McAbs preparation. The immunized splenocytes were fused with the SP2/0 myeloma cells. After the hybridoma cells were subcloned with limited dilution by G250 and pET-32 protein screeing, for the first time, seven hybridomas producing McAbs steadily had been produced. One of the McAbs was IgM isotype, and one was IgG2a, the others were IgG1. Good affinity test results were obtained when the affinity of the McAbs against G250 protein was tested by indirect ELISA, the affinity constant of the McAbs were from 1×10^-8 to 1×10^-10. Western blot shows the high specificity of the McAbs with the G250 fusion protein. Immunohistochemisty test characterized the high specificity of the McAb with the clear cell RCC tissue.3. The preliminary study on radioimmunoimagingMcAb GM005 and control McAb mouse IgG were reduced by 2-mercaptoethanol and then was technetium-99m labeled with the direct method. Then, radio activity and label rate were measured and radiochemical purity was measured by paper chromatography. The immunogenicity of labed McAbs was tested by indirect ELISA method. 10 nude mice bearing with RCC xenograft were devided into 2 groups. 3.7MBq/20ug of McAbs were injected into each mouse via tail vein. 2 hours, 8 hours and 24 hours later respectly, RII were performed with SPECT. After 24 hours, all nude mice were sacrificed and the heart, the liver, the spleen, the lung, the kidney, the intestine, the blood, the musles and tumor were take out. Radio count were measured byγcounter and calculated percentage of injected dose per gram (%ID/g) of every organ and tissue.Conlclusion:1. G250 was highly expressed in prokaryotic expression system. It laid a good foundation for further study such as the study of monoclonal antibody and protein function.2. Based the immunogenic G250 fusion protein, using hybridoma technology, we first prepared specific and high affinity monoclonal antibodies against G250 in China.3. The McAb which we prepared(GM005) was succesfully labled with ^99mTc and specific tumor radioimmunoimaging in nude mice bearing with RCC xenograft was obtained.