When the f1-reactive antibodies in the phage peptide immunized serum were sufficiently absorbed with f1, and if the absorbed serum still has significant reactivity to the corresponding phagotope, it indicates the existence of phage displayed peptide specific antibodies in the immunized serum. In comparison of the reactivity of all three clones, immunized serum from both clone No.1 and clone No.3 showed significnt phagotope specificity but result from No.1 clone seemed to be more consistently and was selected to check its specificity to PSMA on the slides with immunohistochemical staining, and f1 immunized serum was used as negative control. At the same time, several other human cancer samples such as lung cancer, colon cancer and stomach cancer are also tested and no positive staining was observed on the epithelial cells of these samples (Data not shown). However, positive staining was localized on the malignant epithelial cells of human prostate cancer tissue, while, no positive staining was observed on the f1-immunized serum stained slides (Fig 4).

Fig 4. Immunohistochemical staining of PSMA in human prostate cancer. A. Slide of prostate cancer was immunostained with f1 absorbed mimotope-immunized serum. Positive staining was localized to the malignant epithelial cells. B. Control staining with f1-immunized serum serum was negative in all cases. Lightly counterstaining was done with Harris hematoxylin. (400 ?).

DISCUSSION

In our experiment, we did not perform the biopanning with the mixture of 9aa linear and 9aa cys peptide library. Since in many cases, when these two libraries were mixed together in the selection, only the linear peptide clones can be isolated from the last identification (unpublished data, Dr. Paolo Monaci), possibly because the growth and amplification efficiency of the linear phage peptide clones is much higher than that of the circular phage peptide clones. After several rounds of elution and amplification, the ratio of the linear phage peptide clones will dramatically increase in the selected pool, while the positive clones from circular phage peptide library will be lost at last. From our experimental results we can see the enrichment of the positive linear phage peptide is much faster than that of the circular phage peptide library.

Generally, many researchers perform three rounds or more of biopanning in the affinity selection before picking out single clones for identification. However, we just performed two rounds of affinity selection which is sufficient for the isolation of positive clones. In fact, how many rounds of affinity selection is appropriate depend on the aim of the selection and the target used for the selection. In our case, we used the purified mAb as the target for affinity selection. The enrichment of target-specific phage peptide with mAb is much faster than that of the selection with complicated target such as serum antibodies. Thus, less rounds of selection is sufficient for the isolation of positive clones. On the other hand, the aim of our experiment is the epitope location of the PSMA. Generally, only 4-6 amino acids are critical for the interaction between the antigen and antibody. In this case, trying to pick out the interaction motif from a series of different antibody-reactive phage peptides is very important. Therefore, keeping the variety of the isolated positive clones is critical for this point. However, higher rounds of selection will result in the enrichment of the dominant clone (Dr. Li Hua, personal communication). On the contrary, we have to identify many more clones in order to keep the variety of the positive clones.

After the immunization of mice with three mimotopes displayed on phage, we did not use the synthesized peptide to test the binding activity of the mimotope immunized serum antibodies, because to some extent, the interaction between the antibody and phage displayed peptide depends on the peptide's micro environment which is provided by the phage particle during the affinity selection, in this case the free peptide will loose the conformation which can be recognized when it was displayed on phage particle [14]. Therefore, we employed the competitive ELISA to test the mimotope specificity of immune serum. From the competitive ELISA results, we observed that both clone No.1 and No.3 induced significant mimotope specific response. Many researchers have demonstrated that filamentous phage is an excellent immunogen. The easily induced immune response in mice is T-cell dependent and undergoes class switching from IgM to IgG [14]. However, the induction of mimotope-specific antibodies is a much more difficult process and varies considerably from one mimotope to another mimotope. This may be reasonable since the immune system can differentiate different antigens and produces very different responses.

Comparing the conserved sequence motif "VDPA/SK" derived from No.1, 2 and 3 phage clones with the PSMA sequence "ESKVDPSK" which was used in the screening process of mAb 4G5, we found they are of high homology, and the "VDPSK" may play a major role in the interaction between the PSMA and mAb 4G5. In addition, the homology of the peptide to the real antigen may be further promoted through an in vitro evolution strategy (Zhu et al submitted [15]). From our results it showed that if we have not any data about the mAb reactive antigen, the deduced peptide or oligonucleotide from the interaction motif can be used as a probe for the identification of the mAb specific antigen. In fact, since the establishment of monoclonal antibody technology, many monoclonal antibodies have been developed which are specific for many different antigens. However, many of these mAb-specific antigens have not been identified yet, such as many cell specific mAbs. In this case, in addition to its successful and extensive application in the epitope mapping of antigen, phage displayed peptide library technology can be a very effective way worthy to be tried for the identification of antigen, even this strategy is limited to the identification of the linear epitope at present.

Our experimental results have demonstrated that 4G5 specific epitope is located at 719-723aa of PSMA, which belongs to extracellular of PSMA. The highly prostate-specific and membrane-bound character of PSMA makes it an ideal target for clinical diagnostic and therapeutic applications in the treatment and management of prostatic carcinoma. Especially, the antibody induced internalization of PSMA which has been identified recently possibly indicate the biological function of PSMA in vivo. Evidently, the characterization of this PSMA extracellular domain specific mAb will be very useful in the antibody targetting strategy for the diagnosis and therapy of prostate cancer.

Recent researches showed PSMA not only overexpressed in prostate cancer, but also in the neovasculature of a variety of malignant neoplasms, indicating that PSMA may play an important role in the genesis and devlopment of other cancer. The mimotopes screened out presumably might be used as vaccine to induce PSMA specific immune responses in vivo, and maybe hopefully inhibit the development of the cancer.

ACKNOWLEDGEMENTS

This work was supported by the Special Support Fund Stz-2-06 from the Chinese Academy of Sciences and also supported by World Laboratory. We thank Prof. Ricardo Cortese, Dr. Paolo Monaci and Dr. Franco Felici of IRBM, Italy for providing peptide libraries and Dr Minenkova Olga for helpful discussion and advice. We also thank Dr YangJin, Dr. Tao QinHua, Yao Gang, and Huang Junyu for providing specimen slides.

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[15] Zhu ZY, O Minenkova, F Bellintani et al. "In vitro evolution" of ligands for HCV-specific serum antibodies. (Submitted for publication)

Received Nov-13-1999.   Revised Nov-19-1999.  Accepted Nov-24-1999.

^*Corresponding author; E-mail: imceng@server.shcnc.ac.cn

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