Colorectal carcinoma-associated antigen Ca-Hb3 detected by one-dimensional SDS-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry.

AIM: To comprehensively identify the proteins of tumor relative antigen Ca-Hb3 recognized by colorectal carcinoma monoclonal antibody Hb3. METHODS: Ca-Hb3 was isolated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by digestion with trypsin. Trypsin peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins identified by mass spectrometry were analyzed using bioinformatics. RESULTS: Ca-Hb3 was identified as a CKAP4-like protein by Nano HPLC tandem mass spectrometry analysis. The molecular weight of CKAP4-like protein was 62.02 kDa, including one hydrophobic region, one transmembrane domain, five coiled coils, four glycosylation sites and forty-nine phosphorylation sites. CKAP4-like protein had a high homogeneity with DeltaNp63alpha. The characteristic expression of DeltaNp63alpha that is considered a potential oncogene in the isoforms of p63 was similar to that of Ca-Hb3. CONCLUSION: Ca-Hb3 is probably a CKAP4-like protein, belonging to DeltaNp63alpha isoform of p63 family.

[Cloning, expression and purification of interferon-kappa, a novel human interferon, and its antiviral activity].

OBJECTIVE: To prepare human interferon-k (hIFN-kappa) and study its biological activities. METHODS: Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities. RESULTS: The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity. CONCLUSION: Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.

[Construction and screening of human anti-idiotypic single chain antibodies of nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: The potential of anti-idiotypic antibody as a surrogate of tumor antigen for cancer therapy has been demonstrated in clinical investigations. But at present, many anti-idiotypic antibodies are mouse-original antibodies, which can cause human anti-mouse antibody (HAMA) response and decrease the curative effect. The objective of this study was to construct phage human anti-idiotypic antibody library and select beta type anti-idiotypic single chain antibodies bearing the internal image of the nasopharyngeal carcinoma (NPC) associated antigen to overcome human anti- mouse antibody response caused by application of mouse-original anti-idiotypic antibody. METHODS: Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). V(H) and V(L) genes were amplified by RT-PCR and combined to single chain fragments of variable region (scFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into E.coli MC1061 to construct the scFv-displaying phage library. After four rounds of panning with monoclonal antibody (mAb) FC2,the beta type Ab2 scFv were selected by Sandwich ELISA and binding inhibition test. RESULTS: Of 10 NPC patients, 8 patients showed their B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of V(L) genes were obtained by RT-PCR amplification and then connected to form 14 scFv genes. ScFv genes were transducted into E.coli MC1061. The library capacity was 1.5x10(8) clones. After panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. Five clones,which might display beta type Ab2 scFv, were selected by binding inhibition test. CONCLUSION: The strategy for preparing phage anti-idiotypic antibody library and selecting beta type Ab2 scFv by immunization in vitro, EBV transformation, and phage display technique is feasible, which provide a way for preparing cancer vaccine using beta type Ab2 scFv.

[Construction of phage anti-idiotypic antibody library using sensitized in-vitro B-lymphocytes of nasopharyngeal cancer patients].

AIM: To construct phage human anti-idiotypic antibody library. METHODS: Peripheral blood mononuclear cells(PBMCs) of patients with nasopharyngeal cancer(NPC) were sensitized in-vitro and transformed by Epstein-Barr virus(EBV). V(H) and V(L) genes were re-amplified by PCR and combined to single-chain fragment of variable region(ScFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library. RESULTS: Detection of Sandwich ELISA showed that of 10 NPC patients,8 patients' B cells transformed by EBV could produce anti-idiotypic antibodies to NPC. 5 types of V(H) genes and 7 types of V(L) genes were obtained by PCR re-amplification and then connected with (Gly(4)Ser)(3) linker to form 14 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into MC1061 via electroporation. Phage anti-idiotypic antibody library with sink size being 1.1x10(7) was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 70%. CONCLUSION: A strategy for preparing human single chain anti-idiotypic antibody by means of phage antibody library technique in combination with EBV transformation technique is feasible.

Transfection of colorectal cancer cells with chemokine MCP-3 (monocyte chemotactic protein-3) gene retards tumor growth and inhibits tumor metastasis.

AIM: To evaluate the possibility of the induction of anti-tumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically. RESULTS: MCP-3 mRNA expression was detected by RT-PCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021+/-0.253) cm(2) vs (1.769+/-0.371) cm(2), P<0.05) or CMT93/mock cells (1.021+/-0.253) cm(2) vs (1.680 +/-0.643)cm(2), P<0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls. CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.

[Active immunity for anti-colorectal cancer induced by chemokine MCP-3 gene transfection].

BACKGROUND & OBJECTIVE: Chemokines play an important role in the infiltration of immune cells to tumor tissues. Anti-tumor immune response had been elicited in many tumor models by the chemokine gene transfection. The aim of this study was to evaluate the possibility of inducing anti-colorectal cancer active immune response by transfection of mouse colorectal cancer CMT93 cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by chemotaxis assay. In vivo experiments were performed to observe the tumorigenicity of wild type CMT93 and MCP-3 gene modified tumor cells. The immune cell infiltration in tumor tissues and tumor metastasis were detected histopathologically. RESULTS: RT-PCR detection showed MCP-3 was expressed in MCP-3 gene-transfected G418-resistant clones(CMT93/MCP-3), but not in wild type CMT93. In chemotaxis assay, the results showed that the cell culture supernatant of CMT93/MCP-3 possess obviously chemotactic activity. The chemotactic index of the CMT93/MCP-3 supernatant was 5.57(P < 0.05). The supernatants from the control groups did not possessed the chemotactic activity. In vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased significantly compared to wild type CMT93, but the tumors grew more slowly from CMT93/MCP-3 than from the controls (P < 0.05). In the tumor tissue from CMT93/MCP-3, obvious infiltrated immune cells were found, and few immune cells infiltrated in the tumor tissue from the controls. In the mice inoculated with CMT93/MCP3 tumor cells, tumor metastasis was inhibited significantly, its metastasis rate was 0(0/7), lower than that of CMT93 (100%, 4/4) and CMT93/mock (80%, 4/5) (P < 0.05). CONCLUSION: Transfection with chemokine MCP-3 gene can induce anti-colorectal cancer active immune response, but the tumor growth cannot be inhibited completely by merely MCP-3 gene transfection.

[Primary characterization and sequence analysis of anti-colorectal cancer phage fusion antibodies].

OBJECTIVE: To identify 5 phage fusion antibodies against colorectal cancer from in vitro immunized phage library and analyze their sequences. METHODS: Cell ELISA, immunohistochemistry, DNA sequencing and computer analysis were employed. RESULTS: Five clones of phage antibodies were tested by cell ELISA, and all of them reacted to human colorectal cancer cell lines, human embryo kidney endothelial cell line and some tumor cell lines, but not to mouse-original cell lines. They also reacted weakly to human hepatic cell lines. The binding specificity of the phage antibodies for colorectal cancer cells was confirmed by immunohistochemistry with cultured cells and colorectal carcinoma and colon tissue sections. They reacted to colorectal carcinoma cell lines, human embryo kidney endothelial cell lines and nasopharyngeal carcinoma cell lines. CH273 reacted specifically to colorectal cancer cells in human colorectal carcinoma sections but not to any of the cells in human colon sections. The 5 clones were further analyzed after their DNA sequencing. The sequences of CH723, CH209 and CHA12 were identical. The lengths of CH273, CH205 and CH723 were 732 bp, 366 bp and 723 bp, respectively. The VDJ regions of CH273, CH205 and CH723 belonged to VH3-30-D1-26-JH3-linker-V1-13-JL2, VH1-46-D6-13-JH3 and VH3-30-D1-26-JH3-linker-L2-J kappa 2, respectively. CONCLUSIONS: Phage antibodies binding to colorectal tissues and cells are confirmed, on which human anti-tumor ScFv and VH fragments may be further developed and applied to clinical therapy.

Expression of B7 costimulation molecules by colorectal cancer cells reducestumorigenicity and induces anti-tumor immunity.

AIM:To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS:B7 gene was transfected into mouse colon cancer cell line CMT93.The transfectants were selected in DMEM containing 800mg/L G418, and B7 molecules were detected by immunohistochemistry.Experiments in vivo include: (1)5X10(6) B7(+) CMT93 cells were inoculated into the back of C57BL/6 mice subcutanously to determine their tumorigenicity (n = 4). As control, wild type CMT93 cells were inoculated the same as the experimental group (n= 3). (2) The mice primed by B7(+) CMT93 cells whose tumors vanished were rechallenged with wild type CMT93 to observe the immune protection of these mice against the wild type CMT93 (n = 4). Non-primed 4 native mice inoculated with wild type CMT93 were used as control.With in vivo cytotoxicity assay, the mice were immunized with B7 (+) CMT93 or the wild type CMT93 by intraperitoneal injection (n = 4X2). The spleen cells and the abdominal cavity infiltrating lymphocytes were obtained and cultured for two days. Cytotoxicity of these cells against the B7 gene modified or wild type CMT93 was detected by MTT assay.RESULTS:B7 high expression clones were obtained after the transfection of the B7 gene into CMT93 cells by electroporation. Immunohistochemistry results showed mainly membrance staining and partly cytoplasm staining in B7 gene transfected CMT93 cells. in vivo experiments: (1)After the inoculation of the B7(+) CMT93 cells in the back of C57BL/6 mice, they lost their tumorigenicity greatly (P < 0.01). All the small tumors growing in the early period in the experimental group vanished in one month, and the tumors in control group grew progressively. (2) No tumors were found in all 4 mice primed by B7(+) CMT93 cells after they were rechallenged with wild type CMT93. In the control group all mice had grown tumors (P < 0.05). In vitro cytotoxicity assay, the CTLs induced by B7(+) CMT93 had a higher cytotoxity against the wild type CMT93 than that induced by wild type CMT93 (P < 0.05), and the cytotoxity of CTLs induced by B7(+) CMT93 against B7(+) CMT93 cells was higher than that against wild type CMT93 cells (P < 0.05).CONCLUSION:The results suggest that the expression of costimulation B7 molecules by colorectal cancer cells can decrease their tumorigenicity greatly, and the B7 molecule can augment the activation of the CTLs against colorectal cancer, and it plays an important role in CTL effector function as well.

Radioimmunoimaging of colorectal cancer using (99m)Tc labeled monoclonal antibody.

AIM:To determine whether Hb3 and its fragment F(ab')(2) have practical value in radioimmunoimaging of colorectal cancer.METHODS:Intact Hb3 was purified by hydroxylapatite chromatography.The fragment F(ab') (2) was prepared by cold digestion and purified as intact Hb3.Hb3 and its fragment F(ab') (2) were labeled with 99mTc by direct labeling method using SnCl(2) as reducing agent. The radioactive doses ranged from 15 to 40 mCi.The imaging was accomplished by single photon emission computered tomograph (SPECT) with imaging time ranging from 2.5 to 48 hours. In this study, 10 patients were selected. Among them, 7 were administered with intact Hb3, and 3 with F(ab') (2) fragment. All the patients were diagnosed as having colorectal adenocarcinoma.RESULTS:After purification, intact Hb3 and its fragment F(ab') (2) were fit for radioimmunoimaging. The percentage of labeling of (99m)Tc to Hb3 or F(ab') (2) was 80.6%-91.5%. Among the 10 patients, 3 of 7 patients administered with intact Hb3 had positive scans, the other 4 had negative scans, and 2 of 3 patients administered with F(ab') (2)had positive scans, the other 1 had negative scans.CONCLUSION:The results showed that both intact Hb3 and its F(ab') (2) have some practical value in radioimmunoimaging of colorectal cancer, and the effects of imaging with F(ab') (2) was better than that with intact Hb3.