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Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

Pavoni E, MonteriĆ¹ G, Santapaola D, Petronzelli F, Anastasi AM, Pelliccia A, D'Alessio V, De Santis R, Minenkova O - BMC Biotechnol. (2007)

Bottom Line: In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry.The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kenton Srl, c/o Sigma-Tau SpA, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it

ABSTRACT

Background: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.

Methods: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells.

Results: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells.

Conclusion: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

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Fluorescence staining and flow cytometry with anti-MUC1 and anti-CEA antibodies. (a) Fluorescence staining of breast carcinoma cells MCF7 and SkBr3, expressing epithelial mucin MUC1 and normal breast epithelial MCF10-2A cells by using phage antibody anti-MUC1 MB5 (left panels). Right panels show results of flow cytometry analysis of phage displayed MB5 and irrelevant anti-SP2 single-chain antibodies. (b) Staining of LoVo colorectal adenocarcinoma cells expressing CEA protein by phage-displayed anti-CEA CB37 scFv antibody is shown. Staining of negative control MCF10-2A cells is included (left panels). Binding of phage antibody CB37 to LoVo was also measured by flow cytometry (right panels).
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Figure 7: Fluorescence staining and flow cytometry with anti-MUC1 and anti-CEA antibodies. (a) Fluorescence staining of breast carcinoma cells MCF7 and SkBr3, expressing epithelial mucin MUC1 and normal breast epithelial MCF10-2A cells by using phage antibody anti-MUC1 MB5 (left panels). Right panels show results of flow cytometry analysis of phage displayed MB5 and irrelevant anti-SP2 single-chain antibodies. (b) Staining of LoVo colorectal adenocarcinoma cells expressing CEA protein by phage-displayed anti-CEA CB37 scFv antibody is shown. Staining of negative control MCF10-2A cells is included (left panels). Binding of phage antibody CB37 to LoVo was also measured by flow cytometry (right panels).

Mentions: The binding capacity of the anti-MUC1 antibody MB5 and the anti-CEA antibody CB37 were assessed by immunofluorescence staining of tumor cells directly with phage antibodies (Fig. 7). The MB5 antibody intensively stained MCF7 cells, known for high MUC1 expression, and also reacted well with another breast carcinoma cell line, SkBr3. The CB37, an anti-CEA antibody, efficiently bound colon adenocarcinoma cells, LoVo, expressing the carcinoembryonic antigen. No background staining for normal breast epithelium was observed. Binding of the MB5 and CB37 phage-displayed scFvs was also measured by flow cytometry. According to FACS analysis, the MB5 stained 71% of MCF7 and 23.3% of ScBr3 cells. With regard to the anti-CEA CB37 antibody, it bound 44% of LoVo cells.


Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

Pavoni E, MonteriĆ¹ G, Santapaola D, Petronzelli F, Anastasi AM, Pelliccia A, D'Alessio V, De Santis R, Minenkova O - BMC Biotechnol. (2007)

Fluorescence staining and flow cytometry with anti-MUC1 and anti-CEA antibodies. (a) Fluorescence staining of breast carcinoma cells MCF7 and SkBr3, expressing epithelial mucin MUC1 and normal breast epithelial MCF10-2A cells by using phage antibody anti-MUC1 MB5 (left panels). Right panels show results of flow cytometry analysis of phage displayed MB5 and irrelevant anti-SP2 single-chain antibodies. (b) Staining of LoVo colorectal adenocarcinoma cells expressing CEA protein by phage-displayed anti-CEA CB37 scFv antibody is shown. Staining of negative control MCF10-2A cells is included (left panels). Binding of phage antibody CB37 to LoVo was also measured by flow cytometry (right panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175506&req=5

Figure 7: Fluorescence staining and flow cytometry with anti-MUC1 and anti-CEA antibodies. (a) Fluorescence staining of breast carcinoma cells MCF7 and SkBr3, expressing epithelial mucin MUC1 and normal breast epithelial MCF10-2A cells by using phage antibody anti-MUC1 MB5 (left panels). Right panels show results of flow cytometry analysis of phage displayed MB5 and irrelevant anti-SP2 single-chain antibodies. (b) Staining of LoVo colorectal adenocarcinoma cells expressing CEA protein by phage-displayed anti-CEA CB37 scFv antibody is shown. Staining of negative control MCF10-2A cells is included (left panels). Binding of phage antibody CB37 to LoVo was also measured by flow cytometry (right panels).
Mentions: The binding capacity of the anti-MUC1 antibody MB5 and the anti-CEA antibody CB37 were assessed by immunofluorescence staining of tumor cells directly with phage antibodies (Fig. 7). The MB5 antibody intensively stained MCF7 cells, known for high MUC1 expression, and also reacted well with another breast carcinoma cell line, SkBr3. The CB37, an anti-CEA antibody, efficiently bound colon adenocarcinoma cells, LoVo, expressing the carcinoembryonic antigen. No background staining for normal breast epithelium was observed. Binding of the MB5 and CB37 phage-displayed scFvs was also measured by flow cytometry. According to FACS analysis, the MB5 stained 71% of MCF7 and 23.3% of ScBr3 cells. With regard to the anti-CEA CB37 antibody, it bound 44% of LoVo cells.

Bottom Line: In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry.The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kenton Srl, c/o Sigma-Tau SpA, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it

ABSTRACT

Background: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.

Methods: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells.

Results: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells.

Conclusion: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

Show MeSH
Related in: MedlinePlus