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

Pavoni E, Monteriù 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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Immunohistochemical staining of tumor cells. (a) The scFvs MIX7, MIX17 and MIX39 show significant staining of the MCF7, MDA-MB-231 and MDA-MB-468 breast carcinoma cells. No staining is observed with the negative control (irrelevant anti-SP2 antibody). (b) Staining of breast carcinoma cells in comparison with normal breast epithelial cells MCF10-2A. The selected antibodies stain the non-fixed cells more intensively than the fixed MCF7 cells, but not the MCF10-2A cells. Weak background is observed only for MIX39 scFv when it interacts with MCF10-2A cells. No staining is observed for negative control anti-SP2 antibody. (c) Staining non-fixed MCF7 cells, magnification ×60.
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Figure 5: Immunohistochemical staining of tumor cells. (a) The scFvs MIX7, MIX17 and MIX39 show significant staining of the MCF7, MDA-MB-231 and MDA-MB-468 breast carcinoma cells. No staining is observed with the negative control (irrelevant anti-SP2 antibody). (b) Staining of breast carcinoma cells in comparison with normal breast epithelial cells MCF10-2A. The selected antibodies stain the non-fixed cells more intensively than the fixed MCF7 cells, but not the MCF10-2A cells. Weak background is observed only for MIX39 scFv when it interacts with MCF10-2A cells. No staining is observed for negative control anti-SP2 antibody. (c) Staining non-fixed MCF7 cells, magnification ×60.

Mentions: To demonstrate the specificity of selected antibodies we examined three scFvs in soluble form (MIX7, MIX17 and MIX39) by immunohistochemical staining. These antibodies were chosen because of their good reactivity, specificity and stability in soluble form. The first staining was performed on various methanol-fixed breast carcinoma cells, including MCF7, MDA-MB-231 and MDA-MB-468 (Fig. 5a). Different intensity staining was observed for all three antibodies tested, compared to the irrelevant anti-SP2 antibody. In the second experiment, formaldehyde-fixed or non-fixed dried cells were stained (Fig. 5b). All selected antibodies specifically stained both fixed and non-fixed carcinoma MCF7 cells, but did not stain normal epithelial MCF10-2A cells. However, the signal was notably stronger for non-fixed cells. Weak background labeling was registered for MIX39 when it interacted with non-fixed MCF10-2A cells. Intensive staining activity of MIX17 and MIX7 was associated with the cell membrane, cytoplasm and nuclear membrane, while MIX39 staining was of nuclear localization (Fig. 5c).


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

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

Immunohistochemical staining of tumor cells. (a) The scFvs MIX7, MIX17 and MIX39 show significant staining of the MCF7, MDA-MB-231 and MDA-MB-468 breast carcinoma cells. No staining is observed with the negative control (irrelevant anti-SP2 antibody). (b) Staining of breast carcinoma cells in comparison with normal breast epithelial cells MCF10-2A. The selected antibodies stain the non-fixed cells more intensively than the fixed MCF7 cells, but not the MCF10-2A cells. Weak background is observed only for MIX39 scFv when it interacts with MCF10-2A cells. No staining is observed for negative control anti-SP2 antibody. (c) Staining non-fixed MCF7 cells, magnification ×60.
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Related In: Results  -  Collection

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Figure 5: Immunohistochemical staining of tumor cells. (a) The scFvs MIX7, MIX17 and MIX39 show significant staining of the MCF7, MDA-MB-231 and MDA-MB-468 breast carcinoma cells. No staining is observed with the negative control (irrelevant anti-SP2 antibody). (b) Staining of breast carcinoma cells in comparison with normal breast epithelial cells MCF10-2A. The selected antibodies stain the non-fixed cells more intensively than the fixed MCF7 cells, but not the MCF10-2A cells. Weak background is observed only for MIX39 scFv when it interacts with MCF10-2A cells. No staining is observed for negative control anti-SP2 antibody. (c) Staining non-fixed MCF7 cells, magnification ×60.
Mentions: To demonstrate the specificity of selected antibodies we examined three scFvs in soluble form (MIX7, MIX17 and MIX39) by immunohistochemical staining. These antibodies were chosen because of their good reactivity, specificity and stability in soluble form. The first staining was performed on various methanol-fixed breast carcinoma cells, including MCF7, MDA-MB-231 and MDA-MB-468 (Fig. 5a). Different intensity staining was observed for all three antibodies tested, compared to the irrelevant anti-SP2 antibody. In the second experiment, formaldehyde-fixed or non-fixed dried cells were stained (Fig. 5b). All selected antibodies specifically stained both fixed and non-fixed carcinoma MCF7 cells, but did not stain normal epithelial MCF10-2A cells. However, the signal was notably stronger for non-fixed cells. Weak background labeling was registered for MIX39 when it interacted with non-fixed MCF10-2A cells. Intensive staining activity of MIX17 and MIX7 was associated with the cell membrane, cytoplasm and nuclear membrane, while MIX39 staining was of nuclear localization (Fig. 5c).

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