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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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Selection of anti-MCF7 antibodies. (a) Reactivity of phage pools after fourth and fifth rounds of panning, in comparison with original libraries, was tested. Data reported are the average values of assays performed in triplicate. (b) Fingerprinting analysis of antibody clones. PCR-amplified scFv genes were analyzed by using HaeIII and AluI double digestion. The analysis of clones 17–39, selected from mixLIB, is shown at right, and the list of different anti-MCF7 antibodies obtained shown at left. (c) Cell ELISA reactivity of single phage clones. Data reported are the average values of assays performed in triplicate. Cells developing with irrelevant anti-SP2 antibody are included as negative control.
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Figure 3: Selection of anti-MCF7 antibodies. (a) Reactivity of phage pools after fourth and fifth rounds of panning, in comparison with original libraries, was tested. Data reported are the average values of assays performed in triplicate. (b) Fingerprinting analysis of antibody clones. PCR-amplified scFv genes were analyzed by using HaeIII and AluI double digestion. The analysis of clones 17–39, selected from mixLIB, is shown at right, and the list of different anti-MCF7 antibodies obtained shown at left. (c) Cell ELISA reactivity of single phage clones. Data reported are the average values of assays performed in triplicate. Cells developing with irrelevant anti-SP2 antibody are included as negative control.

Mentions: We tested functionality of a single TIL-derived library (scFvB96) by selecting breast cancer-specific antibodies through cell-based panning on living MCF7 breast carcinoma cells. Four additional libraries, including scFvB87, scFvB95, scFvmix and scFvEC23, were pooled together (mixLIB) and panned in similar fashion. Four or five selection rounds on the tumor cells were necessary for mixLIB and scFvB96 libraries, respectively, in order to obtain phage pools enriched by specific cell binders (Fig. 3a). Then, randomly picked clones were analyzed by PCR for presence of complete scFv antibody genes. The full-length scFv phage clones were tested by cell-based ELISA, and analyzed by DNA fingerprinting (Fig. 3b). Table 3 summarizes clone analysis data. All different positive clones were sequenced. Amino acid sequences, deduced from DNA sequences, confirmed correct, in-frame antibody structures.


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)

Selection of anti-MCF7 antibodies. (a) Reactivity of phage pools after fourth and fifth rounds of panning, in comparison with original libraries, was tested. Data reported are the average values of assays performed in triplicate. (b) Fingerprinting analysis of antibody clones. PCR-amplified scFv genes were analyzed by using HaeIII and AluI double digestion. The analysis of clones 17–39, selected from mixLIB, is shown at right, and the list of different anti-MCF7 antibodies obtained shown at left. (c) Cell ELISA reactivity of single phage clones. Data reported are the average values of assays performed in triplicate. Cells developing with irrelevant anti-SP2 antibody are included as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Selection of anti-MCF7 antibodies. (a) Reactivity of phage pools after fourth and fifth rounds of panning, in comparison with original libraries, was tested. Data reported are the average values of assays performed in triplicate. (b) Fingerprinting analysis of antibody clones. PCR-amplified scFv genes were analyzed by using HaeIII and AluI double digestion. The analysis of clones 17–39, selected from mixLIB, is shown at right, and the list of different anti-MCF7 antibodies obtained shown at left. (c) Cell ELISA reactivity of single phage clones. Data reported are the average values of assays performed in triplicate. Cells developing with irrelevant anti-SP2 antibody are included as negative control.
Mentions: We tested functionality of a single TIL-derived library (scFvB96) by selecting breast cancer-specific antibodies through cell-based panning on living MCF7 breast carcinoma cells. Four additional libraries, including scFvB87, scFvB95, scFvmix and scFvEC23, were pooled together (mixLIB) and panned in similar fashion. Four or five selection rounds on the tumor cells were necessary for mixLIB and scFvB96 libraries, respectively, in order to obtain phage pools enriched by specific cell binders (Fig. 3a). Then, randomly picked clones were analyzed by PCR for presence of complete scFv antibody genes. The full-length scFv phage clones were tested by cell-based ELISA, and analyzed by DNA fingerprinting (Fig. 3b). Table 3 summarizes clone analysis data. All different positive clones were sequenced. Amino acid sequences, deduced from DNA sequences, confirmed correct, in-frame antibody structures.

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