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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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Analysis of oligoclonality of TIL-derived antibodies. (a) V(D)J analysis of TIL-derived antibody genes. SMART cDNA derived from ten different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96), normal breast, normal testis and peripheral blood lymphocytes from four healthy donors (L1, L2, L3, L4), was used, as template for amplification of V(D)J antibody regions. Samples of cDNA were normalized by amplification of β-actin housekeeping gene. All V(D)J fragments were well-amplified and gave DNA bands of expected molecular weight in all cases, excluding normal testis cDNA sample. (b) The same PCR products were fractionated by 10% PAGE, giving a higher resolution of DNA bands. (c) Antibody subclass distribution. PCR-amplified normal breast and B84 cDNA samples not showing oligoclonal bands in V(D)J test, have prevalence of IgA bands in comparison to IgG1 and IgG2 (left panels), while three samples, B91, B92 and B93, giving strong oligoclonal bands in previous test, have IgG1 or both IgG1 and IgG2 band prevalence in comparison with IgA (right panels). (d) Clonality of heavy chain antibodies derived from B92 and B93 cDNA samples. Amino acid sequences of variable regions of 30 clones were deduced from randomly sequenced γ-chain antibody genes derived from B92 and B93 cDNA. Peptide sequences are reported in single-letter code. Identical amino acids in similar clones are represented by a dash.
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Figure 1: Analysis of oligoclonality of TIL-derived antibodies. (a) V(D)J analysis of TIL-derived antibody genes. SMART cDNA derived from ten different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96), normal breast, normal testis and peripheral blood lymphocytes from four healthy donors (L1, L2, L3, L4), was used, as template for amplification of V(D)J antibody regions. Samples of cDNA were normalized by amplification of β-actin housekeeping gene. All V(D)J fragments were well-amplified and gave DNA bands of expected molecular weight in all cases, excluding normal testis cDNA sample. (b) The same PCR products were fractionated by 10% PAGE, giving a higher resolution of DNA bands. (c) Antibody subclass distribution. PCR-amplified normal breast and B84 cDNA samples not showing oligoclonal bands in V(D)J test, have prevalence of IgA bands in comparison to IgG1 and IgG2 (left panels), while three samples, B91, B92 and B93, giving strong oligoclonal bands in previous test, have IgG1 or both IgG1 and IgG2 band prevalence in comparison with IgA (right panels). (d) Clonality of heavy chain antibodies derived from B92 and B93 cDNA samples. Amino acid sequences of variable regions of 30 clones were deduced from randomly sequenced γ-chain antibody genes derived from B92 and B93 cDNA. Peptide sequences are reported in single-letter code. Identical amino acids in similar clones are represented by a dash.

Mentions: We analyzed the expression patterns of the antibody fragment genes by semi-quantitative PCR from SMART cDNA template. A panel of cDNAs from ten breast carcinomas and samples of normal breast, testis and peripheral blood lymphocytes from healthy donors were normalized by PCR amplification of β-actin, a housekeeping gene (Fig. 1a). Hypervariable heavy chain antibody regions (V(D)J) were amplified as described in Materials and Methods. After analysis by agarose gel electrophoresis, the PCR samples were also fractionated by high resolving 10% PAGE (Fig. 1b). In applying this technique, we observed that seven of ten tumor-derived samples contained various discrete bands, characterizing oligoclonality of the immune response in these patients, while the well-amplified normal breast and peripheral lymphocyte DNA fragments did not contain intensive bands but formed a smear, consisting of bands of differing length. The observed oligoclonality of the immunoglobulins did not correlate with the age of the patients.


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)

Analysis of oligoclonality of TIL-derived antibodies. (a) V(D)J analysis of TIL-derived antibody genes. SMART cDNA derived from ten different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96), normal breast, normal testis and peripheral blood lymphocytes from four healthy donors (L1, L2, L3, L4), was used, as template for amplification of V(D)J antibody regions. Samples of cDNA were normalized by amplification of β-actin housekeeping gene. All V(D)J fragments were well-amplified and gave DNA bands of expected molecular weight in all cases, excluding normal testis cDNA sample. (b) The same PCR products were fractionated by 10% PAGE, giving a higher resolution of DNA bands. (c) Antibody subclass distribution. PCR-amplified normal breast and B84 cDNA samples not showing oligoclonal bands in V(D)J test, have prevalence of IgA bands in comparison to IgG1 and IgG2 (left panels), while three samples, B91, B92 and B93, giving strong oligoclonal bands in previous test, have IgG1 or both IgG1 and IgG2 band prevalence in comparison with IgA (right panels). (d) Clonality of heavy chain antibodies derived from B92 and B93 cDNA samples. Amino acid sequences of variable regions of 30 clones were deduced from randomly sequenced γ-chain antibody genes derived from B92 and B93 cDNA. Peptide sequences are reported in single-letter code. Identical amino acids in similar clones are represented by a dash.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Analysis of oligoclonality of TIL-derived antibodies. (a) V(D)J analysis of TIL-derived antibody genes. SMART cDNA derived from ten different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96), normal breast, normal testis and peripheral blood lymphocytes from four healthy donors (L1, L2, L3, L4), was used, as template for amplification of V(D)J antibody regions. Samples of cDNA were normalized by amplification of β-actin housekeeping gene. All V(D)J fragments were well-amplified and gave DNA bands of expected molecular weight in all cases, excluding normal testis cDNA sample. (b) The same PCR products were fractionated by 10% PAGE, giving a higher resolution of DNA bands. (c) Antibody subclass distribution. PCR-amplified normal breast and B84 cDNA samples not showing oligoclonal bands in V(D)J test, have prevalence of IgA bands in comparison to IgG1 and IgG2 (left panels), while three samples, B91, B92 and B93, giving strong oligoclonal bands in previous test, have IgG1 or both IgG1 and IgG2 band prevalence in comparison with IgA (right panels). (d) Clonality of heavy chain antibodies derived from B92 and B93 cDNA samples. Amino acid sequences of variable regions of 30 clones were deduced from randomly sequenced γ-chain antibody genes derived from B92 and B93 cDNA. Peptide sequences are reported in single-letter code. Identical amino acids in similar clones are represented by a dash.
Mentions: We analyzed the expression patterns of the antibody fragment genes by semi-quantitative PCR from SMART cDNA template. A panel of cDNAs from ten breast carcinomas and samples of normal breast, testis and peripheral blood lymphocytes from healthy donors were normalized by PCR amplification of β-actin, a housekeeping gene (Fig. 1a). Hypervariable heavy chain antibody regions (V(D)J) were amplified as described in Materials and Methods. After analysis by agarose gel electrophoresis, the PCR samples were also fractionated by high resolving 10% PAGE (Fig. 1b). In applying this technique, we observed that seven of ten tumor-derived samples contained various discrete bands, characterizing oligoclonality of the immune response in these patients, while the well-amplified normal breast and peripheral lymphocyte DNA fragments did not contain intensive bands but formed a smear, consisting of bands of differing length. The observed oligoclonality of the immunoglobulins did not correlate with the age of the patients.

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