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Identification of tumor-binding scFv derived from clonally related B cells in tumor and lymph node of a patient with breast cancer

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Abstract

The purpose of this study was to determine the clonal relationship between B cells within a breast cancer and the B cells in the tumor-draining lymph node (TDLN). We also determined the binding capacity of antibodies derived from these sources to autologous cancer and autologous noncancer breast tissue. Antibody clonality of B cells derived from tumor and lymph node was determined by analyzing heavy and light chain immunoglobulin sequences. The number of shared clonal groups observed between tumor and lymph node antibodies was significant for both heavy (p = 0.004) and light chain (p = 0.012) populations. Panning with phage-displayed single-chain variable fragment libraries derived from the tumor and lymph node B cells resulted in multiple antibodies that bound autologous tumor. Sequence analysis of enriched antibodies recovered after the third round of panning the tumor and TDLN libraries against autologous tumor lysates had a genetic relationship. These results indicate that B cells infiltrating a patient’s breast cancer and B cells present in the tumor-draining lymph node are clonally and functionally related.

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Abbreviations

CDR:

Complementarity determining region

ELISA:

Enzyme-linked immunosorbent assay

HER2:

Human epidermal growth factor receptor 2

IMGT:

ImMunoGeneTics information system

Mean V:

Value of the mean slope

MUC1:

Epithelial mucin

PBMCs:

Peripheral blood mononuclear cells

PBS:

Phosphate-buffered saline

PEG:

Polyethylene glycol

scFv:

Single-chain variable fragment

TBS:

Tris-buffered saline

TDLN:

Tumor-draining lymph nodes

TIL-B cells:

Tumor-infiltrating B lymphocytes

TMB:

3,3′,5,5′-Tetramethylbenzidine

References

  1. Spaner D, Bahlo A (2011) B lymphocytes in cancer immunology. In: Medin J, Fowler D (eds) Experimental and applied immunotherapy. Humana Press, New York, pp 37–57

    Chapter  Google Scholar 

  2. Nielsen JS, Nelson BH (2012) Tumor-infiltrating B cells and T cells: working together to promote patient survival. Oncoimmunology 1:1623–1625. doi:10.4161/onci.21650

    Article  PubMed Central  PubMed  Google Scholar 

  3. Coronella JA, Spier C, Welch M et al (2002) Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast. J Immunol 169:1829–1836

    Article  CAS  PubMed  Google Scholar 

  4. Nzula S, Going JJ, Stott DI (2003) Antigen-driven clonal proliferation, somatic hypermutation, and selection of B lymphocytes infiltrating human ductal breast carcinomas. Cancer Res 63:3275–3280

    CAS  PubMed  Google Scholar 

  5. Simsa P, Teillaud J-L, Stott DI et al (2005) Tumor-infiltrating B cell immunoglobulin variable region gene usage in invasive ductal breast carcinoma. Pathol Oncol Res 11:92–97

    Article  CAS  PubMed  Google Scholar 

  6. Wang Y, Ylera F, Boston M et al (2007) Focused antibody response in plasma cell-infiltrated non-medullary (NOS) breast cancers. Breast Cancer Res Treat 104:129–144. doi:10.1007/s10549-006-9409-3

    Article  CAS  PubMed  Google Scholar 

  7. Hansen MH, Nielsen HV, Ditzel HJ (2002) Translocation of an intracellular antigen to the surface of medullary breast cancer cells early in apoptosis allows for an antigen-driven antibody response elicited by tumor-infiltrating B cells. J Immunol 169:2701–2711

    Article  CAS  PubMed  Google Scholar 

  8. Kotlan B, Simsa P, Teillaud J-L et al (2005) Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes. J Immunol 175:2278–2285

    Article  CAS  PubMed  Google Scholar 

  9. Pavoni E, Monteriù G, Santapaola D et al (2007) Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells. BMC Biotechnol 7:70. doi:10.1186/1472-6750-7-70

    Article  PubMed Central  PubMed  Google Scholar 

  10. Rothe A, Klimka A, Tur MK et al (2004) Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines. Int J Mol Med 14:729–735

    CAS  PubMed  Google Scholar 

  11. Belimezi MM, Papanastassiou D, Merkouri E et al (2006) Growth inhibition of breast cancer cell lines overexpressing Her2/neu by a novel internalized fully human Fab antibody fragment. Cancer Immunol Immunother 55:1091–1099. doi:10.1007/s00262-005-0100-z

    Article  CAS  PubMed  Google Scholar 

  12. Ayat H, Burrone OR, Sadghizadeh M et al (2013) Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients. Biologicals 41(6):345–354. doi:10.1016/j.biologicals.2013.05.004

    Article  CAS  PubMed  Google Scholar 

  13. Krag DN, Anderson SJ, Julian TB et al (2010) Sentinel-lymph-node resection compared with conventional axillary-lymph-node dissection in clinically node-negative patients with breast cancer: overall survival findings from the NSABP B-32 randomised phase 3 trial. Lancet Oncol 11:927–933. doi:10.1016/S1470-2045(10)70207-2

    Article  PubMed Central  PubMed  Google Scholar 

  14. De Cicco C, Chinol M, Paganelli G (1998) Intraoperative localization of the sentinel node in breast cancer: technical aspects of lymphoscintigraphic methods. Semin Surg Oncol 15:268–271

    Article  PubMed  Google Scholar 

  15. Setiadi AF, Ray NC, Kohrt HE et al (2010) Quantitative, architectural analysis of immune cell subsets in tumor-draining lymph nodes from breast cancer patients and healthy lymph nodes. PLoS ONE 5:e12420. doi:10.1371/journal.pone.0012420

    Article  PubMed Central  PubMed  Google Scholar 

  16. Chang AY, Bhattacharya N, Mu J et al (2013) Spatial organization of dendritic cells within tumor draining lymph nodes impacts clinical outcome in breast cancer patients. J Transl Med 11:242. doi:10.1186/1479-5876-11-242

    Article  PubMed Central  PubMed  Google Scholar 

  17. Wei S, Shreiner AB, Takeshita N et al (2008) Tumor-induced immune suppression of in vivo effector T-cell priming is mediated by the B7-H1/PD-1 axis and transforming growth factor beta. Cancer Res 68:5432–5438. doi:10.1158/0008-5472.CAN-07-6598

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Li Q, Lao X, Pan Q et al (2011) Adoptive transfer of tumor reactive B cells confers host T cell immunity and tumor regression. Clin Cancer Res 17(15):4987–4995. doi:10.1158/1078-0432.CCR-11-0207

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Zirakzadeh AA, Marits P, Sherif A, Winqvist O (2013) Multiplex B cell characterization in blood, lymph nodes, and tumors from patients with malignancies. J Immunol 190:5847–5855. doi:10.4049/jimmunol.1203279

    Article  CAS  PubMed  Google Scholar 

  20. Barbas CF III, Burton DR, Scott JK, Silverman GJ (2001) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  21. Brochet X, Lefranc M-P, Giudicelli V (2008) IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 36:W503–W508. doi:10.1093/nar/gkn316

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Mehr R, Sternberg-Simon M, Michaeli M, Pickman Y (2012) Models and methods for analysis of lymphocyte repertoire generation, development, selection and evolution. Immunol Lett 148:11–22. doi:10.1016/j.imlet.2012.08.002

    Article  CAS  PubMed  Google Scholar 

  23. Dixon WJ, Massey FJ (1983) Introduction to statistical analysis, 4th edn. McGraw-Hill Science, Engineering & Mathematics, New York

    Google Scholar 

  24. Kao J, Salari K, Bocanegra M et al (2009) Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS ONE 4:e6146. doi:10.1371/journal.pone.0006146

    Article  PubMed Central  PubMed  Google Scholar 

  25. Shukla GS, Krag DN (2005) A sensitive and rapid chemiluminescence ELISA for filamentous bacteriophages. J Immunoass Immunochem 26:89–95. doi:10.1081/IAS-200051990

    Article  CAS  Google Scholar 

  26. Sun Y, Shukla GS, Kennedy GG et al (2009) Biopanning phage-display libraries on small tissue sections captured by laser capture microdissection. J Biotech Res 1:55–63

    CAS  PubMed Central  PubMed  Google Scholar 

  27. Chang B, Casali P (1994) The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol Today 15:367–373. doi:10.1016/0167-5699(94)90175-9

    Article  CAS  PubMed  Google Scholar 

  28. Muramatsu M, Kinoshita K, Fagarasan S et al (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553–563

    Article  CAS  PubMed  Google Scholar 

  29. Stavnezer J, Guikema JEJ, Schrader CE (2008) Mechanism and regulation of class switch recombination. Annu Rev Immunol 26:261–292. doi:10.1146/annurev.immunol.26.021607.090248

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Thie H, Toleikis L, Li J et al (2011) Rise and fall of an anti-MUC1 specific antibody. PLoS ONE 6:e15921. doi:10.1371/journal.pone.0015921

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Hansen MH, Nielsen H, Ditzel HJ (2001) The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells. Proc Natl Acad Sci USA 98:12659–12664. doi:10.1073/pnas.171460798

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Kotlan B, Simsa P, Foldi J et al (2003) Immunoglobulin repertoire of B lymphocytes infiltrating breast medullary carcinoma. Hum Antibodies 12:113–121

    CAS  PubMed  Google Scholar 

  33. Lavinder JJ, Wine Y, Giesecke C et al (2014) Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc Natl Acad Sci USA 111(6):2259–2264. doi:10.1073/pnas.1317793111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

We acknowledge Stephanie Pero, Girja Shukla, Yujing Sun, and Donald Weaver for helpful discussions and Chelsea Carmen for reagent preparation. We also thank Seth Harlow, Ted James, and Patti Lutton for their help in recruiting patients for this study. Finally, we recognize the administrative support of Shelley Bissonnette, Eileen Caffrey, and Sarah Howe in securing financial support and maintaining regulatory obligations. This research was supported by the SD Ireland Cancer Research Fund, the University of Vermont Department of Surgery, and Department of Defense Predoctoral Training Award #W81XWH-08-1-0756.

Conflict of interest

The authors of this paper report no conflict of interest with regard to this paper.

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Authors and Affiliations

  1. Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building Room E309, Burlington, VT, 05405, USA

    Leah J. Novinger, Takamaru Ashikaga & David N. Krag

  2. Department of Surgery, University of Vermont College of Medicine, Burlington, VT, 05405, USA

    Leah J. Novinger & David N. Krag

  3. Department of Medical Biostatistics, University of Vermont College of Medicine, Burlington, VT, 05405, USA

    Takamaru Ashikaga

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  1. Leah J. Novinger
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Correspondence to David N. Krag.

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Novinger, L.J., Ashikaga, T. & Krag, D.N. Identification of tumor-binding scFv derived from clonally related B cells in tumor and lymph node of a patient with breast cancer. Cancer Immunol Immunother 64, 29–39 (2015). https://doi.org/10.1007/s00262-014-1612-1

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