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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. 2004 Oct; 14(4):729-35.IJ

Abstract

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.

Authors+Show Affiliations

Department I of Internal Medicine, Laboratory of Immunotherapy, University Hospital Cologne, D-50931 Cologne, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15375609

Citation

Rothe, A, et al. "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." International Journal of Molecular Medicine, vol. 14, no. 4, 2004, pp. 729-35.
Rothe A, Klimka A, Tur MK, et al. 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. 2004;14(4):729-35.
Rothe, A., Klimka, A., Tur, M. K., Pfitzner, T., Huhn, M., Sasse, S., Mallmann, P., Engert, A., & Barth, S. (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. International Journal of Molecular Medicine, 14(4), 729-35.
Rothe A, et al. 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. 2004;14(4):729-35. PubMed PMID: 15375609.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Rothe,A, AU - Klimka,A, AU - Tur,M K, AU - Pfitzner,T, AU - Huhn,M, AU - Sasse,S, AU - Mallmann,P, AU - Engert,A, AU - Barth,S, PY - 2004/9/18/pubmed PY - 2005/3/29/medline PY - 2004/9/18/entrez SP - 729 EP - 35 JF - International journal of molecular medicine JO - Int J Mol Med VL - 14 IS - 4 N2 - The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments. SN - 1107-3756 UR - https://www.unboundmedicine.com/medline/citation/15375609/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_ L2 - http://www.spandidos-publications.com/ijmm/14/4/729 DB - PRIME DP - Unbound Medicine ER -