Growth inhibition of breast cancer cell lines overexpressing Her2/neu by a novel internalized fully human Fab antibody fragment

Belimezi, Maria; Papanastassiou, Danai; Merkouri, E; Baxevanis, Constantin N.; Mamalaki, Avgi

doi:10.1007/s00262-005-0100-z

Cited by 15 publications

(11 citation statements)

References 36 publications

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“…Of particular interest are libraries that have been generated from patients with cancer as the source material. Multiple libraries have been evaluated that were derived from patients with neuroblastoma (Uttenreuther-Fischer et al, 2006), breast cancer (Rubinstein et al, 2002;Belimezi et al, 2006), lung cancer (Graus et al, 1998), ovarian cancer (Figini et al, 2009) and multiple other tumor types (Roovers et al, 2001;Baskar et al, 2009;Li et al, 2001;Dantas-Barbosa et al, 2009). The source material from these patients included peripheral blood and tumor-draining lymph nodes.…”

Section: Discussionmentioning

confidence: 99%

“…Using patientderived B cells to generate phage antibody libraries has the potential advantage of including antibodies that have been made by the patient in response to their own cancer. Phage antibody libraries have been generated from patients with breast cancer, colorectal cancer and multiple tumor types (Rubinstein et al, 2002;Belimezi et al, 2006;Graus et al, 1998;Figini et al, 2009;Roovers et al, 2001;Baskar et al, 2009;Li et al, 2001;Dantas-Barbosa et al, 2009) including neuroblastoma (Uttenreuther-Fischer et al, 2006). Application of these libraries has been primarily to screen for ligands to cancer cell lines or heterologous cancer tissue.…”

Section: Introductionmentioning

confidence: 99%

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Autologous antibodies that bind neuroblastoma cells

Sun

Sholler

Shukla

et al. 2015

Journal of Immunological Methods

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Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Autologous antibodies that bind neuroblastoma cells

Sun

Sholler

Shukla

et al. 2015

Journal of Immunological Methods

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“…The procedures for cloning the sequence encoding human EC D HER 2 in vector pRSET , as well as the effective production of the recombinant protein in strain C41 E. coli , isolation, washing off and storage of protein inclusion bodies were described earlier [11]. …”

Section: Methodsmentioning

confidence: 99%

“…In our previous study, we repeated the expression of EC D HER 2 in Escherichia coli using pRSET vector (Life Technologies) under the control of a highly active promoter, bacteriophage T7 [11]. In the construct used, the N-terminal signal peptide responsible for the secretion of HER 2 and cut in a eukaryotic cell in the course of its translocation into the endoplasmic reticulum lumen, was removed.…”

Section: Introductionmentioning

confidence: 99%

Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli

et al. 2014

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Receptor 2 of the human epidermal growth factor (HER2/neu, c-erbB2) is a 185 kDa proto-oncogene protein characterized by an overexpression in some oncological diseases, including 30% of mammary glands cancers, as well as tumors in the ovary, stomach and other organs of the human body. Since HER2- tumor status testing is the essential part of a successful cancer treatment, the expression and purification of substantial amounts of the extracellular domain (ECD) of HER2 is an important task. The production of ECD HER2 in Escherichia coli has several advantages over the use of eukaryotic expression systems, but the bulk of the recombinant product in bacteria accumulates as insoluble protein inclusion bodies. In this study, we obtained ECD HER2 in Escherichia coli as insoluble inclusion bodies and elaborated a simple, efficient, and fast protocol for the solubilization, refolding, and isolation of the protein in soluble form.

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“…The extracellular domain of human Her2/neu receptor (ECD/Her2, aminoacid residues 1 - 627) was previously enzymatically amplified by PCR and subcloned into the expression vector pPICZaC (Invitrogen, Carlsbad, California, USA) for soluble expression in yeast P. pastoris [18]. Protein production was induced with methanol while cells were grown at 30°C for 72 h. The culture supernatant was passed through a 0.22 - μm membrane filter, concentrated using the Minitan Ultrafiltration System (Millipore, Billerica, Massachusetts, USA) equipped with 30 kDa cut-off membrane and then dialyzed extensively against 20 mM sodium phosphate buffer pH 7.4, containing 0.5 M NaCl and 10 mM Imidazole.…”

Section: Methodsmentioning

confidence: 99%

The mannosylated extracellular domain of Her2/neu produced in P. pastorisinduces protective antitumor immunity

et al. 2009

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BackgroundHer2/neu is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Several attempts have been made using the extracellular domain of Her2/neu (ECD/Her2) as a prophylactic vaccine in mice with no success in tumor prevention.MethodsThe extracellular domain of Her2/neu (ECD/Her2) was expressed in yeast P. pastoris, in a soluble highly mannosylated form. The immune response of the immunization with this recombinant ECD/Her2 was analyzed using immunoprecipitation and western blot analysis, proliferation and cytotoxicity assays as well as specific tumor growth assays.ResultsMannosylated ECD/Her2 elicited a humoral response with HER2/neu specific antibodies in vaccinated mice, which were able to reduce the proliferation rate of cancer cells in vitro. Moreover, it elicited a cellular response with Her2/neu-specific CTL capable of lysing tumor cells, in vitro. When immunized Balb/c and HHD mice were challenged with Her2/neu-overexpressing cells, tumor growth was inhibited.ConclusionHere we report on the efficacy of the extracellular domain of human Her2/neu produced in yeast P. pastoris, which confers mannosylation of the protein, to act as a potent anti-tumor vaccine against Her2/neu overexpressing tumors. Specific cellular and humoral responses were observed as well as efficacy.

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Growth inhibition of breast cancer cell lines overexpressing Her2/neu by a novel internalized fully human Fab antibody fragment

Cited by 15 publications

References 36 publications

Autologous antibodies that bind neuroblastoma cells

Autologous antibodies that bind neuroblastoma cells

Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli

The mannosylated extracellular domain of Her2/neu produced in P. pastorisinduces protective antitumor immunity

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