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A novel fully human antitumour immunoRNase targeting ErbB2-positive tumours.

Borriello M, Laccetti P, Terrazzano G, D'Alessio G, De Lorenzo C - Br. J. Cancer (2011)

Bottom Line: Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment.Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb.Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia Strutturale e Funzionale, Università Federico II, via Cinthia, Napoli 80126, Italy.

ABSTRACT

Background: ErbB2 is an attractive target for immunotherapy, as it is a tyrosine kinase receptor overexpressed on tumour cells of different origin, with a key role in the development of malignancy. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment.

Methods: A novel human immunoRNase, called anti-ErbB2 human compact antibody-RNase (Erb-hcAb-RNase), made up of the compact anti-ErbB2 antibody Erbicin-human-compact Antibody (Erb-hcAb) and human pancreatic RNase (HP-RNase), has been designed, expressed in mammalian cell cultures and purified. The immunoRNase was then characterised as an enzymatic protein, and tested for its biological actions in vitro and in vivo on ErbB2-positive tumour cells.

Results: Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb. Moreover, this novel immunoRNase is endowed with an effective and selective antiproliferative action for ErbB2-positive tumour cells both in vitro and in vivo. Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb.

Conclusion: Erb-hcAb-RNase could be a promising candidate for the immunotherapy of ErbB2-positive tumours.

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Related in: MedlinePlus

Schematic representation of the chimeric ImmunoRNase, Erb-hcAb-RNase, obtained by fusing the human compact antibody Erb-hcAb and the HP-RNase. Erbicin=the human anti-ErbB2 scFv; H=hinge; CH2-CH3=the heavy constant domains of the human IgG1 Fc; S=the spacer peptide AAASGGPEGGS linking the scFv-Fc and the RNase moieties; HP-RNase=the RNase moiety.
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fig1: Schematic representation of the chimeric ImmunoRNase, Erb-hcAb-RNase, obtained by fusing the human compact antibody Erb-hcAb and the HP-RNase. Erbicin=the human anti-ErbB2 scFv; H=hinge; CH2-CH3=the heavy constant domains of the human IgG1 Fc; S=the spacer peptide AAASGGPEGGS linking the scFv-Fc and the RNase moieties; HP-RNase=the RNase moiety.

Mentions: A new human anti-ErbB2 immunoagent was generated by fusing HP-RNase with the fully human anti-ErbB2 compact antibody (Erb-hcAb) (De Lorenzo et al, 2004b). The cDNAs coding for the human compact antibody Erb-hcAb and HP-RNase were amplified by PCR and cloned into the eukaryotic pCMV-ER-myc expression vector. In particular, the cDNA encoding HP-RNase was cloned downstream to the sequence encoding the carboxy terminus of the scFv-Fc (Erb-hcAb) by adding a spacer encoding a 11-amino-acid residue peptide linker (AAASGGPEGGS) to minimise the steric hindrance between the two moieties of the chimeric protein (Figure 1). The recombinant plasmid, sequenced to confirm faithful cloning, was stably transfected in 293 T (human embryonic kidney) cells, and the recombinant construct was expressed as a secretion product into the culture medium. Once selected by quantitative ELISA assays, the clone producing the highest levels of Erb-hcAb-RNase was used for the production of the chimeric immunoagent, which was then purified by affinity chromatography on a protein A-Ceramic Hyper DF column. The immunoagent was named Erb-hcAb-RNase .


A novel fully human antitumour immunoRNase targeting ErbB2-positive tumours.

Borriello M, Laccetti P, Terrazzano G, D'Alessio G, De Lorenzo C - Br. J. Cancer (2011)

Schematic representation of the chimeric ImmunoRNase, Erb-hcAb-RNase, obtained by fusing the human compact antibody Erb-hcAb and the HP-RNase. Erbicin=the human anti-ErbB2 scFv; H=hinge; CH2-CH3=the heavy constant domains of the human IgG1 Fc; S=the spacer peptide AAASGGPEGGS linking the scFv-Fc and the RNase moieties; HP-RNase=the RNase moiety.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111160&req=5

fig1: Schematic representation of the chimeric ImmunoRNase, Erb-hcAb-RNase, obtained by fusing the human compact antibody Erb-hcAb and the HP-RNase. Erbicin=the human anti-ErbB2 scFv; H=hinge; CH2-CH3=the heavy constant domains of the human IgG1 Fc; S=the spacer peptide AAASGGPEGGS linking the scFv-Fc and the RNase moieties; HP-RNase=the RNase moiety.
Mentions: A new human anti-ErbB2 immunoagent was generated by fusing HP-RNase with the fully human anti-ErbB2 compact antibody (Erb-hcAb) (De Lorenzo et al, 2004b). The cDNAs coding for the human compact antibody Erb-hcAb and HP-RNase were amplified by PCR and cloned into the eukaryotic pCMV-ER-myc expression vector. In particular, the cDNA encoding HP-RNase was cloned downstream to the sequence encoding the carboxy terminus of the scFv-Fc (Erb-hcAb) by adding a spacer encoding a 11-amino-acid residue peptide linker (AAASGGPEGGS) to minimise the steric hindrance between the two moieties of the chimeric protein (Figure 1). The recombinant plasmid, sequenced to confirm faithful cloning, was stably transfected in 293 T (human embryonic kidney) cells, and the recombinant construct was expressed as a secretion product into the culture medium. Once selected by quantitative ELISA assays, the clone producing the highest levels of Erb-hcAb-RNase was used for the production of the chimeric immunoagent, which was then purified by affinity chromatography on a protein A-Ceramic Hyper DF column. The immunoagent was named Erb-hcAb-RNase .

Bottom Line: Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment.Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb.Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia Strutturale e Funzionale, Università Federico II, via Cinthia, Napoli 80126, Italy.

ABSTRACT

Background: ErbB2 is an attractive target for immunotherapy, as it is a tyrosine kinase receptor overexpressed on tumour cells of different origin, with a key role in the development of malignancy. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment.

Methods: A novel human immunoRNase, called anti-ErbB2 human compact antibody-RNase (Erb-hcAb-RNase), made up of the compact anti-ErbB2 antibody Erbicin-human-compact Antibody (Erb-hcAb) and human pancreatic RNase (HP-RNase), has been designed, expressed in mammalian cell cultures and purified. The immunoRNase was then characterised as an enzymatic protein, and tested for its biological actions in vitro and in vivo on ErbB2-positive tumour cells.

Results: Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb. Moreover, this novel immunoRNase is endowed with an effective and selective antiproliferative action for ErbB2-positive tumour cells both in vitro and in vivo. Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb.

Conclusion: Erb-hcAb-RNase could be a promising candidate for the immunotherapy of ErbB2-positive tumours.

Show MeSH
Related in: MedlinePlus