Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction

Coloma, M. J.; Hastings, Alice; Wims, Letitia A.; Morrison, Sherie L.

doi:10.1016/0022-1759(92)90092-8

Cited by 172 publications

(127 citation statements)

References 20 publications

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“…With a degenerate primer HL1 deduced from the signal peptide sequence of heavy chain [15], and C , designed to be complementary to the N-terminus of gamma constant region, a PCR product of V H gene including a leader peptide was obtained. Amplification of V L gene A21 were performed using the primers VL, deduced from the framework region 1 (FR1) sequence of the light chain, and Ck, designed to be complementary to C-terminal of the C kappa region.…”

Section: Isolation Of Antibody Variable Region Genesmentioning

confidence: 99%

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

et al. 2003

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The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.

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Section: Isolation Of Antibody Variable Region Genesmentioning

confidence: 99%

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

et al. 2003

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“…The 5' primers for amplification of mouse V genes were synthesized according to Coloma et al [3] . An extra VH5' primer was added according to newly published sequences data [4] (VHL4 in Table 1).…”

Section: Design Of Pcr Primersmentioning

confidence: 99%

Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light chain

Li¹,

Wang²,

Li³

et al. 1998

WJG

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AIM To clone mouse anti-human gastric cancer mAb (3H11) variable genes and to construct 3H11 humanmouse chimericantibody. METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RT-PCR method from 3H11 hybridoma cells, using 5' primers for leader sequences. The 3H11 VL gene was then inserted into human-mouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.

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“…The subcloned huEndo and huEndo-P125A genes were ligated in frame to the carboxyl end of the heavy chain constant domain of human IgG3 in the vector pAT135 (24,25). The endostatin heavy chain constant region was joined to the C-terminus of the anti-HER2 heavy chain of a recombinant humanized monoclonal antibody 4D5-8 (Genentech) in the expression vector (pSV2-his) containing a HisD gene for eukaryotic selection (23,26).…”

Section: Cell Lines Materials and Animalsmentioning

confidence: 99%

Targeted Delivery of an Antibody–Mutant Human Endostatin Fusion Protein Results in Enhanced Antitumor Efficacy

Shin¹,

Cho²,

Merchan³

et al. 2011

Molecular Cancer Therapeutics

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The antiangiogenic protein endostatin showed considerable preclinical antitumor activity, but limited efficacy in phase I/II trials. Prior studies using an anti-HER2 antibody-murine endostatin fusion showed enhanced antitumor activity compared to anti-HER2 antibody or endostatin given alone, or in combination. We have generated two anti-HER2 human endostatin fusion proteins by fusing either wildtype or a mutant human endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2 IgG3 antibody. Antitumor efficacy was examined in murine and human breast tumor models. HuEndo-P125A antibody fusion protein (aHER2-huEndo-

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Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction

Cited by 172 publications

References 20 publications

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light chain

Targeted Delivery of an Antibody–Mutant Human Endostatin Fusion Protein Results in Enhanced Antitumor Efficacy

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