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dc.contributor.authorShaw, David M
dc.contributor.authorEmbleton, Jim
dc.contributor.authorWestwater, C
dc.contributor.authorRyan, Matthew G
dc.contributor.authorMyers, Kevin A
dc.contributor.authorKingsman, Susan M
dc.contributor.authorCarroll, M W
dc.contributor.authorStern, Peter L
dc.date.accessioned2009-11-13T13:03:00Z
dc.date.available2009-11-13T13:03:00Z
dc.date.issued2000-12-15
dc.identifier.citationIsolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4. 2000, 1524 (2-3):238-46 Biochim. Biophys. Actaen
dc.identifier.issn0006-3002
dc.identifier.pmid11113573
dc.identifier.urihttp://hdl.handle.net/10541/86122
dc.description.abstractThe oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.
dc.language.isoenen
dc.subjectCancer Antigensen
dc.subjectStomach Canceren
dc.subject.meshAmino Acid Sequence
dc.subject.meshAntibodies, Monoclonal
dc.subject.meshAntigens, Neoplasm
dc.subject.meshBase Sequence
dc.subject.meshCloning, Molecular
dc.subject.meshEnzyme-Linked Immunosorbent Assay
dc.subject.meshFemale
dc.subject.meshGene Therapy
dc.subject.meshHumans
dc.subject.meshImmunoglobulin Fragments
dc.subject.meshImmunoglobulin Variable Region
dc.subject.meshImmunohistochemistry
dc.subject.meshMembrane Glycoproteins
dc.subject.meshMolecular Sequence Data
dc.subject.meshMutation
dc.subject.meshPlacenta
dc.subject.meshRecombinant Fusion Proteins
dc.subject.meshStomach Neoplasms
dc.subject.meshSurface Plasmon Resonance
dc.titleIsolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.en
dc.typeArticleen
dc.contributor.departmentCRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.en
dc.identifier.journalBiochimica et Biophysica Actaen
html.description.abstractThe oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.


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