Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

2.50
Hdl Handle:
http://hdl.handle.net/10541/86122
Title:
Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.
Authors:
Shaw, David M; Embleton, Jim; Westwater, C; Ryan, Matthew G; Myers, Kevin A; Kingsman, Susan M; Carroll, M W; Stern, Peter L
Abstract:
The 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.
Affiliation:
CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Citation:
Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4. 2000, 1524 (2-3):238-46 Biochim. Biophys. Acta
Journal:
Biochimica et Biophysica Acta
Issue Date:
15-Dec-2000
URI:
http://hdl.handle.net/10541/86122
PubMed ID:
11113573
Type:
Article
Language:
en
ISSN:
0006-3002
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorShaw, David Men
dc.contributor.authorEmbleton, Jimen
dc.contributor.authorWestwater, Cen
dc.contributor.authorRyan, Matthew Gen
dc.contributor.authorMyers, Kevin Aen
dc.contributor.authorKingsman, Susan Men
dc.contributor.authorCarroll, M Wen
dc.contributor.authorStern, Peter Len
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.en
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

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