Linked Life Data

8568873

Source:http://linkedlifedata.com/resource/pubmed/id/8568873

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1996-3-1
pubmed:abstractText
The use of antibodies to target tumor antigens has had limited success, partially due to the large size of IgG molecules, difficulties in constructing smaller single chain Fv (scFv) antibody fragments, and immunogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the VH and VL genes of a binding scFv are replaced with repertoires of V genes (chain shuffling). The applicability of this approach to protein binding scFv is unknown. For this work, chain shuffling was used to increase the affinity of a non-immune human scFv, which binds the glycoprotein tumor antigen c-erbB-2 with an affinity of 1.6 x 10(-8) M. The affinity of the parental scFv was increased sixfold (Kd = 2.5 x 10(-9) M) by light-chain shuffling and fivefold (Kd = 3.1 x 10(-9) M) by heavy-chain shuffling, values comparable to those for antibodies against the same antigen produced by hybridomas. When selections were performed on antigen immobilized on polystyrene, spontaneously dimerizing scFv were isolated, the best of which had only a slightly lower Kd than wild type (Kd = 1.1 x 10(-8) M). These scFv dimerize on phage and are preferentially selected as a result of increased avidity. Compared to scFv which formed only monomer, dimerizing scFv had mutations located at the VH-VL interface, suggesting that VH-VL complementarity determines the extent of dimerization. Higher-affinity monomeric scFv were only obtained by selecting in solution using limiting concentrations of biotinylated antigen, followed by screening mutant scFv from bacterial periplasm by koff in a BIAcore. Using the proper selection and screening conditions, protein binding human scFv with affinities comparable to murine hybridomas can be produced without immunization.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0022-2836
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
255
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
28-43
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection.
pubmed:affiliation
Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco 94110, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't