Linked Life Data

19022801

Source:http://linkedlifedata.com/resource/pubmed/id/19022801

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2009-2-19
pubmed:abstractText
An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1741-0134
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
22
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
175-88
pubmed:meshHeading
pubmed-meshheading:19022801-Amino Acid Sequence, pubmed-meshheading:19022801-Animals, pubmed-meshheading:19022801-Antibodies, Bispecific, pubmed-meshheading:19022801-Antibody Affinity, pubmed-meshheading:19022801-Antigens, CD30, pubmed-meshheading:19022801-Base Sequence, pubmed-meshheading:19022801-Cloning, Molecular, pubmed-meshheading:19022801-Enzyme-Linked Immunosorbent Assay, pubmed-meshheading:19022801-Escherichia coli, pubmed-meshheading:19022801-Flow Cytometry, pubmed-meshheading:19022801-Humans, pubmed-meshheading:19022801-Immunoglobulin Fab Fragments, pubmed-meshheading:19022801-Immunoglobulin kappa-Chains, pubmed-meshheading:19022801-Immunoglobulin lambda-Chains, pubmed-meshheading:19022801-Killer Cells, Natural, pubmed-meshheading:19022801-Mice, pubmed-meshheading:19022801-Models, Molecular, pubmed-meshheading:19022801-Molecular Sequence Data, pubmed-meshheading:19022801-Mutation, pubmed-meshheading:19022801-Myeloma Proteins, pubmed-meshheading:19022801-Protein Folding, pubmed-meshheading:19022801-Receptors, IgG, pubmed-meshheading:19022801-Recombinant Fusion Proteins, pubmed-meshheading:19022801-Sequence Analysis, DNA, pubmed-meshheading:19022801-Solubility
pubmed:year
2009
pubmed:articleTitle
Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.
pubmed:affiliation
Lehrstuhl für Biologische Chemie, Technische Universität München, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't