Biomedical Engineering Reference
In-Depth Information
treatment of rosacea.
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This is probably the basis for the observed reduction in
inflammation in rosacea after application of azelaic acid-containing creams.
9.5 Antibody-Derived Inhibitors
Monoclonal antibodies can be specifically designed and engineered to become
powerful and highly specific inhibitors (or activators). For therapeutic pur-
poses, human antibodies isolated by antibody phage display methodology
should be used ideally, as they do not elicit immune responses.
9.5.1 Potential of Antibody Phage Display Strategy
Antibody phage display involves the expression and display of a fragment of an
antibody molecule on the surface of a filamentous phage, usually M13 or the fd.
RNAs are extracted from human volunteer B-cells and reverse-transcribed to
cDNA. Then, cDNAs are converted to DNAs and cloned into the genome of
the filamentous phage.
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Each phage will carry only one copy of genetic
material and its representative antibody. Phages can be engineered to express
only a single copy of the antibody (monovalent) or multiple copies (poly-
valent).
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Monovalent methodology is more common, since it is not expected
to interfere with the ability of phages to infect bacteria. Phages with displayed
antibodies are then screened over an immobilized target. Figure 9.5 summarizes
the antibody phage display method. Each phage carries only one distinct
antibody. At the end of the experiment that includes multiple rounds of
selection, the obtained phages displaying high-anity antibodies are amplified
in E. coli and sequenced. The antibody-encoding gene is transferred into bac-
teria to produce large quantities of the antibody.
9.5.2 Antibodies Directed Against KLKs
Dyax has developed a series of human monoclonal antibodies based on highly
diverse phage display library and a high-throughput surface plasmon resonance
(SPR) anity screening method.
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One of the KLK1-specific antibodies
(DX-2300) exhibited competitive inhibition with a very low K
i
of 130 pM.
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Since DX-2300 shows competitive inhibition and lack of binding to KLK1-
complexed with active site inhibitors, it is concluded that it likely binds to the
active site or next to it.
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KLK1 was reported as the primary kininogenase in
airways, and its activity is upregulated in bronchoalveolar lavage fluid from
patients with asthma and allergic rhinitis, indicating that this antibody may
become a valuable tool for the therapy of asthma. In an allergic sheep model of
asthma, DX-2300 inhibited the allergen-induced late-phase bronchoconstric-
tion and the airway hyper-responsiveness to carbachol.
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Another interesting approach for the production of antibodies involves
active immunization. It was mentioned earlier that in EAE,
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a mouse model
of multiple sclerosis, administration of rat Klk6 resulted in the production of
