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Avimers hold their own

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Abstract

Antibody surrogates based on a class of human receptor domains bind protein targets with high affinity.
NATURE BIOTECHNOLOGY VOLUME 23
NUMBER 12
DECEMBER 2005 1493
Avimers hold their own
Ki Jun Jeong, Robert Mabry & George Georgiou
Antibody surrogates based on a class of human receptor domains bind protein targets with high affinity.
Success in designing proteins with strong
binding capacities, much like good fortune in
poker, depends on knowing how to fold ’em
and how to hold ’em. In this issue, Stemmer
and coworkers
1
have met this challenge with
a new class of antibody surrogates that have
high ligand affinity, low immunogenicity and
the capacity for high expression in bacteria.
Although antibodies and antibody frag-
ments remain the most intensely pursued
class of protein pharmaceuticals
2,3
, inter-
est in proteins that can serve as antibody
surrogates is greater than ever before
4
. In
part, this can be attributed to the technical
limitations of immunoglobulins. Despite
processing advances over the last 15 years,
the production of full-length, properly gly-
cosylated antibodies by mammalian cells
remains costly. Although bacterially pro-
duced antibody fragments can in principle
be used when glycosylated Fc regions are
not required for biological activity
5
(for
example, to bind and neutralize target mol-
ecules), many have a propensity to aggregate,
are expressed in low yields or have poor ther-
modynamic stability. Another often cited but
less important limitation of antibodies is
that their disulfide bonds preclude cytoplas-
mic expression, which is required for gene
therapy applications
4
. Finally, it is difficult
to engineer antibodies with altered biodis-
tribution, for example, the capacity to cross
the blood-brain barrier.
At least some of these limitations can be
addressed by protein engineering. For example,
expression in ‘humanized’ yeast holds promise
for low-cost manufacturing of homogenous
preparations of properly glycosylated proteins
6
,
whereas second-generation display technolo-
gies may be used to select for well-expressed
antibody fragments in bacteria or for folding
and stability in the absence of disulfides
7
(K.J.J.
and G.G., unpublished data).
In light of such improvements in antibody
technology, where do protein surrogates fit
in? The answer lies equally in pragmatic
concerns and in technical advantages. The
intellectual property landscape for antibody
manufacturing and antibody engineering,
especially using techniques such as phage
display, is very complex. As a result, the
development of new antibody-based reagents
for therapy, diagnosis or even purification
purposes is burdened by the obligation to
commit a significant portion of any potential
revenue stream to paying royalties
2
. Although
scientists may find such an intrusion of
commercial considerations distasteful, the
fact remains that business considerations in
some instances hinder the development of
therapeutic antibodies.
Protein scaffolds that can be engineered for
ligand binding present a way to circumvent
intellectual property restrictions and may
also facilitate efficient, inexpensive manu-
facturing in bacteria
4
. These scaffolds are
typically short polypeptides that assume a
simple, thermodynamically stable fold and are
expressed at respectable levels in Escherichia
coli. They must also be able to tolerate amino
acid substitutions within contiguous regions
that define a binding surface. Typically, bind-
ers are generated by screening combinatorial
libraries in which multiple positions have
been randomized. Over 30 naturally existing
protein domains have been engineered for
ligand binding, including α-helical proteins
(such as affibodies), β-barrels (for example,
lipocalins), β-sandwich proteins (fibronectin
type III) and α2/β2 designed ankyrin-repeat
proteins
4
. A recent paper describes the engi-
neering of high-affinity designed ankyrin
repeat proteins with a varying number of
repeat units
8
.
Stemmer and coworkers now report a
new type of nonimmunoglobulin binding
protein—or avimer’ (from avidity multi-
mer)—derived from human A-domains that
are found in various cell surface receptors,
George Georgiou and Ki Jun Jeong are in the
Department of Chemical Engineering and
Institute for Cellular and Molecular Biology,
University of Texas at Austin, 2500 Speedway,
Austin, Texas 78712, USA. Robert Mabry is at
the Institute for Cellular and Molecular Biology,
University of Texas at Austin, 2500 Speedway,
Austin, Texas 78712, USA.
e-mail: gg@che.utexas.edu
Figure 1. Avimer construction. (a) A prototypical A-domain comprises ~30–35 amino acids (~4 kDa)
including six cysteine residues (purple), four residues for calcium binding (yellow) and two structural
scaffold residues (dark blue). Nonconserved residues are indicated in gray. (b) After exon shuffling,
a library of assorted A-domains that share only universally conserved residues is generated. The
remaining residues are assembled randomly. (c) Multiple A domains are linked by sequential panning
and screening by phage display (domain walking). (d) Fusion of an IgG binding domain to the N
terminus of multimerized avimers prolongs the serum half-life of multimeric avimers.
Screening of
monomer
specificity
N
C
Target ligand
IgG
C
C
Ca
C
C
C
C
Exon
shuffling
A-domain
ab
cd
Bob Crimi
NEWS AND VIEWS
© 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology
1494 VOLUME 23
NUMBER 12
DECEMBER 2005 NATURE BIOTECHNOLOGY
including low density lipoprotein receptors
9
.
Avimers are multidomain proteins, analo-
gous to designed ankyrin repeats. However,
avimers are generated by sequential selection
of individual binding domains, each of which
recognizes a different epitope, thereby gener-
ating a protein that can bind multiple sites on
a ligand (Fig. 1) or even multiple targets.
The authors convincingly showed that
linking multiple, individually selected A-
domains generates avimers with ligand
affinities in the picomolar and even sub-
picomolar range. Remarkably, although
A-domains contain six cysteines that form
three disulfide bonds, avimers containing
up to eight A-domains with a total of 48
cysteines can be produced in the cytoplasm
of E. coli and then properly folded upon
air oxidation to give yields >1 g/L in high
cell-density fermentations—a bioprocess
engineer’s dream come true! According to
the authors, such yields were observed with
several avimers to different ligands. In addi-
tion, avimers are highly stable after weeks of
incubation at elevated temperature in buffer
and for several days in serum.
Of course, the small size of avimers should
lead to rapid renal clearance in animals.
Stemmer and colleagues solved this prob-
lem with a hitchhiker’ approach that takes
advantage of IgG as a carrier to enhance
pharmacokinetics. For instance, to increase
the half-life of a three-domain protein that
binds interleukin 6 (IL-6) trivalently, they
obtained a domain that binds both cynomol-
gus monkey and human IgG. Attachment of
this IgG-binding domain to the trimer gen-
erated a 19-kDa avimer with a half-life of
~90 h, which compares favorably with the
half-lives of certain FDA-approved protein-
based therapeutics.
Presumably, this stabilization arises from
the recycling of the IgG-avimer complexes
through interactions with the Fc receptor
FcRn. Armed with a prolonged half-life, the
anti-IL6-anti-Fc avimer showed potent anti-
inflammatory activity in mice. Remarkably, it
did not appear to induce significant antibody
responses in the rodent model. Whether low
immunogenicity is an intrinsic, general prop-
erty of avimers—related to their high stabil-
ity, as the authors speculate—and whether
no or minimal anti-avimer responses will
occur in humans remain open questions.
Repeat proteins, such as designed ankyrin
repeats and now A-domains, provide a level
of modularity that is very advantageous for
generating high-affinity binders and for
tailoring their pharmacokinetic proper-
ties. The stitching together of individually
selected binding domains demonstrated by
Stemmer and colleagues capitalizes fully
on this property of repeat proteins. Their
approach generated proteins that have the
requisite binding affinity, are sufficiently
small to ensure rapid systemic distribu-
tion after subcutaneous administration and
show prolonged persistence in circulation.
Through their comprehensive evaluation of
the properties of avimers in vitro and in vivo,
the authors have provided some of the most
convincing arguments to date in favor of
engineered antibody surrogates. Ultimately,
however, the degree to which avimers and
other scaffolds will replace antibodies hinges
on their therapeutic efficacy and the absence
of side effects in humans. We now eagerly
await the results of clinical trials.
1. Silverman, J. et al. Nat. Biotechnol. 23, 1556–1561
(2005).
2. Baker, M. Nat. Biotechnol. 23, 1065–1072
(2005).
3. Hoogenboom, H.R. Nat. Biotechnol. 23, 1105–
11016. (2005)
4. Binz, H.K., Amstutz, P. & Pluckthun, A. Nat.
Biotechnol. 23, 1257–1268 (2005).
5. Mabry, R. et al. Infect. Immun. (in the press).
6. Hamilton, S.R. et al. Science. 301, 1244–1246
(2003).
7. Harvey, B.R. et al. Proc. Natl. Acad. Sci. USA 101,
9193–9198 (2004).
8. Binz, H.K. et al. Nat. Biotechnol. 22, 575–582
(2004).
9. North, C.L. & Blacklow, S.C. Biochemistry 38, 3926–
3935 (1999).
Bacterial lessons in sausage
making
Vincent G H Eijsink & Lars Axelsson
The genome sequence of a meat-borne lactic acid bacterium sheds light on
its food-preserving abilities.
Lactic acid bacteria have long been associ-
ated with the fermentation, acidification and
preservation of food. People consume large
amounts of these bacteria every day in prod-
ucts such as yogurt, cheese and fermented
meats. In this issue, Chaillou et al.
1
report the
genome sequence of a highly specialized lac-
tic acid bacterium, Lactobacillus sakei, whose
principal habitat is the surface of meats, where
it prevents growth of unwanted organisms. In-
depth analysis of the L. sakei genome reveals
unique features that reflect a range of adapta-
tions to this particular niche.
As its name implies, L. sakei was first isolated
from saké, a Japanese rice wine (or, perhaps
more accurately, rice beer) that is produced
partly by lactic acid fermentation. The spe-
cies was not considered of particular interest
until the mid-1980s, when it was shown that
the spontaneous fermentation of meat, as in
the manufacture of salami and other dry fer-
mented sausages, was dominated by strains of
L. sakei and its close relative, L. curvatus. Similar
strains were also found to dominate the flora
in vacuum-packed meat and processed meat
products stored at cold temperatures. Curiously,
before the advent of modern classification tools
for bacteria, these meat strains were considered
difficult to classify and designated atypical
lactobacilli”
2
—a judgement that is only con-
firmed by the work of Chaillou et al.
Early research on L. sakei focussed on its use
as a starter culture in meat fermentations
3
.
It was shown to be a robust species that is
highly competitive in the meat environment
and that tolerates harsh conditions (includ-
ing oxygen). For the lactic acid bacteria group
as a whole, the 1990s also saw a tremendous
effort in fundamental and applied research
on bacteriocins, small antimicrobial peptides
that inhibit growth of important food-borne
pathogens such as Listeria monocytogenes
4
. L.
sakei is important in this regard because many
strains produce potent bacteriocins, which
contribute to biopreservation. Coincidently,
and atypically, the sequenced strain of L. sakei
is not a bacteriocin producer. However, the
genome does contain genes related to the
production of several bacteriocins as well
as several ‘immunity genes’ that may pro-
vide protection against these compounds.
Chaillou et al. note that the presence of these
immunity genes may contribute to the com-
petitiveness of this L. sakei strain
4
.
Vincent G.H. Eijsink is in the Department of
Chemistry, Biotechnology and Food Science,
Norwegian University of Life Sciences, Chr.
M. Falsensvei 1, N-1432 Ås, Norway, and Lars
Axelsson is at Matforsk, The Norwegian Food
Research Institute, Osloveien 1, N-1430 Ås,
Norway. e-mail: vincent.eijsink@umb.no or lars.
axelsson@matforsk.no
NEWS AND VIEWS
© 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology
... Avimers are approximately 4 kDa in size [38], based on the conserved A-domain of different cell surface receptors [36]. Avimers are produced by a selection of distinct binding sites, thereby providing a protein that is capable of binding different sites on the same target or even binding to different targets simultaneously [39]. Avimers can be expressed at considerable amounts without perceptible inclusion body formation in Escherichia coli after the selection of optimal binders by phage display [38]. ...
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There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed.
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Background Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding sequence. Methods The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature peptide encoding the traceable avimer molecule was carried out in Escherichia coli strain BL21 using IPTG (Isopropyl β-D-1-thiogalactopyranoside) induction process. The expression level of the 11 kDa traceable avimer was studied by SDS-PAGE, western blot and ELISA analysis. Results Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11 kDa C426 avimer molecule was detectable without any degradation compared with the control group. Conclusion Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity and traceability features.
... Additionally, avimers are relatively small in comparison to antibody mimetics scaffolds, such as tetranectin (20 kDa), protein A (7 kDa), and lipocalin (20 kDa). Avimers are developed by the sequential selection of individual binding domains that enable generation of multidomain scaffolds to recognize different epitopes in the same ligand, single ligands, or multiple target ligands (44). Avimers have shown high affinity and specificity against their ligands with picomolar binding constants as well as high therapeutic efficiency with IC 50 in subpicomolar concentrations (125,126). ...
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The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 10 is June 12, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Amgen (USA) www.amgen.com (Jeong et al., 2005) Fynomers N/A (Qiu et al., 2007) *The italic words show ''indications'', while the targets are shown in parentheses. ...
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  • H K Binz
  • P Amstutz
  • A Pluckthun
Binz, H.K., Amstutz, P. & Pluckthun, A. Nat. Biotechnol. 23, 1257-1268 (2005).
  • J Silverman
Silverman, J. et al. Nat. Biotechnol. 23, 1556-1561 (2005).