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Avimers hold their own



Antibody surrogates based on a class of human receptor domains bind protein targets with high affinity.
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
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
, inter-
est in proteins that can serve as antibody
surrogates is greater than ever before
. 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
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
. 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
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
and G.G., unpublished data).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
. 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
. 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
. A recent paper describes the engi-
neering of high-affinity designed ankyrin
repeat proteins with a varying number of
repeat units
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.
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
Target ligand
Bob Crimi
© 2005 Nature Publishing Group
1494 VOLUME 23
including low density lipoprotein receptors
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
2. Baker, M. Nat. Biotechnol. 23, 1065–1072
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
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
9. North, C.L. & Blacklow, S.C. Biochemistry 38, 3926–
3935 (1999).
Bacterial lessons in sausage
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.
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
—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
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
. 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
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: or lars.
© 2005 Nature Publishing Group
... 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]. ...
Full-text available
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.
... The avidity generated by combining multiple bind-ing domains is a powerful approach to increase their affinity and specificity 4 . These molecules have several advantageous compared to common antibodies including small size, high stability, strong binding affinity to the target molecule, specificity and high resistance against heat denaturation and enzymatic degradation 5,6 . In addition, the avimer molecules can be produced based on bacterial expression systems 7 . ...
Full-text available
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). ...
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 for revised estimates.
... Amgen (USA) (Jeong et al., 2005) Fynomers N/A (Qiu et al., 2007) *The italic words show ''indications'', while the targets are shown in parentheses. ...
Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.
Monoclonal antibodies (mAbs) are a swiftly growing class of targeted therapeutics for malignancies. After their first advent, the antibody (Ab) engineering trail has shown an evolutionary trajectory – from the rodent-derived Abs to the chimeric, humanized and fully human Abs with higher efficacy and lower/no immunotoxicity. Despite possessing great clinical potentials, several reports have highlighted that monospecific mAbs, even with high-affinity, often fail to induce sufficient immunologic responses. The full activation of the immune system demands cooperative interactions of immunotherapies with target antigen (Ag) towards functional avidity. Although the monospecific mAbs show affinity to a target Ag, they often fail to render sufficient avidity necessary for the activation of intracellular signaling mechanisms and the provocation of the immune system. Thus, various Ab/non-Ab scaffolds with much greater therapeutic impacts have been engineered based on the adjustment of their affinity and avidity balance. Novel multivalent Ab scaffolds (e.g., MDX-447, MT110, CD20Bi, TF2, and FBTA05) and mimetic Abs (e.g., Adnectin, DARPins, Ecallantide) offer improved pharmacokinetic and pharmacodynamic properties. Here, we discuss the avidity and multivalency and provide comprehensive insights into advanced Ab scaffolds used for immunotargeting and therapy of cancer.
Safety and efficacy constitute the major criteria governing regulatory approval of any new drug. The best method to maximize safety and efficacy is to deliver a proven therapeutic agent with a targeting ligand that exhibits little affinity for healthy cells but high affinity for pathologic cells. The probability of regulatory approval can conceivably be further enhanced by exploiting the same targeting ligand, conjugated to an imaging agent, to select patients whose diseased tissues display sufficient targeted receptors for therapeutic efficacy. The focus of this Review is to summarize criteria that must be met during design of ligand-targeted drugs (LTDs) to achieve the required therapeutic potency with minimal toxicity. Because most LTDs are composed of a targeting ligand (e.g., organic molecule, aptamer, protein scaffold, or antibody), spacer, cleavable linker, and therapeutic warhead, criteria for successful design of each component will be described. Moreover, because obstacles to successful drug design can differ among human pathologies, limitations to drug delivery imposed by the unique characteristics of different diseases will be considered. With the explosion of genomic and transcriptomic data providing an ever-expanding selection of disease-specific targets, and with tools for high-throughput chemistry offering an escalating diversity of warheads, opportunities for innovating safe and effective LTDs has never been greater.
The number of disease-associated protein targets has significantly increased over the past decade due to advances in molecular and cellular biology technologies, human genetic mapping efforts and information gathered from the human genome project. The identification of gene products that appear to be involved in supporting the underlying cause of disease has offered the biopharmaceutical industry an opportunity to develop compounds that can specifically target these molecules to improve therapeutic responses and lower the risk of unwanted side effects that are commonly seen in traditional small chemical-based medicines. An overview of targeted drug therapies is presented in this review. We include a review of the various classes of targeted therapeutic agents, the types of disease-associated molecules being targeted by these agents and the challenges currently being encountered for the successful development of these various platforms for the treatment of disease. An understanding of the current targeted therapy landscape, the discovery and selection of disease-specific gene products that are being targeted, and an overview of targeted therapies in preclinical and clinical studies. A description of the various targeted therapeutic platforms, target selection criteria and examples of each are discussed in order to provide the reader with the current status of the field and emerging areas of targeted therapy discovery and development. Novel medications are in demand for the treatment of serious medical conditions including cancer, autoimmune, infectious and metabolic diseases. Targeted therapies offer a way to develop very specific treatments for serious medical conditions while concomitantly resulting in little to no off-target toxicity. Targeted therapies provide an opportunity to develop personalized medicines with superior treatment modalities for the patient and a better quality of life.
A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.
Full-text available
Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of α2-macroglobulin (α2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity α2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for α2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nm) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.
Full-text available
We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.
Full-text available
Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final K(D) of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning.
Full-text available
During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.
Full-text available
Not all adaptive immune systems use the immunoglobulin fold as the basis for specific recognition molecules: sea lampreys, for example, have evolved an adaptive immune system that is based on leucine-rich repeat proteins. Additionally, many other proteins, not necessarily involved in adaptive immunity, mediate specific high-affinity interactions. Such alternatives to immunoglobulins represent attractive starting points for the design of novel binding molecules for research and clinical applications. Indeed, through progress and increased experience in library design and selection technologies, gained not least from working with synthetic antibody libraries, researchers have now exploited many of these novel scaffolds as tailor-made affinity reagents. Significant progress has been made not only in the basic science of generating specific binding molecules, but also in applications of the selected binders in laboratory procedures, proteomics, diagnostics and therapy. Challenges ahead include identifying applications where these novel proteins can not only be an alternative, but can enable approaches so far deemed technically impossible, and delineate those therapeutic applications commensurate with the molecular properties of the respective proteins.
Full-text available
We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.
The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.
We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.
The current generation of antibodies has done more than make a few companies rich. It has laid the groundwork for ambitious companies to move to maturity.
  • 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).