Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.     

Automated screening procedure for high-throughput generation of antibody fragments

In the emerging field of proteomics, there is an urgent need for catcher molecules such as antibodies for detecting the proteome or parts of the proteome in a microarray format. A suitable source for providing a large diversity of binders is obtained by combinatorial libraries, such as phage display libraries of single chain antibody fragments (scFv) or Fab fragments. To find novel binders from the n-CoDeR libraries with a high throughput, we have automated the screening process with robotics. The automated system is configured to screen tens of thousands of clones per day to target antigens in various formats, including peptides and soluble proteins, as well as cell-bound targets; thus, it is well designed to meet demands from the proteomics area.     

Protein microarrays for antibody profiling: specificity and affinity determination on a chip

Protein microarray technology facilitates the detection and quantification of hundreds of binding reactions in one reaction from a minute amount of sample. Proof-of-concept studies have shown that the set-up of sensitive assay systems based on protein arrays is possible, however, the lack of specific capture reagents limits their use. Therefore, the generation and characterisation of capture molecules is one of the key topics for the development of protein array based systems. Recombinant antibody technologies, such as HuCAL (human combinatorial antibody library; MorphoSys, Munich, Germany), allow the fast generation of highly specific binders to nearly any given target molecule. Although antibody libraries comprise billions of members, it is not the selection process, but the detailed characterisation of the pre-selected monoclonal antibodies that presents the bottleneck for the production of high numbers of specific binders. In order to obtain detailed information on the properties of such antibodies, a microarray-based method has been developed. We show that it is possible to define the specificity of recombinant Fab fragments by protein and peptide microarrays and that antibodies can be classified by binding patterns. Since the assay uses a miniaturised system for the detection of antibody-antigen interactions, the observed binding occurs under ambient analyte conditions as defined by Ekins (J. Pharm. Biomed. Anal. 1989, 7, 155-168). This allows the determination of a relative affinity value for each binding event, and a ranking according to affinity is possible. The new microarray based approach has an extraordinary potential to speed up the screening process for the generation of recombinant antibodies with pre-defined selection criteria, since it is intrinsically a high-throughput technology.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Evaluation of an inverse molecular design algorithm in a model binding site

Computational molecular design is a useful tool in modern drug discovery. Virtual screening is an approach that docks and then scores individual members of compound libraries. In contrast to this forward approach, inverse approaches construct compounds from fragments, such that the computed affinity, or a combination of relevant properties, is optimized. We have recently developed a new inverse approach to drug design based on the dead-end elimination and A* algorithms employing a physical potential function. This approach has been applied to combinatorially constructed libraries of small-molecule ligands to design high-affinity HIV-1 protease inhibitors (Altman et al., J Am Chem Soc 2008;130:6099-6013). Here we have evaluated the new method using the well-studied W191G mutant of cytochrome c peroxidase. This mutant possesses a charged binding pocket and has been used to evaluate other design approaches. The results show that overall the new inverse approach does an excellent job of separating binders from nonbinders. For a few individual cases, scoring inaccuracies led to false positives. The majority of these involve erroneous solvation energy estimation for charged amines, anilinium ions, and phenols, which has been observed previously for a variety of scoring algorithms. Interestingly, although inverse approaches are generally expected to identify some but not all binders in a library, due to limited conformational searching, these results show excellent coverage of the known binders while still showing strong discrimination of the nonbinders.     

Selection of streptavidin binders from a DNA-encoded chemical library

DNA-encoded libraries of small organic molecules facilitate the construction of large, encoded self-assembling chemical libraries for the identification of high-affinity binders to protein targets. We have constructed a library of 477 chemical compounds, coupled to 48mer-oligonucleotides, each containing a unique six-base sequence serving as "bar-code" for the identification of the chemical moiety. The functionality of the library was confirmed by selection and amplification of both high- and low-affinity binding molecules specific to streptavidin.     

Single domain camel antibodies: current status

The antigen-binding capacity of the paired variable domains of an antibody is well established. The observation that the isolated heavy chains of anti-hapten antibodies retain some antigen-binding capacity in the absence of light chains led to attempts to obtain an even smaller antigen-binding unit in a VH format. Unfortunately, the poor solubility, the reduced affinity for the antigen and the irreproducible outcome showed that additional protein engineering would be required to successfully generate single-domain antibody fragments. By serendipity, it was discovered that this engineering is already performed continuously in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH-VL combinatorial diversity, these heavy-chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions. Methods are described to tap the VHH repertoire of an immunised dromedary or llama. These VHH libraries contain a high titre of intact antigen-specific binders that were matured in vivo. Synthetic libraries of a 'camelised' human VH, a mouse VH or a camelid VHH scaffold with a randomised CDR3 could constitute a valid alternative to immune libraries to retrieve useful single-domain antigen binders. The recombinant VHH that are selected from such libraries are well expressed, highly soluble in aqueous environments and very robust. Some in vivo matured VHH were also shown to be potent enzyme inhibitors, and the low complexity of the antigen-binding site is an asset in the design of peptide mimetics. Because of their smaller size and the above properties, the VHH clearly offer added-value over conventional antibody fragments. They are expected to open perspectives as enzyme inhibitors and intrabodies, as modular building units for multivalent or multifunctional constructs, or as immuno-adsorbents and detection units in biosensors.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library

Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 μm and acted as a CXCR4 antagonist (IC(50) = 23 μm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.     

Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million‐fold improvements in binding affinity by alternating rounds of diversification and flow cytometry‐based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads—allowing isolation of extremely weak binders from billions of non‐binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000‐fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin‐biotin that do not cross‐react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.     

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries.Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.     

Single domain camel antibodies: current status

The antigen-binding capacity of the paired variable domains of an antibody is well established. The observation that the isolated heavy chains of anti-hapten antibodies retain some antigen-binding capacity in the absence of light chains led to attempts to obtain an even smaller antigen-binding unit in a VH format. Unfortunately, the poor solubility, the reduced affinity for the antigen and the irreproducible outcome showed that additional protein engineering would be required to successfully generate single-domain antibody fragments. By serendipity, it was discovered that this engineering is already performed continuously in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH–VL combinatorial diversity, these heavy-chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions. Methods are described to tap the VHH repertoire of an immunised dromedary or llama. These VHH libraries contain a high titre of intact antigen-specific binders that were matured in vivo. Synthetic libraries of a ‘camelised’ human VH, a mouse VH or a camelid VHH scaffold with a randomised CDR3 could constitute a valid alternative to immune libraries to retrieve useful single-domain antigen binders. The recombinant VHH that are selected from such libraries are well expressed, highly soluble in aqueous environments and very robust. Some in vivo matured VHH were also shown to be potent enzyme inhibitors, and the low complexity of the antigen-binding site is an asset in the design of peptide mimetics. Because of their smaller size and the above properties, the VHH clearly offer added-value over conventional antibody fragments. They are expected to open perspectives as enzyme inhibitors and intrabodies, as modular building units for multivalent or multifunctional constructs, or as immuno-adsorbents and detection units in biosensors.     

High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization,Binding and Tumor Targeting

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with ⁶⁴Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.     

High-affinity binders selected from designed ankyrin repeat protein libraries

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.     

Encoded self-assembling chemical libraries

The isolation of molecules capable of high-affinity and specific binding to biological targets is a central problem in chemistry, biology and pharmaceutical sciences. Here we describe the use of encoded self-assembling chemical (ESAC) libraries for the facile identification of molecules that bind macromolecular targets. ESAC technology uses libraries of organic molecules linked to individual oligonucleotides that mediate the self-assembly of the library and provide a code associated with each organic molecule. After panning ESAC libraries on the biomolecular target of interest, the 'binding code' of the selected compounds can be 'decoded' by a number of experimental techniques (e.g., hybridization on oligonucleotide microarrays). The potential of this technology was demonstrated by the affinity maturation (>40-fold) of binding molecules to human serum albumin and bovine carbonic anhydrase, leading to binders with dissociation constants in the nanomolar range.     

从噬菌体文库中筛选潜在的GMCSF的拮抗剂(英文)

以粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ） 为筛选文库的靶分子, 通过高效筛选（Ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ, ＨＴＳ） 方法来筛选多种多肽噬菌体文库, 在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭＣＳＦ结合的多肽, 并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ） 实验的证实。这些多肽先导化合物经过进一步的优化, 可能成为ＧＭＣＳＦ细胞因子的拮抗剂