Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

新型四价抗体的优势与基因工程制备方法

近年来基因工程抗体技术发展迅速,将单价小分子抗体改建为双价或多价,成为抗体工程中活跃的领域之一.四价抗体相比于单价或双价抗体在增加抗体亲和力、降低解离率、延长半寿期等药代动力学,受体交联效应等方面具有优势.因此,具有较好的研究和临床应用前景.制备四价抗体的方法主要有化学偶联法和基因工程法,后者主要包括：①通过改变单链抗体VH和VL之间的连接肽的长短对单链抗体之间的聚合作用产生影响;②利用连接肽将scFv进行连接而形成多价抗体分子;③通过寡聚化的结构域,如亮氨酸拉链结构、链亲和素、人p53四聚化功能域、子宫球蛋白等介导多价抗体的形成.     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Quantitative specificity‐based display library screening identifies determinants of antibody‐epitope binding specificity

Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three‐fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

X‐ray crystal structure localizes the mechanism of inhibition of an IL‐36R antagonist monoclonal antibody to interaction with Ig1 and Ig2 extra cellular domains

Cellular signaling via binding of the cytokines IL‐36α, β, and γ along with binding of the accessory protein IL‐36RAcP, to their cognate receptor IL‐36R is believed to play a major role in epithelial and immune cell‐mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL‐36R and a Fab derived from a high affinity anti‐IL‐36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL‐36R, reveals similarities with other structurally characterized IL‐1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL‐36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.     

Using affinity chromatography to engineer and characterize pH‐dependent protein switches

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione‐S‐transferase to convert this protein into a pH‐dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH‐dependent GSH‐binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector‐specific allostery into a protein structure.     

pH‐selective mutagenesis of protein–protein interfaces: In silico design of therapeutic antibodies with prolonged half‐life

Understanding the effects of mutation on pH‐dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein–protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH‐dependent energy profiles demonstrate excellent agreement with experimentally measured pH‐dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH‐dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH‐selective binding. The method can be used to select mutations that change the pH‐dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH‐dependent binding behavior of IgG to FcRn with the aim of improving the half‐life of therapeutic antibodies in the target organism. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low‐cost and scalable antibody capturing in solutions. Immunoglobulin‐binding Protein A is widely used in affinity‐based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two‐phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35‐fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB‐induced protein body formation. We also demonstrated production of HFBI–Protein A fusion protein in tobacco BY‐2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody‐binding capacity of the Protein A block was similar to the nonfused Protein A. The best‐performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two‐phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.     

The length of the linker between the epidermal growth factor‐like domains in factor VIIa is critical for a productive interaction with tissue factor

Formation of the factor VIIa (FVIIa)‐tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure‐based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)‐like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ‐carboxyglutamic acid (Gla)‐containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly‐Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF‐mediated stabilization of the active conformation of FVIIa and abrogated TF‐induced activity enhancement. In addition, FVIIa variants with short linkers associated 80‐fold slower with soluble TF (sTF) as compared with wild‐type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca²⁺‐free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)‐FVIIa reaching the level of Gla‐domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo‐FVIIa activity.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.     

IgG‐single chain Fv fusion protein therapeutic for alzheimer's disease: Expression in CHO cells and pharmacokinetics and brain delivery in the rhesus monkey

Monoclonal antibodies (MAb) directed against the Abeta amyloid peptide of Alzheimer's disease (AD) are potential new therapies for AD, since these antibodies disaggregate brain amyloid plaque. However, the MAb is not transported across the blood–brain barrier (BBB). To enable BBB transport, a single chain Fv (ScFv) antibody against the Abeta peptide of AD was re‐engineered as a fusion protein with the MAb against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the ScFv therapeutic antibody across the BBB. Chinese hamster ovary (CHO) cells were stably transfected with a tandem vector encoding the heavy and light chains of the HIRMAb–ScFv fusion protein. A high secreting line was isolated following methotrexate amplification and dilutional cloning. The HIRMAb–ScFv fusion protein in conditioned serum‐free medium was purified by protein A affinity chromatography. The fusion protein was stable as a liquid formulation, and retained high‐affinity binding of both the HIR and the Abeta amyloid peptide. The HIRMAb–ScFv fusion protein was radiolabeled with the ¹²⁵I‐Bolton–Hunter reagent, followed by measurement of the pharmacokinetics of plasma clearance and brain uptake in the adult Rhesus monkey. The HIRMAb–ScFv fusion protein was rapidly cleared from plasma and was transported across the primate BBB in vivo. In conclusion, the HIRMAb–ScFv fusion protein is a new class of antibody‐based therapeutic for AD that has been specifically engineered to cross the human BBB. Biotechnol. Bioeng. 2010; 105: 627–635. © 2009 Wiley Periodicals, Inc.     

Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes IIMtl in the presence and absence of a connecting linker

The mannitol transporter enzyme II^Mtl of the bacterial phosphotransferase system is a multi‐domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II^Mtl, which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ～28‐fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.     

Engineering high affinity superantigens by phage display

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa light chains. PpL from P.magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1-B5) of PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.     

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti‐c‐Met scFv, which was a cytoplasmic non‐foldable protein, was redesigned as the model system. This study confirmed that the engineered anti‐c‐Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c‐Met antigen. The structures of the wild‐type anti‐c‐Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.     

Mechanical properties of BiP protein determined by nano‐rheology

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N‐terminal Nucleotide‐Binding Domain (NBD), and the C‐terminal Substrate‐Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano‐rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides‐ ATP, ADP, and AMP‐PNP‐, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K_D) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.