Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.

Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations.

The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al. (2009) Proc Natl Acad Sci USA 105:14808-14813] with ultradeep 454 sequencing, we determined the amino acid preference in every position and identified several positions in the natural sequence that restrict functional expression. A strong accumulation of shifts, i.e., a residue preference different from wild type, is detected for helix 1, suggesting a key role in receptor biosynthesis. Furthermore, under selective pressure we observe a shift of the most conserved residues of the N-terminal helices. This unique data set allows us to compare the in vitro evolution of a GPCR to the natural evolution of the GPCR family and to observe how selective pressure shapes the sequence space covered by functional molecules. Under the applied selective pressure, several positions shift away from the wild-type sequence, and these improve the biophysical properties. We discuss possible structural reasons for conserved and shifted residues.

T-cell receptor gene transfer exclusively to human CD8(+) cells enhances tumor cell killing.

Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8(+) cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8(+) cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8(+) T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and basic research requiring CD8(+) cell-specific gene delivery.

Complexes of the neurotensin receptor 1 with small-molecule ligands reveal structural determinants of full, partial, and inverse agonism.

Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.

Engineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action.

The receptor tyrosine kinase HER2 acts as oncogenic driver in numerous cancers. Usually, the gene is amplified, resulting in receptor overexpression, massively increased signaling and unchecked proliferation. However, tumors become frequently addicted to oncogenes and hence are druggable by targeted interventions. Here, we design an anti-HER2 biparatopic and tetravalent IgG fusion with a multimodal mechanism of action. The molecule first induces HER2 clustering into inactive complexes, evidenced by reduced mobility of surface HER2. However, in contrast to our earlier binders based on DARPins, clusters of HER2 are thereafter robustly internalized and quantitatively degraded. This multimodal mechanism of action is found only in few of the tetravalent constructs investigated, which must target specific epitopes on HER2 in a defined geometric arrangement. The inhibitory effect of our antibody as single agent surpasses the combination of trastuzumab and pertuzumab as well as its parental mAbs in vitro and it is effective in a xenograft model.

Rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts.

Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.

Structural Basis for the Selective Inhibition of c-Jun N-Terminal Kinase 1 Determined by Rigid DARPin-DARPin Fusions.

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control.

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells.

A number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library.

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

Rigidly connected multispecific artificial binders with adjustable geometries.

Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.

Rigidity of the extracellular part of HER2: Evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions.

The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.

Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability.

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.

DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 ß-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.

Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy.

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition.

Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

CD19-specific triplebody SPM-1 engages NK and γδ T cells for rapid and efficient lysis of malignant B-lymphoid cells.

Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells.SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors.These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies.

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.

Biophysical properties of human antibody variable domains.

There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.

Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen.

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.