Annemarie Honegger

University of Zurich | UZH · Biochemisches Institut

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 bipar...

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 ago...

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 i...

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...

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 microscop...

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 e...

Crystals of sufficient quality in terms of size and intrinsic order are still indispensable to determine the structures of biological macromolecules by X-ray diffraction techniques. Despite large progress in instrumentation and screening throughput, many proteins still resist yielding diffraction-quality crystals. To overcome this bottleneck, strat...

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms we need tools which 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 bin...

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 extrace...

Slides for a Course in Macromolecular Visualization (1/3) The course is part of BCH 306: ”Biochemical and Biophysical Methods”, targeted at biochemistry students at the end of the 3rd year, just before they enter the research labs to work on their master’s thesis projects. I therefore do not have to teach the underlying biochemistry at the same tim...

Slides for a Course in Macromolecular Visualization (2/3) The course is part of BCH 306: ”Biochemical and Biophysical Methods”, targeted at biochemistry students at the end of the 3rd year, just before they enter the research labs to work on their master’s thesis projects. I therefore do not have to teach the underlying biochemistry at the same tim...

Slides for a Course in Macromolecular Visualization (3/3) The course is part of BCH 306: ”Biochemical and Biophysical Methods”, targeted at biochemistry students at the end of the 3rd year, just before they enter the research labs to work on their master’s thesis projects. I therefore do not have to teach the underlying biochemistry at the same tim...

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...

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 ?? T cells. SPM-1 is an optimized version of triplebody ds(19-16-1...

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...

Induction of Apoptosis by Bi-paratopic DARPins in Comparison to Trastuzumab Treatment. BT474 cells were seeded 24 h before treatment in RPMI1640 containing 10 % FBS in a 12- well dish. Annexin V-Alexa488 (Life Technologies) was added to give a 1:50 final dilution and propidium iodide (2 g/l) was added to give a 1:2000 final dilution. Afterwards, ce...

Supplementary Figures 1-6, Supplementary Methods and Supplementary References

Induction of Apoptosis by Bi-paratopic DARPins in Comparison to Trastuzumab Treatment. (S1-S2) BT474 cells were seeded 24 h before treatment in RPMI1640 containing 10 % FBS in a 12- well dish. Annexin V-Alexa488 (Life Technologies) was added to give a 1:50 final dilution and propidium iodide (2 g/l) was added to give a 1:2000 final dilution. Afterw...

An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into...

Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated virtually against all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compo...

Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins...

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 hig...

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 tran...

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...

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...

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...

Although nowadays many different libraries for the selection of antibodies are available, hybridoma cells and immunized animals are still an important source for the production and selection of specific binders. Therefore, this protocol describes the isolation of the coding mRNA, PCR amplification of VL and VH domains and the final assembly of thes...

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutio...

Antibody variable domains differ considerably in stability. Single-chain Fv (scFv) fragments derived from natural repertoires frequently lack the high stability needed for therapeutic application, necessitating reengineering not only to humanize their sequence, but also to improve their biophysical properties. The human V(H)3 domain has been identi...

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody reperto...

Antibody variable domains vary widely in their intrinsic thermodynamic stability. Despite the mutual stabilization of the domains in the scFv fragment, most scFv derived from monoclonal antibodies without further engineering show poor to moderate stability. The situation gets more complex for Fab fragments and full-sized antibodies: while the disul...

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...

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,...

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient fold...

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). Hu...

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intra- bodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity...

In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of...

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 se...

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or...

complexed with the hapten at 2.6 A resolution. The crystal structure showed that only van der Waals interactions are involved in binding. The somatic Ala H93 Val mutation in M02/05/01 lls up an empty cavity in the binding pocket. This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its der...

Introduction When analyzing an individual protein structure, sequential numbering of the amino acid residues within each chain, combined with a unique chain label for each chain in multi-chain structures, is the most convenient method to Abbreviations used: CDR, complementaritydetermining region; FR, framework region of immunoglobulin variable doma...

this paper. Residue numbers with a white background correspond to CDR regions

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an...

Introduction Disulfide bonds are conserved across almost the whole immunoglobulin superfamily. In antibody variable domains (VH and VL ), they connect the two b-sheets of the immunoglobulin domain, from strand b to strand f. The contributing cysteine residues L23 and L88 in VL , and H22 and H92 in VH (numbering according to Kabat et al., 1991) are...

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 hydrop...

A common residue numbering scheme for all immunoglobulin variable domains (immunoglobulin light chain lambda (V(lambda)) and kappa (V(kappa)) variable domains, heavy chain variable domains (V(H)) and T-cell receptor alpha (V(alpha)), beta (V(beta)), gamma (V(gamma)) and delta (V(delta)) variable domains) has been devised. Based on the spatial align...

Immunoglobulin V(H) domain frameworks can be grouped into four distinct types, depending on the main-chain conformation of framework 1. Based on the analysis of over 200 X-ray structures representing more than 100 non-redundant V(H) domain sequences, we have come to the conclusion that the marked structural variability of the V(H) framework 1 regio...

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyz...

The N-terminal segment (FR-H1) of the heavy chain (VH) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to imp...

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or the...

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loadin...

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Esch...

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of β-...

The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed t...

Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance. Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated. The antibodies M02/01/01 and M02/05/01 are high...

Introduction Recombinant antibody fragments produced in microorganisms have a great potential as specific targeting reagents and may replace traditional animal immunization. This is a technology based entirely on microbial biotechnology, which is amenable to high throughput generation of reagents. There has been growing interest especially in the s...

The β-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer β-sheet (away from the VH-VL interface) and the second part to the inner β-sheet. This kink differs in length and sequence between the Vκ, Vλ and VH domains and y...

Introduction The disulde bond signicantly contributes to the stability of antibody domains. Most antibodies will not tolerate its loss and will react to its removal with a dramatic loss of free energy of folding. However, there are a number of interesting applications for antibody fragments in environments that are not compatible with efcient disul...

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln),...

We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and...

We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding cons...

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Flox) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Flred)] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These ‘flavo...

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. I...

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplificatio...

While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequ...

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-displa...

The folding kinetics of the variable domains of the phosphorylcholine-binding antibody McPC603, combined into a scFv fragment [V H-(Gly 4 Ser) 3-V L ], were investigated by the use of fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). All three methods gave evidence for the occurrence of a major kinetic interme...