08:55 Comments by Session Chairperson
Joost A. Kolkman, Ph.D., Associate Director, Molecular Biology, Ablynx NV

09:00 Phylomers: Synthetic Peptides Derived from Protein Fragments Suitable for Intracellular as Well as Extracellular Targets
Paul Watt, Ph.D., Vice President, Drug Discovery, Phylogica Ltd.
Antibodies are powerful therapeutic agents which can exhibit high specificity, low toxicity and block targets which are difficult to block by conventional means. However, due to their large size and the presence of disulphide bonds, they are not ideally suited to blocking intracellular targets. Phylomers are a new class of peptide derived from biodiverse natural protein fragments. These peptides can have high affinities and high specificity for targets, even before affinity maturation or optimisation. Phylomer peptides can also exhibit superior functional hit-rates, when compared to randomly derived peptides, presumably due to an evolutionary selection for structure and stability. Synthesised Phylomers against intracellular targets have been shown to function in ex vivo and in vivo models of ischemia and wound healing of severe burns, when fused to a protein transduction domain. These new peptides may offer major advantages in the ability to access targets such as transcription factors which are challenging to block with small molecules or with larger proteins such as antibodies or protein-based scaffolds. Libraries of Phylomers also offer greater structural diversity since they represent multiple classes of protein folds.

09:30 Moving Beyond Antibodies: Anticalins®, A Novel Class of Therapeutic Proteins
Andreas Hohlbaum, Ph.D., Director. of Science and Preclinical Development, Pieris AG
Anticalins® as engineered human binding proteins derived from the lipocalin scaffold exhibit great potential for the development of novel therapeutics. Recent preclinical data from the PRS-050 program targeting VEGF will be presented to highlight how guided selection and rational protein engineering of Anticalins® can be readily achieved yielding candidates with drug development potential. Furthermore, selected case studies will demonstrate the adaptability of different product candidates regarding custom designed functionality and pharmacokinetic parameters including dual targeting.

10:00 In Vitro Selection and Characterization of DARPins and Fab Fragments for the Co-Crystallization of Membrane Proteins
Thomas Huber, MSc., Laboratory of Prof. A. Plueckthun, Department of Biochemistry, University of Zurich
The determination of 3D structures of membrane proteins is still extremely difficult. Co-crystallization with binding proteins may be an important aid to obtain crystals suitable for high resolution structures. The in vitro selection of binding proteins specific for integral membrane proteins and an efficient way to screen their potency for co-crystallization will be presented.

11:00 DARPins for Targeted Tumor Therapy and Diagnosis
Kaspar Binz, Ph.D, Executive Director Molecular Partners
Designed ankyrin repeat proteins (DARPins) are a novel class of highly stable binding proteins, which can be selected against a variety of different target proteins with high selectivity and affinity. We show data on the engineering of multimeric DARPin constructs binding specifically to several targets at the same time without losing their favorable properties. Also, we present data on the excellent tumor accumulation and fast body clearance of radiolabeled DARPins in a xenograft mouse model. Our data suggest that DARPins are ideal candidates for targeted therapy and diagnosis in vivo.

11:30 Aptamers - An Antibody Alternative
Anthony Keefe, Ph.D, Senior Principal Investigator, Aptamer Discovery, Archemix Corp
Therapeutic aptamers are discovered using a genetic selection technique and function by binding to and inhibiting extracellular protein targets. In these respects they can be considered highly analogous to therapeutic antibodies. However, because they are ultimately chemically synthesized and do not elicit an immune response they also offer some profound advantages over antibodies. Currently there are several aptamers in the clinic for oncology and cardiovascular indications. Additionally an anti-VEGF therapeutic aptamer has been approved by the FDA for macular degeneration. A broad overview of the discovery and optimization process will be given along with an update of progress in the clinic with this technology.

12:00 Computer Aided Multi-Parameter Gene Design: Impact of Synthetic DNAs on Antibody Expression in CHO Cell Lines
Marcus Graf, Ph.D, Co-Founder, Manager of Operations, Management, GENEART AG
Protein production in cells is dependent on various factors including the underlying nucleotide sequence. Gene optimization is dedicated to improving the expression properties of transgenes by codon adaptation to the individual host, increasing RNA production, stability and nuclear export. However, most gene optimization strategies depend on codon usage adaptation only, whereas RNA optimization relies on optimization of many different parameters such as removal of RNA secondary structures, adjustment of CG-values, avoidance of splice sites and elimination of instability elements. With the help of a multi parameter optimizing algorithm, degeneration of the genetic code provides a powerful tool to identify, analyze as well as utilize parameters and respective motifs to increase and/or adjust expression yields or other important properties of a gene such as its safety or genetic stability. Together with Lonza, a study was conducted where heavy and light chain encoding antibody genes were optimized for expression in CHO and were synthetically constructed. Natural and optimized genes were stably transfected into CHO cells using Lonza’s GS system and expression was compared in a head to head study by testing 100 transfected cell lines of each. An increase of mean expression values by a factor of 1.4 could be observed when using optimized antibody encoding genes.

12:30 Lunch on Your Own
(Luncheon Workshop Sponsorship Available)

13:40 Comments by Session Chairperson
Joost A Kolkman, Ph.D., Associate Director, Molecular Biology, Ablynx NV

13:45 Tumor Targeting with Chimeric Heavy Chain Antibodies
Jianbing Zhang, Ph.D., Research Officer, Institute for Biological Sciences, National Research Council
IgG has been chosen as the major format of antibody drug due to its ability to induce ADCC and CDC and its long serum half life. However, its large molecular size is considered the major factor preventing it from having a better tumor penetration. We describe here the generation of chimeric heavy chain antibodies (cHCAb) consisting of llama single domain antibody as the binding domain and human Fc as the effector domains. Three such cHCAb, targeting EGFR, Clusterin and CEACAM6, were constructed and tested in vitro and in vivo. cHCAbs are approximately half the size of full IgG and yet have a complete human Fc fragment. Its ease of construction and high expression yield make it an attractive antibody format for in vivo tumor imaging and potentially tumor therapy.

14:15 Single Chain Fab (scFab) Fragment and Human Targeted RNases
Stefan Dübel, Ph.D., Director, Department of Biotechnology, Technical University of Braunschweig
We demonstrate the introduction of a polypeptide linker between Fd and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab). We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. This new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments, with particular value for high throughput proteome binder generation projects. We further show the generation and production of the first entirely human targeted RNase (huTR) directed against CD30+ lymphomas. The scFv-Fc-RNase construct was produced in HEK293T cells, yielding of up to 4 mg/L soluble protein after purification. Size exclusion chromatography revealed a homodimer with RNase activity insensitive to the RNase inhibitor RNasin. Surface plasmon resonance analysis revealed an affinity to CD30 of KD < 1 nM for both the scFv-Fc and the scFv-Fc-RNase protein. Internalization of the scFv-Fc-RNase protein by CD30+ Karpas-299 cell was demonstrated by confocal microscopy. Proliferation of CD30+ lymphoma cell line Karpas-299 was strongly inhibited by CD30 specific huTR protein (IC50 = 3,3 nM). The huTR is a promising candidate for the immunotherapy of CD30+ lymphomas, because of its expected low immunogenicity, good production yields and potent effector function upon target cell binding and internalization, and its insensitivity to RNase inhibitor. Its modular design is set to target other internalizing tumor antigens using different antibody domains.

14:45 Plant Seeds as Bioreactor for the Production of scFv-Fc Antibody Variants
Ann Depicker, Ph.D., Department of Plant Systems Biology, Flanders Institute for Biotechnology, Ghent University
The use of transgenic plants for the production of high value recombinant proteins is a promising alternative to conventional systems such as animal and insect cell cultures, yeast and bacteria. Recent advances in this field have shown that plant-based production systems offer many practical, economic and safety advantages compared with other systems. Among the many plant-based production systems that have been developed for molecular farming, seeds have the particular advantage of accumulating high levels of recombinant proteins in a relatively small volume and stable environment in which they are protected from degradation. In addition, downstream protein extraction from seeds is easier than from vegetable or leaf tissue, which will inevitably reduce production costs and benefit the consumer. In our group, best results so far were obtained with expression cassettes containing 5’ and 3’ expression signals of seed storage protein genes of Phaseolus vulgaris. The use of these regulatory sequences has boosted seed-specific heterologous expression of a model single chain Fv (scFv) in Arabidopsis thaliana seeds to accumulation levels of 36% of TSP (De Jaeger et al. 2002) . Recently, this expression cassette was used to express more complex antibody-derived fragments, i.e. scFv-Fc fusion proteins (Van Droogenbroeck et al. 2007). We show that functional scFv-Fc antibodies, expressed with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels in Arabidopsis thaliana seeds (scFv-Fc’s up to14% of TSP or 2% of seed weight). Furthermore, results will be presented showing that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. These results illustrate the value of a seed-specific expression platform for the safe and inexpensive production of antibodies in the scFv-Fc format, which are easier to produce and have the potential to replace IgGs in specific detection, purification, diagnostic, and even therapeutic applications.

15:45 Antibodies as Protease Inhibitors
Laetitia Devy, Ph.D., Principal Scientist, Cell Biology, Dyax sa
Proteases represent 2-3% of the human genome and are important regulators of inflammation and tumor growth. They have been extensively pursued with small molecule drugs but with varying success. Relatively few protease inhibitors have been approved. Antibodies provide the potential for a new generation of protease inhibitors with high levels of potency and selectivity. Combining our human antibody phage display library with automated selection and screening strategies, we have isolated inhibitors of a range of metallo- and serine proteases. We will illustrate this success using DX-2300 and DX-2400. DX-2300 potently inhibits tissue kallikrein 1 (Ki=39 pM) and has demonstrated activity in preclinical models of airway inflammation. DX-2300 has been shown to block kinin generation by tissue kallikrein in vivo and therefore, may be efficacious in various inflammatory diseases. DX-2400 selectively inhibits MMP-14 (Ki=0.9nM), a membrane bound metalloproteinase and has been shown to inhibit tumor progression in different tumor models (MDA-MB-231, MDA-MB-435, BT-474 and PC3). DX-2400 also significantly decreases the incidence of metastases. These antibodies represent innovative approaches for the inhibition of key protease regulators of inflammation and cancer.

16:15 Adecatumumab: A Human Antibody for Tumor Therapy
Tobias Raum, Ph.D., Director, Immunotherapy, Micromet AG
Adecatumumab (MT201) is a fully human IgG1 antibody against the pancarcinoma antigen EpCAM (CD326). The antibody was derived via phage display - guided selection from an expressed human antibody repertoire. The antibody proved to be efficacious in mouse tumour models and was further explored in clinical Phase I and II studies in prostate and breast cancer patients. Clinical activity of adectumumab will be presented with a focus on its very low immunogenicity.