12:45 pm Registration

1:55 Chairperson's Remarks
Lutz Jermutus, Ph.D., Director, Oncology Discovery, Cambridge Antibody Technology Ltd.

2:00 Improved Performance of an Antibody Expressed in Glyco-Engineered Moss
Gilbert Gorr, Ph.D., CSO, greenovation Biotech GmbH
Antibodies expressed in glyco-engineered moss assembled correctly, showed the identical molecular weight and the same specificity in comparison to its parent antibody and also showed the expected glyco-optimized N-linked oligosaccharide pattern lacking core fucosylation. A more than 30-fold increased ADCC activity of such a fucose-deficient therapeutic IgG1 antibody recognizing the carbohydrate antigen Lewis Y (over-expressed on epithelial cancers) was achieved independently of the FcgRIII genotype of the patient. This improved ADCC effector function is not impaired by normal human serum up to 40%, in contrast to the ADCC activity mediated by the parental antibody. Moreover, variations of the very C-terminal part of the heavy chain normally detected on recombinantly produced antibodies are not present on moss-derived antibodies resulting not only in highly reduced heterogeneity but also represents a peptide structure which is more similar to the situation found on human IgG molecules. Taken together, the use of glyco-engineered moss as host for antibody production is highly advantageous when compared to other expression technologies.

2:30 The Role of Hypervariable Loops for Antigen Recognition - Insights From a Comparison Between Antibodies, Alternative Scaffolds, and Engineered Lipocalins
Arne Skerra, Ph.D., Professor, Department of Biological Chemistry, Technical University of Munich
The role of structurally hypervariable and, often, conformationally flexible loops (CDRs) is well known for the generation of antibodies by the immune system. In the last years the concept of using alternative protein scaffolds in order to engineer new binding proteins has emerged, whereby also rigid secondary structural elements were exploited to introduce side chain diversity. In this context the contribution of peptide loop flexibility to properties such as target affinity and specificity has become less clear. Novel insight is provided by the crystal structure of an engineered lipocalin, a so-called "anticalin", that binds the T-cell coreceptor CTLA-4 with pM affinity in an antagonistic manner and thus provides a promising drug candidate for immune stimulation. Comparison between the structures of the anticalin both in the free state and in complex with the protein target reveals an induced fit in its loop region, which allows an interesting comparison with the binding sites of antibodies.

3:00 Antibody Engineering for Improved Specificity
Terry Nakagawa, Ph.D., Senior Scientist, Antibody Research, Abbott Laboratories
CDR (complementarity-determining region) mutagenesis and yeast surface display technology were applied to the engineering of an anti-cyclosporine antibody with the objective of improving recognition of cyclosporine (CsA) parent drug in the presence of one of its major metabolites, AM1(M17). The anti-cyclosporine mouse hybridoma 29-56-14 was the model system from which a single chain construct (scFv) comprised of the immunoglobulin heavy and light chains was expressed on the surface of yeast cells. scFv yeast libraries encoding mutations at multiple antigen binding sites utilizing a CDR scanning approach were screened by flow cytometry for improved binding to biotinylated CsA in the presence of 10~100 fold molar excess of AM1(M17) metabolite. Four distinct yeast clones encoding mutations in CDRs H2, H3, L2, and L3 were isolated and exhibited a greater than 80 fold increase of IC50 for AM1(M17) compared to wildtype yeast control. Mutants were then expressed as mouse IgG2a Abs and tested for affinity for AM1(17) metabolite and CsA. Mutant Abs exhibited ~10 fold lower affinity for AM1(M17) compared to wildtype Ab (~140nM vs. 14nM KD), with minimal change to the affinity for CsA.

Friday, May 18 8:30 am Conference Registration and Morning Coffee

8:50 Chairperson's Remarks
Hennie Hoogenboom, Ph.D., Chief Scientific Officer, Ablynx NV

9:00 Monoclonal Antibody Classification Based on Epitope-Binding Using Differential Antigen Disruption
Ergang Shi, Ph.D., Senior Scientist, Protein Science, Regeneron Pharmaceuticals, Inc.
The ability to process a large number of mAbs to an antigen based on their epitope diversity is essential for systematically screening desirable clones. This demand is challenging the traditional antigen screening assays as well as the traditional epitope mapping methodologies on speed, efficiency and throughput. To overcome these challenges, we’ve developed a novel antigen-structure-based screening approach, named as differential antigen disruption (DAD), to profile a large number of mAbs according to their epitope bindings. The presentation will focus on the development and applications of the DAD technology for VelocImmune® human therapeutic antibody programs in Regeneron.

9:30 Use of CHO Pools for Rapid Generation of Material for in Vivo Testing
Ray Field, Ph.D., Cambridge Antibody Technologies

10:00 Maintaining Product Quality While Developing Production Cell Lines Expressing Peptide-Antibody Fusion Proteins in Mammalian Cells
Haimanti Dorai, Ph.D., Research Fellow, Pharmaceutical Development, Centocor R&D Inc.
N-terminal proteolytic degradation and product aggregation are two negative characteristics that challenge expression of fusion proteins in mammalian cells. N-terminal proteolytic degradation results in an inactive product whereas aggregation of the product might result in an increase in its immunogenicity and make formulating the product more difficult. In an attempt to develop production cell lines expressing antibody fusion proteins in mammalian systems, we examined a number of parameters that effect product quality while keeping in mind issues such as productivity and ease of manufacture. Results from our analyses suggest that the parameters that had a significant influence on product quality include: 1) primary structure of the fusion protein that incorporates engineered glycosylation sites at the peptide-antibody junction region and 2) choice of mammalian expression systems that have a dramatic influence on product quality as well as productivity.

11:00 Rapid High Yield Production System for Fully Human and Glyco-Optimised Antibodies and Biopharmaceuticals
Steffen Goletz, Ph.D., Chief Executive Officer, GLYCOTROPE GmbHThe Glyco
Express technology for new generations of biopharmaceuticals is based on a toolbox of glycoengineered human cell lines. The toolbox with in-process control and adjustment of the glycosylation enables the selection and generation of proteins with optimised fully human glycosylation addressing various key aspects of glycosylation like sialylation, fucosylation, galactosylation and branching. New generations of antibodies and other biopharmaceuticals with large increases in bioactivity, improved pharmaceutical properties and new patent protection are generated. GlycoExpress is a biotechnologically superior rapid high yield expression system, comprising own amplification, secretion signal peptides and vector systems, showing a series of biotechnological advantages over CHO production systems including a very high expression yield, doubling time, speed, robustness, stability and handling efficiencies. The talk will also focus on a own cancer antibody glycoimproved in respect to antigen recognition, antibody activity and bioavailability.

11:30 Improving Antibody Effector Function through Fc Optimization
Ezio Bonvini, M.D., Vice President, Research, and Jeffrey Stavenhagen, Ph.D., Director, Molecular Immunology, MacroGenics, Inc.
Monoclonal antibodies have become a significant part of the armamentarium for the treatment of malignancies and autoimmune diseases. In many cases, the response to monoclonal antibodies is shaped by the engagement of their Fc region with Fc receptors on immune effector cells. The activation of these effector cells is in turn controlled by the balance between activating and inhibitory Fc receptors. By a combination of yeast surface display, high throughput screening, and rational design, we have engineered the IgG1 Fc region to impart monoclonal antibodies with specific binding and effector characteristics, including enhanced in vitro antibody-dependent cell cytotoxicity and in vivo tumor elimination in xenograft mouse tumor models. Fc optimization is a robust means of enhancing therapeutic monoclonal antibodies whose mechanism of action depends, at least in part, on the engagement of effector cells.

12:00 pm Luncheon Workshop (Sponsorship Available) or Lunch on Your Own

1:30 High Concentration Monoclonal Antibody Suspensions for Subcutaneous Injection 
Larry R. Brown, Sc.D., Chief Technology Officer, Epic Therapeutics, Inc., A wholly owned subsidiary of Baxter Healthcare
Monoclonal antibodies (mAbs) are being targeted against specific disease related antigens to treat cancer, rheumatoid arthritis etc. An important challenge for the delivery of mAbs is that they usually require intravenous injection of several hundred-milligram doses. A critical medical need in the formulation of this drug class is finding an alternative to the IV route of administration. We have incorporated monoclonal and polyclonal antibodies into our PROMAXX microsphere formulation technology. Microspheres were formulated by mixing monoclonal anti-factor VIII and anti-CD34 antibodies (Baxter Biosciences, Hayward, CA), with water-soluble polymer solutions, such as Poloxamer. The solution was cooled and a turbid suspension formed. The polymer was removed, yielding an essentially excipient free mAb microsphere visualized by scanning electron microscopy. The microsphere formation process preserved antibody integrity, as demonstrated by fluorescence spectroscopy and by size exclusion chromatography. Light scattering measurements of particle size showed monodispersed mAb microspheres with a diameter of 1.92 microns. X-ray powder diffraction showed that the protein in the microsphere was amorphous. Microsphere suspensions at concentrations of 300 mg/mL, were shown to be injectable through 26 gauge needle sizes in vitro and in vivo. Pharmacokinetic studies of mAb microspheres and solutions delivered by the subcutaneous route will be presented.

2:00 Expression of Glyco-Optimized Monoclonal Antibodies in the LEX System
Dr. Lynn Dickey, Vice President, Research, Biolex Therapeutics
Monoclonal antibodies (mAbs) are one of the fastest growing classes of protein therapeutics with N-glycosylation playing a significant role in mAb function. The structure and extent of heterogeneity of N-glycans attached to recombinant mAbs is one of the distinguishing features in selecting a protein expression platform. Here we report the expression of human glyco-optimized mAbs in the small aquatic plant Lemna. Lemna provides the basis of an attractive expression platform for the cost effective, manufacture of therapeutic proteins free of zoonotic pathogens. MAb glyco-optimization was accomplished by co-expression with an interference RNA (RNAi) construct targeting the endogenous expression of alpha-1,3-fucosyltransferase (Fuct) and beta-1,2-xylosyltransferase (Xylt) genes. The resultant mAb contained a single major glycoform without any detectable levels of plant-specific N-glycans. In addition to the glycoform homogeneity, glyco-optimized mAbs were also shown to have significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity through increased effector cell Fc-receptor binding affinity when compared to non glyco-optimized CHO or Lemna expressed mAbs.

2:30 Expression of Antibodies in Mammalian Cells – Areas to Explore for Improvement and Alternative Approaches
Yune Kunes, Ph.D., Senior Scientist II, Project Leader, Biologics, Abbott Bioresearch Center
As more and more antibody therapeutics become approved for clinical applications we have seen steady improvement in the methods for manufacturing these therapeutic proteins over the last 20 years. However, more efficient and reliable production methods are desired. Some of the desired features in new technologies include higher level of antibody secretion into the cultured media, stability of manufacturing cell lines, and reduced time in the cell line generation. In this presentation some of Abbott Bioresearch Center’s current efforts in the field, including work in CHO and HEK293, will be discussed. Data will be presented that describe vector design, and transcription, translation, and secretion mechanisms, antibody characterization in mammalian cells. Considerations in antibody quality, production yield, etc, will be discussed.

3:00 Monoclonal Antibodies Derived from Chimpanzees: A New Approach to Immunoprophylaxis and Immunotherapy
Zhaochun Chen, Staff Scientist, NIAID/NIH
Since chimpanzee globulins are virtually identical to human globulins, they can be used without further manipulation. We have used phage display libraries of chimpanzee bone marrow to provide a repertoire of antibody genes of interest and have generated neutralizing MAbs to a variety of pathogens, including hepatitis viruses, flaviviruses, vaccinia/smallpox viruses and the toxins of Bacillus anthracis and Clostridium botulinum. Typically, the MAbs have had affinities in the subnanomolar to picomolar range and they are promising candidates for immunoprophylaxis and immunotherapy of pathogens with emerging disease and biodefense interest.

3:30 One-Step Clonepixfl Applications for Accelerating the Discovery of Therapeutic Antibodies and Their Production in Mammalian Cells
Julian F. Burke, Ph.D., Chief Scientific Officer, Genetix Limited
In a single step, this new technology screens thousands of cell clones for specific monoclonal antibody expression and quantitatively collects only the best secretors. The technology offers a powerful alternative to current procedures such as limiting dilution and cell sorting. Each analysis results in a small number of high value, high viability (up to greater than 95%) clonal populations that can be expanded rapidly for further assay or processing. A typical timescale of 60 days for generating new candidate clones can be reduced to less than 7 days. The technology is compatible with a wide range of host cells including hybridoma fusions, transfected myelomas and serum-free CHO-S, adherent CHO and HEK, as well as stem cell lines. Co-Authors: Sky Jiang, James Colehan, Christopher J. Mann.