分子重組抗體

5月 1日 (四)

8:00 am - 5:00 pm Registration Open 

SCREENING AND ENGINEERING FOR BETTER CHARACTERISTICS

8:30 am Chairperson's Remarks

8:35 Panning a Human VH Phagemid-Based Phage Display Library for Non-Aggregating Binders
Jamshid Tanha,Ph.D., Institute for Biological Sciences, National Research Council-Canada
The incentive for using human VHs as opposed to their more easily obtainable natural counterparts, VHHs from camelid heavy chain antibodies, is their expected lower im-munogenicity in immunotherapy. Libraries constructed by CDR randomization using non-aggregating human VHs as scaffolds can serve as a source of VHs that bind target antigens. However, a significant portion of VHs in these libraries aggregate because of CDR randomization; this tends to obscure the isolation of soluble binders. Here we present the results of a couple of different selection approaches we have undertaken in order to isolate non-aggregating binders from a human VH phage display library.

9:05 HT Selection and Screening of scFv Antibodies
Peter Pavlik, Ph.D., Project Leader, Bioscience Division, Los Alamos National Laboratory
We have developed a HTS of single-chain antibodies using multiplexed flow cytometry (J. Ayriss et.al., (2007) Journal of Proteome Research, Mar;6(3):1072-82) By using multiplexed flow cytometry, relative expression levels, the identification of non-specific binding and the discrimination of fine specificities can all be determined simultaneously to generate a complete functional profile for each clone. The quality and quantity of data, combined with significant reductions in analysis time and antigen consumption pro-vide notable advantages over standard ELISA methods, and yield much information in the primary screen that is usually only obtained in later screens. By combining high-throughput screening capabilities with multiplex technology we have redefined the parameters for the initial identification of affinity reagents recovered from combinatorial libraries and removed a significant bottleneck in the generation of affinity reagents.

9:35 Metabolic Biotinylation of Recombinant Antibody by Biotin Ligase Retained in the Endoplasmic Reticulum
Bhaswati Barat, Ph.D., Crump Institute for Molecular Imaging, Department of Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA
We have developed an efficient system to metabolically biotinylate secretory proteins in the presence of endoplasmic reticulum-retained biotin ligase. Host cells were engi-neered by transfection with biotin ligase (BPL) containing KDEL, an ER-retention signal. An engineered antibody fragment, anti-CEA diabody, was fused to either 123 amino acid biotin acceptor domain from Propionibacterium shermanii or a smaller 15 amino acid biotin acceptor tag. Analysis of secreted protein demonstrated efficient metabolic biotinylation of the diabody, particularly when the short peptide tag was used. This method provides a general approach for efficiently secreting biotinylated recombinant pro-teins for a variety of streptavidin-biotin technologies.

10:05 Coffee Break in the Exhibit Hall

10:50 The Role of Transcription and Protein Misfolding in Sub-Optimal Expression: The Case of an Antibody-Interleukin Fusion Molecule
Missag Parseghian, Ph.D., Director, Research & Development, Peregrine Pharmaceuticals, Inc.
Vasopermeation Enhancement Agents (VEAs) are antibody-interleukin fusion molecules designed to increase the uptake of cancer therapeutics at the tumor site by the gen-eration of targeted vasopermeability, resulting in greater efficacy of existing therapies. VEAs incorporate a tumor targeting antibody with a fragment of interleukin-2 (IL-2). The trick is to identify an appropriate sized fragment of IL-2 that is structurally sound and provides optimal potency in combination with high protein expression. As a con-tinuation to this past year's presentation on VEAs at the CHI 8th Annual Recombinant Antibodies meeting, we extend our systematic analysis to determine the cause of significant variation in protein expression seen when the length of a protein fragment changes. We have been investigating what differentiates an optimal fragment from one that is sub-optimal and if the differences in protein expression are at the level of transcription or protein misfolding.

11:20 Synergistic CDR Mutagenesis and Clonal High-Throughput Screening Improves Antibody Binding Affinity without Display Panning 
Christian Votsmeier, Ph.D., Project Manager - Antibody Optimization, Pharma, DIREVO Biotech AG
A synergistic CDR mutagenesis approach was applied to D2E7, an anti-TNF-alpha antibody, which already shows ~ 200 pM binding affinity towards its target and which was optimized (to Kd ~ 8 pM) using a yeast display approach in an independent study. Using this system we examined 120,000 variants in a competition ELISA format and identified more than ten double substitution sites that resulted in higher binding affinities. The complete combinatorial complexity of selected advantageous mutations was generated in a subsequent DNA library and completely screened with oversampling for improved dissociation rates using a competitive Time Resolved-FRET assay. This strategy yielded variants with dissociation constants in the low pM range and were further shown to neutralize TNF-alpha more efficiently than the original D2E7 Fab in an cell-based bioassay. Comparisons will be presented to affinities and mutations found using yeast display. These results demonstrate that clonal and quantitative high throughput assays can be used to screen the complete complexity of double-saturation libraries involving every CDR residue position and that this approach results in efficient optimization of antibody binding, even to the low pM range of binding affinities, without the need for display screening.

11:50 End of Recombinant Antibodies Conference