2. Target CDR Hypervariabile Regions
• Ag receptors Fab region
• 2 variable domains (L+H)
• 3 CDRs per domain
• 6 CDRs per Ag receptor
• Ab molecule = 12 CDRs
• IgM pentamer molecule?
3. CDR-H3 Remodeling
• 6 CDR region in variable domain
• CDR-H3 (greatest variability heavy chain)
• CDR-H3 center of Ag binding site
• Variable in Length, Sequence and Structure
• Characterize CDR-H3
• CDR-H3 in antibody drugs
4. Gold Standard
• HuCAL Gold (1st
generation)
– synthetic human Fab library
– Trinucleotide Synthesis
– Bete-lactamase selection
– Fragments displayed on the tip of the phage
– All 6 CDRs (2 Fv domains / 3 CDRs each)
– Uniform Diversity (sequence & length)
– Ag+ tested with high affinity
10 billion functional Fab fragments achieved!!!
8. 3D Structure and Design
Fab fragment of F3P4 rAb Protein Crystals
Bio 505 Molecular Structure
9. Standard HuCAL Price
• AbD Serotech (division of Morphosys)
• Custom Monoclonal Antibody Generation
• Panning and Screening library on a single
protein antigen $10,920.00
• 250 ug each up to 10 unique ELISA positive
Fab antibodies $950.00
• 1 mg of 1 Fab antibody $260.00
• 2 x dry ice overnight S+H to the USA $580.00
10. Additional Services
• Peptide synthesis
• Fc fusion protein expression
• Western blot testing on customer provided lysates
• Bacterial protein expression
• Conversion of Fabs into full immunoglobulins (isotypes)
• Generation of new Fab formats and tag combinations
• Cross reactivity profiling
• Flow cytometry testing of antibodies
• Affinity measurements
• Labeling of antibodies (HRP, biotin, RPE, AP, etc.)
11. HuCAL Clinical Trials
• Drug candidates accelerating
• human clinical trials accelerating
• 2010 MorphoSys milestone payment from Novartis
• P1 clinical trial of a HuCAL-derived fully human Ab
• 3rd HuCAL-derived antibody into human clinical trials
• Novartis, Biogen, Centocor Ortho, Xencor Inc.
• MorphoSys now expects up to 15 proprietary and
partnered antibody programs
Editor's Notes
Fab = Fragment Ag binding region (region on Ab that binds to Ags) Fab – Fc and Fv (2 different H and L chains) Fv = Paratope NH2 region In the amino acid sequence of variable domain of Ag receptor = 3 CDRs CDR1, CDR2,CDR3 The "upper" part (Fab) of an antibody. The complementarity determining regions of the heavy chain are shown in red Ribbon diagrams , AKA Richardson Diagrams , are 3D schematic representations of protein structure and are one of the most common methods of protein depiction used today. The ribbon shows the overall path and organization of the protein backbone in 3D, and serves as a visual framework on which to hang details of the full atomic structure, such as the balls for the copper and zinc atoms at the active site of superoxide dismutase in the image at the right. Ribbon diagrams are generated by interpolating a smooth curve through the polypeptide backbone. Alpha-helicies are shown as coiled ribbons or thick tubes, Beta-stands as arrows, and lines or thin tubes for non-repetitive coils or loops.
Just to illustrate how amazing these regions are are for manipulating and the potential is for design….60 CDRs found per IgM moloecule
tertiary structure of a protein or any other macromolecule is defined by its three-dimensional structure through its the atomic coordinates. Tertiary structure is formed by the packing of protein secondary structure (beta-sheets and alpha-helicies) elements into compact globular units called protein domains. A whole protein might comprise one or several such domains, and its tertiary structure can refer to each individual domain as well as to the complete configuration of the whole protein, provided it contains a single, contiguous polypepetide chain backbone. Proteins that are formed by the assembly of separate, folded polypeptide chains give rise to quaternary structure.
High-resolution homology models are useful in structure-based protein engineering applications, especially when a crystallographic structure is unavailable. development and implementation of Rosetta Antibody, a protocol for homology modeling of antibody variable regions. comparative modeling complementarity determining region (CDR) loop conformations de novo loop modeling of CDR H3 conformation simultaneous optimization of V L -V H rigid-body orientation and CDR backbone and side-chain conformations. The protocol was tested on a benchmark of 54 antibody crystal structures. Founded in 1992, the Company's proprietary Human Combinatorial Antibody Library (HuCAL(R)) technology is used by researchers worldwide for human antibody generation. The U.S. patent (U.S. 6,653,068) entitled "Generation of Specific Binding Partners to (Poly)Peptides Encoded by Genomic DNA Fragments or ESTs" covers methods for the high-level production of expressed sequence tags (ESTs) as fusion proteins and subsequent generation of HuCAL(R) antibodies against these proteins. HuCAL(R) EST is a unique technology, supporting researchers in the area of target validation and genomics research and we apply this technology in collaborations with several partners including Biogen, Centocor, Oridis Biomed and Schering."
The structure of the antibody Fab fragment f3p4, which was selected from a subset of the synthetic HuCAL antibody library 2008 Deducing protein 3D structure from its amino acid sequence