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Hu cal platnimm alis adds
The document provides an overview of HuCAL Platinum, a synthetic antibody library produced by Morphosys. It describes the 7 step process for generating monoclonal antibodies from the library, which includes immobilizing the antigen, panning the library, subcloning and screening colonies, sequencing antibodies, and purification. Key advantages of HuCAL Platinum are that it does not require animals, has a diverse selection of 45 billion antibody genes produced in E. coli, and has over a 90% success rate. Applications include drug discovery, structural analysis, and Morphosys has several antibodies in clinical trials for diseases like cancer and rheumatoid arthritis.
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Hu cal platnimm alis adds
- 1. HuCAL Platinum Ali Baig DominiqueBurns Brandon Chackel June 17, 2013 Frey Daniel et all Structure of the recombinant antibody Fab fragment f3p4 (2008) Department of Biochemistry, University of Zürich, Switzerland. Acta Crystallographica Section D Biological Crystallography 07/2008 :636-43
- 2. Overview • Introduction/Brief Background. •Experimental Details • Results • Applications • Conclusions • Q and A
- 3. Intro What is anAntibody? • Glycoprotein • 2 Heavy Chains + 2 Light Chains • Antigen Binding Site • Variable Region • FAB Domain • Fc Domain • Disulfide Bonds Ylera, Francisco. "HuCAL Antibodies Technical Manual" AbD Serotec 2nd edition. P. 10
- 4. HuCAL Platinum Synthetic AntibodyLibrary – 3rd Generation • 45 Billion Human Antibody Genes • Uses 6 CDRs with Length Dependent Designs • High Specificity • Produced within Bacteria • 8 week Turn-around Time • 90+% Success Rate
- 5. 7 Steps toSuccess 1. Antigen Immobilization 2. Panning 3. Subcloning 4. Primary Screening 5. Sequencing 6. Expression & Purification 7. QC Ylera, Francisco. "HuCAL Antibodies Technical Manual" AbD Serotec 2nd edition. P. 12
- 6. Steps 1-2: Immobilization/Panning •Antigen immobilized on solid support via covalent coupling to magnetic beads. • HuCAL library incubated with the immobilized antigen and nonspecific antibodies removed via washing and the specific antibody phage eluted by addition of a reducing agent. • E. coli culture infected with eluted phage and helper phage to make the purified antibody phage library for subsequent panning round • Usually 3 panning rounds Ylera, Francisco. "HuCAL Antibodies Technical Manual" AbD Serotec 2nd edition. P. 31
- 7. Steps 3-4: Subcloningand Screening. • Enriched antibody isolated and subcloned into Fab expression vector. Vector formats vary between monovalent and bivalent Fab fragments with epitope choice tags. • E. coli transformed via ligation and plated on agar plates. Each colony that grows is a monoclonal antibody. • These colonies are selected and grown in a 384 well-microliter plate. Expression is induced and culture is lysed to release antibodies. Screening is done for specific antigen binding via ELISA/FMAT. , Arrayit Corporation ARYC-Products-DNA/PCR, Purification http://arrayit.com/Products/DNA_Purification/PCR_Purification/PCR_384-Well_20/PCR38420_superfilter_600c.jpg
- 8. Steps 5-7:Sequencing/Purification/QC • Sequencedto identify unique antibodies guaranteeing uniqueness of any antibody and as an ideal antibody storage backup. If needed to be made again, can be easily down by synthesis. • Expressed and purified using one- step affinity chromatography. • Purified antibodies tested by ELISA, or in a bead-based FMAT. Purity is seen by Coomassie staining of SDS-PAGE and yield is measured by UV absorbance at 280 nm. Ylera, Francisco. "HuCAL Antibodies Technical Manual" AbD Serotec 2nd edition. P. 32
- 9. Safety Issues. Standard procedureso safety not a real concern. As with cloning and any other technique, proper protocols must be followed. See slide 6 *
- 10. Pros and Cons Pros •Mouse/Rabbit not needed and done in E coli making antibody process faster change of format. • Very diverse selection. Guided antibody selection allows to block library against related proteins assay • Conversion of antibodies via DNA cloning into different formats: Addition of peptide tags for purification, detection, labeling, a nd immobilization • More than 90% success rate for antibody generation Cons • Always read when it comes to specificity and can always be refined. • Not ideal for quick use and long duration for ordering (8 weeks). • Human error can effect the experiment on transfer.
- 11. Gold vs Platinum •7 test selections were done under same conditions with HuCAL GOLD vs. HuCAL PLATINUM. These numbers of unique sequences isolated against indicated antigens from both libraries. Pressler, Josef, et all HuCAL PLATNIUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems, Journal, of Molecular Biology, vol 413, issue 1, 14 OCtober 2011, p. 261-278
- 12. Comparison of Fabaffinities from HuCAL GOLD and PLATINUM against 2 antigens • (a). Affinities (nM) of Fabs from the HuCAL PLATINUM library against 15 different protein antigens. (b) A comparison of affinities derived from HuCAL PLATINUM and GOLD. Affinity distribution (%) of 300 Fabs from GOLD and 250 Fabs derived from PLATINUM. Pressler, Josef, et all HuCAL PLATNIUM, a Synthetic Fab Library Optimized for Sequence Diversity and Siperior Perofmance in Mammalian Expression Systems, Journal of Molecular Biology, vol 413, issue 1, 14 OCtober 2011, p. 261-278
- 13. Research, Diagnostic, & TherapeuticApplications • Drug discovery • High throughput screening • 3˚ Structure Design • Homology Modeling from sequences • CDR-H3 template • “Antibody Druggability” • MorphoSys HuCAL – Therapeutic Abs
- 14. Crystallography Applications Nahary L.and Benhar I. Design of a Human Synthetic Combinatorial Library of Single-Chain Antibodies (2009) Methods Mol Biol. 525:61-80,
- 15. 3D Structure andDesign Fab fragment of F3P4 rAb Protein Crystals Bio 505 Molecular Structure Nahary L. and Benhar I. Design of a Human Synthetic Combinatorial Library of Single-Chain Antibodies (2009) Methods Mol Biol. 525:61-80,
- 16. 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
- 17. Additional Services • Peptidesynthesis • 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.)
- 18. 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
- 19. Currently in theworks. • MOR103: Rheuoid Arthritis, Fully Human antibody, GM-CSI, neutrophils. • MOR208: B Cell malignant autoimmune CD- 19, Phase II clinical trials • MOR202: Myeloma, Leukemia • 70 Drug candidates: Alzheimer's, cardiovascular dysfunction.
- 20. Conclusions HuCAL Platnium isa synthetic antibody 3rd generation library that has 45 Billion Human Antibody Genes. It is made from the 7 steps shown on the right. The pros are animals are not needed and done in E coli making antibody process faster change of format and it is very diverse. Cons are not ideal for quick use and it can be affected by human error • Results/Applications • Improvements: HuCAL is constantly being refined and improved. See Slide 6*
- 21. Citations. • Chiniaff, Kristinand Nigro, Victoria. "Morphosys." Biology 601: Seminar Series in Biotechnology and Bioinformatics. California State University Channel Islands Extended University. Online Lecture. 16 May 2013 • Kapasova, Nelly and Osario, Arthela. "HuCAL." Biology 601: Seminar Series in Biotechnology and Bioinformatics. California State University Channel Islands Extended University. Online Lecture. 9 May 2013 • Knappik A, et all. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides (2000 11 Feb). Journal of Molecular Biology 57-86 • Frey Daniel et all Structure of the recombinant antibody Fab fragment f3p4 (2008) Department of Biochemistry, University of Zürich, Switzerland. Acta Crystallographica Section D Biological Crystallography 07/2008 :636-43 • Morphosys. HuCAL-the Leading Antibody Technology. Morphosys Annual Research and Development Report. (2002) p. 14-21 • Nahary L. and Benhar I. Design of a Human Synthetic Combinatorial Library of Single-Chain Antibodies (2009) Methods Mol Biol. 525:61-80, • Pressler, Josef, et all HuCAL PLATNIUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems, Journal of Molecular Biology, vol 413, issue 1, 14 October 2011, p. 261-278 • Rauchenberger R. et all Human Combinatorial Fab Library Yielding Specific and Functional Antibodies against the Human Fibroblast Growth Factor Receptor 3*(2003) Journal of Biological Chemistry (40), p. 205 • Rothe, Christine. The Human Combinational Antibody Library (HuCAL) GOLD combines diversification of all Six CDRs according to Natural Immune System with a Novel Display Method for Efficent Selection of High Affinity Antibodies. Journal of Molecular Biology (2008) 376, 1187-1200 • Ylera, Francisco. "HuCAL Antibodies Technical Manual" AbD Serotec 2nd edition. p. 10-41, 43-57
- 22. Q and A •Thank you!
Editor's Notes
- #2 (Ali)
- #3 (Ali)
- #4 (Dominique): if you want to add brandons stuff: 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 regionIn the amino acid sequence of variable domain of Ag receptor = 3 CDRsCDR1, CDR2,CDR3The "upper" part (Fab region) of an antibody. The complementarity determining regions of the heavy chain are shown in red
- #5 (Dominique): see if you want to add brandons stuff in the following: Synthetic human Fab library, sequence optimization, 4 fold unique sequenes + higher affinity, Avoids N-glycosylations sites, Gene optimization / IgGs expression levels,Length dependent Diversity Natural CDR-H3 distribution in human repertoire
- #6 (Dominique)
- #7 (Ali)
- #8 (Ali)
- #9 (Dominique)
- #10 (Ali and Dominique)
- #11 (Dominique)
- #12 (Ali)
- #14 (Brandon)
- #15 (Brandon)
- #16 ( Brandon)The structure of the antibody Fab fragment f3p4, which was selected from a subset of the synthetic HuCAL antibody library 2008Deducing protein 3D structure from its amino acid sequence
- #17 (Brandon) 2 $ 290.00 $ 580.0010 $ 95 $ 950LIBRARY SCREENING 1 $ 10,920
- #18 (Brandon)
- #19 (Brandon) Alliances with Novartis, Biogen, Centocor Ortho, Xencor Inc.
- #20 (Ali)
- #21 (Ali)
- #22 (Ali)