HuCAL Platinum

Ali BaigDominique BurnsBrandon Chackel

June 17, 2013

Frey Daniel et all Structure of the recombinant antibody Fab fragment f3p4 (2008) Department of Biochemistry, University of Zürich, Switzerland. ActaCrystallographica Section D Biological Crystallography 07/2008 :636-43

Overview

• Introduction/Brief Background.

• Experimental Details

• Results

• Applications

• Conclusions

• Q and A

Intro

What is an Antibody?

• 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

HuCAL Platinum

Synthetic Antibody Library – 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

7 Steps to Success

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

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

Steps 3-4: Subcloning and 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

Steps 5-7:Sequencing/Purification/QC

• Sequenced to 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

Safety Issues.

Standard procedure so safety not a real concern. As with cloning and any other technique, proper protocols must be followed.

See slide 6 *

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, and 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.

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

Comparison of Fab affinities 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, Journalof Molecular Biology, vol 413, issue 1, 14 OCtober 2011, p. 261-278

Research, Diagnostic, & Therapeutic Applications

• Drug discovery

• High throughput screening

• 3˚ Structure Design

• Homology Modeling from sequences

• CDR-H3 template

• “Antibody Druggability”

• MorphoSys HuCAL – Therapeutic Abs

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,

3D Structure and Design

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,

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

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.)

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

Currently in the works.

• 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.

Conclusions

HuCAL Platnium is a 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*

Citations.

• Chiniaff, Kristin and 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

Q and A

• Thank you!