Engineering the Medicines of Tomorrow The Path to Platinum: The Evolution of Human Combinatorial Antibody Libraries (HuCAL )
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1 Engineering the Medicines of Tomorrow The Path to Platinum: The Evolution of Human Combinatorial Antibody Libraries (HuCAL ) Antibody Engineering December 2008, San Diego Dr. Stefanie Urlinger, Associate Director R&D Morphosys AG, Munich, Germany MorphoSys AG
2 Contents MorphoSys Technology Synthetic antibody genes Cys Phage Display Trinucleotide Technology History: Evolution of HuCAL Libraries Presenting HuCAL Platinum December 2008, San Diego MorphoSys AG Page 2
3 MorphoSys Technology is Unique HuCAL : Human Combinatorial Antibody Library -S-S- Synthetic antibody libraries made with TRIM: Trinucleotide Mutagenesis CysDisplay TM December 2008, San Diego MorphoSys AG Page 3
4 HuCAL is Unique in its Design: Master Genes Cover the Structural Diversity of Human Antibodies Structures / Folds 132 Ig Variable Domain Structures HuCAL Master Frameworks Germline Genes ~ 51 VH Genes ~ 40 Vk Genes ~ 30 Vl Genes Bioinformatics Analysis of human antibody germline and rearranged genes Analysis Grouping into VH/VL subfamilies according to sequence homologies Gene synthesis Combination of 7 VH and 7 VL HuCAL Master Genes covers structural diversity Source: Knappik et al. J Mol Biol 2000, 296: 57ff December 2008, San Diego MorphoSys AG Page 4
5 HuCAL : Sequence Diversity of Human CDRs Bioinformatics Rearranged Sequences G N G N T K N S G N T A G G S N T K N S G N T A N S G N T G N T G G T N S G V T T K N S G G T N Y N G N T N N S G N T G Y N G N T N N S G G T N Positional Frequency (Statistic) CDR Design 60N 50S 80G 70N 100T 50N 15G 25N 10S 20G 25K 15Y 15G 10V 10T 15A 5S 15T 10G TRIM Oligo Synthesis HuCAL Frameworks + CDR Design Library Construction Taking care of structural residues Using diversity where it matters Capturing the natural repertoire Minimizing the risk of immunogenicity Knappik et al., JMB (2000) 296, Page 5
6 HuCAL s Modularity Facilitates Efficient, Targeted and Controlled Affinity Optimization Selection of HuCAL Fab with functional activity Selective and controlled diversification of CDRs HCDR2 Optimized drug candidate LCDR1 LCDR3 CDR optimization without altering framework regions Highly diverse CDRs designed according to natural distribution in man Optimization of a single Fab as well as a pool of Fabs CDRs can be optimized sequentially or in parallel Combination of optimized heavy and light chain possible Page 6
7 CysDisplay Uniquely Enables Selection of High Affinity Antibodies Fab DTT V H -SS- V L Display of Fab via disulfide linkage to phage (piii) No genetic fusion between Fab and phage protein Cysteines at N-terminus of piii as well as C-terminus of Fab Disulfide bond formation between Fab and piii in the periplasm ensures linkage of phenotype and genotype Phage Specific elution of phage Independent of antibody affinity Especially suited for selection of high affinity antibodies Easy and fast procedure, independent of type of antigen Applicable for high throughput December 2008, San Diego MorphoSys AG Page 7
8 Contents MorphoSys Technology Synthetic antibody genes Cys Phage Display Trinucleotide Technology History: Evolution of HuCAL Libraries Presenting HuCAL Platinum December 2008, San Diego MorphoSys AG Page 8
9 The TRIM Technology Ensures HuCAL s High Quality and Functionality TRIM: Trinucleotide Mutagenesis TRIM uses 20 pre-assembled trinucleotides in standard DNA synthesis Codon based TRIM technology provides solely the desired amino acids Trp A Cys...CAT TG ACT......His Stop Thr... T TG Tyr Phe Leu Mono: TRIM: TGG...CAT TAT ACT... TTT Trp...His Tyr Thr... Phe HuCAL CDRs are highly diverse and human No stop codons or frameshifts No undesired or rare codons Length variation Ratio (bias) of amino acids at any position Mastergene specific CDR subsets Page 9
10 Trinucleotide Technology Trinucleotide-directed mutagenesis (TRIM) [Exclusive license from Johns Hopkins University in Baltimore, MD, U.S.A.] Excellent coupling yields of 96%-98.5% Controlled relative coupling rates of each individual trinucleotide Page 10
11 Limits of Trinucleotide Oligo Synthesis High coupling efficiency in trinucleotide cassette synthesis is mandatory Working example: TRIM cassette with TRIM coupling steps to generate a aa length HCDR3 Coupling Efficiency Synthesis Yield 15 aa 23 aa 95% 46% 30% 90% 20% 9% 85% 8% 2% 80% 3.5% 0.6% HCDR3 Length 15aa 16aa 17aa 18aa 19aa 20aa 21aa 22aa 23aa No. sequences analyzed No. correct sequences % without error No. functional seq. (+/-TRIM) TRIM Insertion TRIM Deletion Nucleotide Insertion/Deletion % functional seq. (+/-TRIM) Page 11
12 TRIM Diversification Strategies Diversification of amino acid composition Diversification of CDR Regions T 1 T 2 T 3 D 5% 4.4% E 5% 4.4% K 5% 4.4% R 5% 20% 4.4% H 5% 20% 4.4% T 5% 4.4% S 5% 15% N 5% 4.4% Q 5% 4.4% G 5% 15% A 5% 4.4% C P 5% 4.4% V 5% 4.4% I 5% 4.4% L 5% 50% 4.4% M 5% 4.4% F 5% 5% 4.4% Y 5% 15% W 5% 5% 4.4% T 1: Fully randomized T 2: Biased mixture T3: Fully randomized but weighted T = TRIM Mixture Single Position atg-ggc-t1-ccg-atc Multiple Positions (e.g. CDR1/2) ggc-t1-ccg-t2-t3-ccg- Continuous Sequences (e.g. HCDR3) T1-(T2) 4 -(T3) 3 -T4 -T5 - T6 -T7 - Page 12
13 Contents MorphoSys Technology Synthetic antibody genes Cys Phage Display Trinucleotide Technology History: Evolution of HuCAL Libraries Presenting HuCAL Platinum December 2008, San Diego MorphoSys AG Page 13
14 HuCAL Libraries The Path to Platinum HuCAL-scFv 1 HuCAL-scFv 2 HuCAL-scFv 3 HuCAL-Fab 1 HuCAL-Fab 2 HuCAL GOLD HuCAL Platinum December 2008, San Diego MorphoSys AG Page 14
15 History of HuCAL Libraries Antibody format Phage display system Diversified CDR regions HuCAL-scFv 1 scfv genetic fusion 2 HuCAL-scFv 3 scfv CysDisplay 2 HuCAL-Fab 1 Fab genetic fusion 2 HuCAL GOLD Fab CysDisplay 6 HuCAL Platinum Fab CysDisplay 6 Major steps in HuCAL Technology Fab antibody format CysDisplay Increased diversity in CDR-1 and CDR-2 Page 15
16 Contents MorphoSys Technology Synthetic antibody genes Cys Phage Display Trinucleotide Technology History: Evolution of HuCAL Libraries Presenting HuCAL Platinum December 2008, San Diego MorphoSys AG Page 16
17 HuCAL Platinum Making GOLD Brighter HuCAL Platinum Concept Key Features: Increased number of functional HCDR3 sequences Optimized selection of master genes Minimized numbers of non-germline positions Gene optimization of master genes Massively reduced number of potential N- linked glycosylation sites Increased library size over HuCAL GOLD Page 17
18 A New HCDR3 Design to Cover Human Structure and Functional Diversity FREQUENCY % 20,0 15,0 10,0 Length distribution HCDR3 Zemlin et al. showed a length dependent amino acid composition of HCDR3 Increased in long HCDR3: Y, S, P, K (and C) Decreased in long HCDR3: G, R 5,0 0, Loop length Nature GOLD Design GOLD Found Zemlin et al., JMB_2003_334(4)_ Page 18 Zemlin et al., JMB_2003_334(4)_
19 HuCAL Platinum: HCDR3 Length Distribution Design and composition of Library HCDR3 selected from Library Lenght Distribution HCDR3 14,0 50 Frequency % 12,0 10,0 8,0 6,0 4,0 Planned Library Unique binde er HuCAL Gold (N=100) HuCAL Platinum (N=300) 2,0 5 0, aa HCDR3 length (aa) Composition of unselected library = Design Increased number of long HCDR3 sequences HuCAL Platinum delivers a higher number of sequences with long HCDR3 Page 19
20 Controlled Diversity to Avoid Undesired Motifs HCDR2 Nat. Seq. GOLD Platinum Kabat % V 90% I 63% S 20% V I 66% S 24% V I 66% S 15% A. 12% A. 14% A. 11% S. 12% S. 14% S. 11% Y. 12%Y. 14% Y. 10% N 12% N 9 % G 12% G 14% G N x S/T 6,3% 7,9% 0% Potential N-linked glycosylation sites (motif NxS/T) can be avoided by omitting either N or S/T in TRIM mixtures of known hot spots Page 20
21 Reduced Number of Potential N-Linked Glycosylation Sites NxS/T could not be eleminated completely without losing significant sequence diversity Hot spots of NxS/T sites found in GOLD were removed or at least dramatically reduced in Platinum % Glycosylation sites LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 Library Platinum Number of clones analysed Glycosylation sites Lambda VH1A % VH1B % VH % VH % VH % VH % VH % kappa VH1A % VH1B % VH2 78 5% VH % VH % VH5 94 5% VH6 86 6% % Potential N-linked glycosylation sites: GOLD (30%) vs. Platinum (5%) December 2008, San Diego MorphoSys AG Page 21
22 Library Sequence Optimization HuCAL master genes optimized in collaboration with GENEART AG Optimized codons for E. coli and mammalian expression system Unfavorable mrna secondary structures were removed Negative regulatory sequence motifs in prokaryotic and eukaryotic system were avoided Page 22
23 HuCAL Platinum: Optimized on DNA Level Genes optimized by GeneOptimizer Software (no amino acid changes introduced) Prokaryotic inhibitory motif VH1A 1 Removed problematic sites Consensus splice site Cryptic splice site RNA instability motif VH1B 1 1 VH2 1 VH VH VH5 1 1 VH6 1 Vl-1 1 Vl Vl-3 1 Alternative start codon Codon usage optimized for E. coli Rare mammalian codons are avoided Vk Vk-2 2 Vk-3 2 CH1 1 3 CL-k 1 1 CL-l 1 Number of potentially problematic motifs significantly reduced Page 23
24 HuCAL Platinum: Size and Correctness Platinum Library Library size Gold λ-libraries Library size Platinum Number of correct clones, Sequencing (95 clones) [%] pm30_vl1-3_vh1a 3,8E+09 72% 6,1E+08 pm30_vl1-3_vh1b 1,4E % pm30_vl1-3_vh2 5,4E+08 7,7E % pm30_vl1-3_vh3-23 3,4E+09 3,7E+09 70,4% pm30_vl1-3_vh ,2E+09 64% pm30_vl1-3_vh5 6,9E+08 4,3E+09 70% pm30_vl1-3_vh6 1,7E+09 3,8E+09 72% κ-libraries pm30_vk1-3_vh1a 5,1E+09 83% 5,0E+08 pm30_vk1-3_vh1b 9,0E+08 76% pm30_vk1-3_vh2 2,9E+09 1,2E+09 79% pm30_vk1-3_vh3-23 3,3E+09 1,7E+09 72% pm30_vk1-3_vh3-23 II 3,3E+09 5,0E+09 75% pm30_vk1-3_vh ,6E+09 76% pm30_vk1-3_vh5 1,2E+09 2,0E+09 73% pm30_vk1-3_vh6 7,7E+08 1,8E+09 65% HuCAL Platinum is around three times as large as HuCAL GOLD HuCAL GOLD contains 64% correct clones, Platinum even 74% Combined Library 1,6E+10 4,5E+10 74% * More rigid criteria were applied for Platinum vs. GOLD e.g. missing TRIMs were counted as errors Page 24
25 HuCAL Platinum: Test Selections Antigen Type Primary Hit rate Platinum > 5 x bgd Primary Hit rate GOLD > 5 x bgd Platinum GOLD GFP soluble CD20 AgX CD25 Fc Fusion TrailR2 Fc Fusion MOR103 anti Id MOR202 anti Id Total: Unique Sequences Platinum GOLD 0 soluble AgX Fc Fusion Fc Fusion anti Id anti Id gfp CD20 CD25 TrailR2 MOR103 MOR202 HuCAL Platinum shows on average a 3.6 fold increased hit diversity compared to HuCAL GOLD Page 25
26 Expression Rates Platinum Antibodies Fab Fab Expression mg/l after after purification [mg/l] IgG1 IgG Expression Expression mg/l after after purification [mg/l] mg/l mg/ cu ulture /L mg/l mg/ cu ulture /L VH1A VH1B VH2 VH3_15 VH3_23 VH5 VH6 0 GFP TrailR2 CD25 AgX Fab expression levels from four different projects Average 14.2 mg/l (n=104) IgG1 expression levels from four different projects Average 28.1 mg/l (n=38); HuCAL GOLD 14 mg/l HuCAL Platinum shows twice the expression rate on IgG level compared to GOLD Page 26
27 HuCAL Platinum Delivers Extraordinarily High Affinities: Parental vs. RapMat KD in [pm] Best affinities naïve library: 22 pm Affinity [pm] Best affinities RapMAT: 12 pm (determined by solution equilibrium titration) Preselected by affinity screening Platinum delivers extraordinarily high affinities from the naïve library which can be quickly improved by RapMAT Page 27
28 HuCAL Platinum TRIM Technology (plus in-frame selection system) applied to generate a high quality library with over 74% correct clones Library size: 4.5 x fold higher diversity of selected antibodies vs. HuCAL GOLD on average HCDR3 length distribution and composition mimicking nature Low number of potential N-linked glycosylation sites: 5% Improved expression rate on IgG and Fab level Page 28
29 Thank you! December 2008, San Diego MorphoSys AG Page 29
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