the path to platinum: the evolution of human combinatorial antibody libraries (hucal€¦ · ·...
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Engineering the Medicinesof Tomorrow
© MorphoSys AG© MorphoSys AG© MorphoSys AG
The Path to Platinum: The Evolution of Human Combinatorial Antibody Libra ries (HuCAL ®)
Antibody Engineering December 2008, San DiegoDr. Stefanie Urlinger, Associate Director R&DMorphosys AG, Munich, Germany
Contents
� MorphoSys‘ Technology� Synthetic antibody genes� Cys Phage Display� Trinucleotide Technology
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� History: Evolution of HuCAL Libraries
� Presenting HuCAL Platinum
Page 2December 2008, San Diego
MorphoSys Technology is Unique
HuCAL: Human Combinatorial Antibody Library
© MorphoSys AG© MorphoSys AG© MorphoSys AG
-S-S-
CysDisplayTM
Synthetic antibody libraries made with TRIM: Trinucleotide Mutagenesis
Page 3December 2008, San Diego
HuCAL is Unique in its Design: Master Genes Cover the Structural Diversity of Human Antibodies
Germline Genes
Structures / Folds HuCAL MasterFrameworks
132 Ig VariableDomain Structures
© MorphoSys AG© MorphoSys AG© MorphoSys AG
Gene synthesis
Germline Genes
~ 51 VH Genes~ 40 Vk Genes~ 30 Vl Genes
Bioinformatics Analysis
Analysis of human antibody germline and rearranged genes
Grouping into VH/VL sub-families according to sequence homologies
Combination of 7 VH and 7 VL HuCAL Master Genes covers structural diversity
Source: Knappik et al. J Mol Biol 2000, 296: 57ff
Page 4December 2008, San Diego
HuCAL Frameworks
TRIM Oligo Synthesis Library Construction
Rearranged Sequences
Bioinformatics
HuCAL: Sequence Diversity of Human CDRs
Taking care of structural residues
Using diversity where
G N G N T KN S G N T AG G S N T KN S G N T AN S G N T GN T G G T NS G V T T KN S G G T N
© MorphoSys AG© MorphoSys AG© MorphoSys AG
CDR Design
December 2008, San Diego Page 5
Knappik et al., JMB (2000) 296, 57-86
Using diversity where it matters
Capturing the natural repertoire
Minimizing the risk of immunogenicity
N S G G T NY N G N T NN S G N T GY N G N T NN S G G T N
Positional Frequency (Statistic)
CDR Design60N 50S 80G 70N 100T 50N15G 25N 10S 20G 25K15Y 15G 10V 10T 15A 5S 15T 10G
+
HuCAL’s Modularity Facilitates Efficient, Targeted and Controlled Affinity Optimization
Selection of HuCAL Fabwith functional activity
Selective and controlleddiversification of CDRs
Optimized drugcandidate
HCDR2
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 6
LCDR1 LCDR3
� CDR optimization without altering framework regions
� Highly diverse CDRs designed according to natural d istribution in man
� Optimization of a single Fab as well as a pool of F abs
� CDRs can be optimized sequentially or in parallel
� Combination of optimized heavy and light chain poss ible
� 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
CysDisplay Uniquely Enables Selection of High Affinity Antibodies
DTT
VH VL
-SS
-Fab
© MorphoSys AG© MorphoSys AG© MorphoSys AG
periplasm ensures linkage of phenotype and genotype
� 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
Phage
Page 7December 2008, San Diego
Contents
� MorphoSys‘ Technology� Synthetic antibody genes� Cys Phage Display� Trinucleotide Technology
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� History: Evolution of HuCAL Libraries
� Presenting HuCAL Platinum
Page 8December 2008, San Diego
The TRIM Technology Ensures HuCAL’s High Quality and Functionality
� No stop codons or frameshifts
� No undesired or rare codons
TRIM uses 20 pre-assembled trinucleotides in standard DNA synthesisCodon based TRIM technology provides solely the desired amino acids
HuCAL CDRs are highly diverse and human
TRIM: Trinucleotide Mutagenesis
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� Length variation
� Ratio (bias) of amino acids at any position
� Mastergene specific CDR subsets
December 2008, San Diego Page 9
TRIM:
TAT...CAT ACT...TGG
TTT
...His Tyr Thr...Trp
Phe
TrpMono:
...CAT ACT... ...His Tyr Thr...Stop
Phe
Cys
Leu
GTA
T TG
solely the desired amino acids
Trinucleotide Technology
Trinucleotide-directed mutagenesis (TRIM) [Exclusive license from Johns Hopkins University in Ba ltimore, MD, U.S.A.]
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 10
� Excellent coupling yields of 96%-98.5% � Controlled relative coupling rates of each individual trinucleotide
Limits of Trinucleotide Oligo Synthesis
� High coupling efficiency in trinucleotide cassette synthesis is mandatory
Coupling Efficiency
15 aa 23 aa95% 46% 30%90% 20% 9%85% 8% 2%80% 3.5% 0.6%
Synthesis Yield
� Working example: TRIM cassette with 15 - 23 TRIM coupling steps to generate a 15 – 23 aa length HCDR3
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 11
HCDR3 Length 15aa 16aa 17aa 18aa 19aa 20aa 21aa 22aa 23aa
No. sequences analyzed 33 42 20 35 39 36 36 31 32
No. correct sequences 27 32 14 30 26 21 26 19 19
% without error 82 76 70 86 67 58 72 61 59
No. functional seq. (+/-TRIM) 31 37 20 34 36 30 33 29 26
TRIM Insertion 2 4 6 3 9 8 7 7 7
TRIM Deletion 2 1 0 0 1 1 0 3 0
Nucleotide Insertion/Deletion 2 5 0 2 3 5 3 2 6
% functional seq. (+/-TRIM) 94 88 100 97 92 83 92 94 81
generate a 15 – 23 aa length HCDR3
TRIM Diversification Strategies
� Single Position
atg-ggc-T1-ccg-atc
Diversification of CDR RegionsDiversification of amino acid composition
T 1 T 2 T 3D 5% 4.4%E 5% 4.4%K 5% 4.4%R 5% 20% 4.4%
T 1: Fully randomized
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� 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 -
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%CP 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%
Fully randomized
T 2:Biased mixture
T3:Fully randomized but weighted
T = TRIM Mixture
Contents
� MorphoSys‘ Technology� Synthetic antibody genes� Cys Phage Display� Trinucleotide Technology
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� History: Evolution of HuCAL Libraries
� Presenting HuCAL Platinum
Page 13December 2008, San Diego
HuCAL Libraries – The Path to Platinum
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� HuCAL-scFv 1
� HuCAL-scFv 2
� HuCAL-scFv 3
� HuCAL-Fab 1
� HuCAL-Fab 2
� HuCAL GOLD
� HuCAL Platinum
Page 14December 2008, San Diego
History of HuCAL Libraries
Antibody format
Phage display system
Diversified CDR regions
HuCAL-scFv 1 scFv genetic fusion 2
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 15
Major steps in HuCAL Technology � Fab antibody format� CysDisplay� Increased diversity in CDR-1 and CDR-2
HuCAL-scFv 3 scFv CysDisplay 2
HuCAL-Fab 1 Fab genetic fusion 2
HuCAL GOLD Fab CysDisplay 6
HuCAL Platinum Fab CysDisplay 6
Contents
� MorphoSys‘ Technology� Synthetic antibody genes� Cys Phage Display� Trinucleotide Technology
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� History: Evolution of HuCAL Libraries
� Presenting HuCAL Platinum
Page 16December 2008, San Diego
HuCAL Platinum – Making GOLD Brighter
HuCAL Platinum Concept
Key Features:
� Increased number of functional HCDR3
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� 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
December 2008, San Diego Page 17
A New HCDR3 Design to Cover Human Structure and Functional Diversity
� Zemlin et al. showed a length dependent amino acid composition of HCDR3
� Increased in long HCDR3: Y, S, P, K (and C)
Length distribution HCDR3
15,0
20,0
© MorphoSys AG© MorphoSys AG© MorphoSys AGZemlin et al. , JMB_2003_334(4)_733-479
December 2008, San Diego Page 18
Y, S, P, K (and C)
� Decreased in long HCDR3: G, R
Zemlin et al. , JMB_2003_334(4)_733-479
0,0
5,0
10,0
4 6 8 10 12 14 16 18 20 22
Loop length
FR
EQ
UE
NC
Y %
Nature GOLD Design GOLD Found
HuCAL Platinum: HCDR3 Length Distribution
Design and composition of Library HCDR3 selected from Library
Lenght Distribution HCDR3
10,0
12,0
14,0
35
40
45
50
Un
iqu
e b
ind
er
HuCAL Gold (N=100)
HuCAL Platinum (N=300)
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 19
� Composition of unselected library = Design
0,0
2,0
4,0
6,0
8,0
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
aa
Fre
quen
cy %
Planned
Library
� Increased number of long HCDR3 sequences
0
5
10
15
20
25
30
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Un
iqu
e b
ind
er
HCDR3 length (aa)
HuCAL Platinum delivers a higher number of sequence s with long HCDR3
Controlled Diversity to Avoid … Undesired Motifs
Kabat 50 51 52 50 51 52 50 51 5225% V 90% I 63% S 20% V I 66% S 24% V I 66% S15% A … …. 12% A …. 14% A ….11% S … …. 12% S …. 14% S ….
GOLDNat. Seq. Platinum
HCDR2
© MorphoSys AG© MorphoSys AG© MorphoSys AG
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
December 2008, San Diego Page 20
11% S … …. 12% S …. 14% S ….11% Y …. 12%Y …. 14% Y ….10% N 12% N9 % G 12% G 14% G…. …. ….…. …. ….…. …. ….
N x S/T 6,3% 7,9% 0%
� 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
Reduced Number of Potential N-Linked Glycosylation Sites
2.0
Library Platinum
Number of clones
analysed
Glycosylation sites
LambdaVH1A 83 4.8%VH1B 89 2.2% VH2 87 5.7%
© MorphoSys AG© MorphoSys AG© MorphoSys AG Page 21December 2008, San Diego
0.0
0.5
1.0
1.5
LCDR1 HCDR3HCDR2HCDR1LCDR3LCDR2
% G
lyco
syla
tion
site
s
VH2 87 5.7% VH3-23 142 4.9%VH3-15 80 3.7%VH5 67 4.8% VH6 88 3.4%kappaVH1A 85 3.5%VH1B 106 5.4%VH2 78 5%VH3-23 141 5.7%VH3-15 85 3.5%VH5 94 5%VH6 86 6%
1311 5%
Potential N-linked glycosylation sites: GOLD (30%) vs . Platinum (5%)
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
© MorphoSys AG© MorphoSys AG© MorphoSys AG
avoided
December 2008, San Diego Page 22
HuCAL Platinum:Optimized on DNA Level
Removed problematic sites
Prokaryoticinhibitory
motifConsensus splice site
Crypticsplice site
RNAinstability
motifAlternativestart codon
VH1A 1VH1B 1 1VH2 1
Genes optimized by GeneOptimizer® Software (no amino acid changes introduced)
Codon usage
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 23
VH2 1VH3-23 1VH3-15 1VH5 1 1VH6 1
Vl-1 1Vl-2 1 1Vl-3 1
Vk-1 1 2Vk-2 2Vk-3 2
CH1 1 3CL-k 1 1CL-l 1
Codon usage optimized for E. coli
Rare mammalian codons are avoided
Number of potentially problematic motifs significan tly reduced
HuCAL Platinum: Size and Correctness
� HuCAL Platinum is around three times as large as HuCAL
Platinum LibraryLibrary size
GoldLibrary size
Platinum
Number ofcorrect clones,Sequencing (95
clones) [%]
λλλλ-Libraries
pM30_VL1-3_VH1A6,1E+08
3,8E+09 72%
pM30_VL1-3_VH1B 1,4E+09 76.4%
pM30_VL1-3_VH2 5,4E+08 7,7E+08 74.7%
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 24
as large as HuCAL GOLD
� HuCAL GOLD contains 64% correct clones, Platinum even 74%
* More rigid criteria were applied for Platinum vs. GOLD e.g. missing TRIMs were counted as errors
pM30_VL1-3_VH2 5,4E+08 7,7E+08 74.7%
pM30_VL1-3_VH3-23 3,4E+09 3,7E+09 70,4%
pM30_VL1-3_VH3-15 - 5,2E+09 64%
pM30_VL1-3_VH5 6,9E+08 4,3E+09 70%
pM30_VL1-3_VH6 1,7E+09 3,8E+09 72%
κκκκ-Libraries
pM30_Vk1-3_VH1A5,0E+08
5,1E+09 83%
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_VH3-15 - 4,6E+09 76%
pM30_Vk1-3_VH5 1,2E+09 2,0E+09 73%
pM30_Vk1-3_VH6 7,7E+08 1,8E+09 65%
Combined Library 1,6E+10 4,5E+10 74%
HuCAL Platinum: Test Selections
Antigen Type
Primary Hit rate
Platinum > 5 x bgd
Primary Hit rate GOLD > 5 x bgd
Platinum GOLD
GFP soluble 352 75 54 8CD20 AgX 36 10 12 4CD25 Fc Fusion 702 526 105 20
TrailR2 Fc Fusion 651 468 72 19MOR103 anti Id 251 346 18 8
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 25
HuCAL Platinum shows on average a 3.6 fold increased hit diversity compared to HuCAL GOLD
0
20
40
60
80
100
120
soluble AgX Fc Fusion Fc Fusion anti Id anti Id
gfp CD20 CD25 TrailR2 MOR103 MOR202
Uni
que
Se
que
nces
Platinum
GOLD
MOR103 anti Id 251 346 18 8MOR202 anti Id 646 662 39 25
Total: 300 84
Expression Rates Platinum Antibodies
Fab Expression mg/Lafter purification
20
30
40
mg/
l cul
ture
Fab Expression after purification [mg/L]
mg/
L
IgG1 Expression mg/Lafter purification
60
80
mg/
l cul
ture
IgG Expression after purification [mg/L]
mg/
L
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 26
IgG1 expression levels from four different projects Average 28.1 mg/l (n=38); HuCAL GOLD 14 mg/l
Fab expression levels from four different projectsAverage 14.2 mg/l (n=104)
HuCAL Platinum shows twice the expression rate on I gG level compared to GOLD
VH1AVH1B VH2
VH3_15
VH3_23
VH5
VH6
0
10
20
mg/
l cul
ture
mg/
L
GFP
TrailR
2
CD25
AgX
0
20
40
mg/
l cul
ture
mg/
L
HuCAL Platinum Delivers Extraordinarily High Affinities: Parental vs. RapMat
Best affinities naïve library: 22 pM
Best affinities RapMAT: 12 pM
(determined by solution equilibrium
KD in [pM]
Affi
nity
[pM
]
© MorphoSys AG© MorphoSys AG© MorphoSys AGDecember 2008, San Diego Page 27
Platinum delivers extraordinarily high affinities f rom the naïve library which can be quickly improved by RapMAT
(determined by solution equilibrium titration)
Preselected by affinity screening
Affi
nity
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 1010
© MorphoSys AG© MorphoSys AG© MorphoSys AG
� 3.6-fold higher diversity of selected antibodies vs. HuCAL GOLD on average
� HCDR3 length distribution and composition mimicking nature
� Low number of potential N-linked glycosylationsites: 5%
� Improved expression rate on IgG and Fab level
December 2008, San Diego Page 28