Antibody Engineering Conference, San Diego - MorphoSys

A New Antibody Library Concept (AB Engineering, San Diego, Dec. 2011) 

Stefanie Urlinger, Director R&D, MorphoSys AG 

Page 1 2011 Antibody Engineering 

© MorphoSys AG

Why a New Antibody Library? 

From: „Building Better Antibody Therapeutics“ by Patrick McGee, Drug Discovery 

and Development, 2007 (quotation Stephen Demarest, Biogen Idec): 

„The construction of nearly every human or humanized antibody drug candidate 

necessitates some unnatural protein engineering.“ […] 

„The only companies with success in this area are those that included stability and 

solubility as a key component of their library designs.“ […] 

„And while there have been many recent successes in this area, there have been 

numerous and costly failures over the past 15 years because stability was not always 

considered a key issue.“ 

Building quality directly into the antibody library: 



AGENDA 

1. Features of Ylanthia 

2. Realization of the Concept 

3. Quality Control 

4. Library Performance 

Page 3 2011 Antibody Engineering © © MorphoSys AG AG

Analyzing the Natural Human Antibody 

Repertoire 

Ranking of most prominent 

variable region genes and 

VH/VL pairings: 

� Extraction of V gene usage and 

VH/VL pairing frequencies from 

own B-cell sequencing efforts and 

several publications analyzing 

both, autoimmune and healthy 

people*. 

� Analysis of VH/Vk as well as 

VH/Vλ pairings. 

Ranking according to natural 

occurrence. 

* Wardemann H. et al. Science (2003); Yurasov S. et al. JEM (2005); Tsuiji M. et al. JEM (2006); Yurasov S. et al. JEM (2006); 

Tiller T. et al. Immunity (2007); Mietzner B. PNAS (2008); Kofer J. et al: unpublished/ personal communication; 

Brezinschek H.P. et al. JCI (1997); Demaisson C. et al. Immunogenetics (1995); Foster S.J. at al. JCI (1997) 

VH 


© MorphoSys AG 

V�

Selection of Top 20 HC and 20 LC Germline 

Genes 

Human antibody repertoire: 

� 40 functional Vkappa 

� 30 Vlambda 

� 50 VH segments germline encoded. 

In silico pre-selection of 20 HC & LC: 

� Natural prevalence in human rearranged 

antibodies 

� Diversity in variable region gene families 

� Diversity in canonical CDR structures 

� Number of potential post-translational 

modification sites (PTMs) 

� Isoelectric point (pI) 

In vitro testing of 400 HC/LC pairs to 

select for pairs with most favorable 

biophysical properties 

* strong T-cell epitope only with rare DRB1*0411 allele 



Ylanthia: Distinct Heavy/Light Chain Pairing 

� Single framework libraries: 

HC/LC pair selected for biophysical properties; 

limited structural repertoire 

� Multiple framework synthetic and PCR-based 

libraries: High structural diversity; but random 

HC/LC pairing might yield to, e.g., antibody 

instability 

� Ylanthia: 36 distinct HC/LC pairs 

pre-selected for favorable biophysical 

properties 

� High structural diversity through main canonical 

CDR conformations of HC & LC 

� Only stable, well expressing HC/LC combinations 



Reduction of Potential Post-Translational 

Modification Sites (PTMs) 

� Fully germline human antibodies contain a relevant number of potential PTMs, 

especially in CDR-H1 and CDR-H2 

� Ylanthia features fully germline and “no PTM” CDR-H1 and -H2 sequences 

� Completely removed from CDR-H1 & -H2: 

� Deamidation sites: NS, NG, NH 

� Isomerization sites: DS, DG, DD 

� Cleavage sites: DP, DQ, NS 

� Oxidation sites: M 

� Glycosylation sites: NxT, NxS 

� CDR-H3: Asn (N) is omitted and Asp (D), Gln (Q) and Met (M) are decreased 



AGENDA 






Screening 400 HC/LC Pairs for Biophysical 

Properties 

Fab (20 HC x 20 LC = 400 combinations) 

Pool cloning 

Fab display vector Fab expression vector 

(1) Phage display (2) Expression yield 

(3) Thermal stability 

(4) Serum stability 

Ranking; Exclusion of unfavorable HC/LC pairs or frameworks 

Pre-selection of ~100 HC/LC combinations 

� Fab and IgG1 expression & purification 

� Biophysical characterization 

� Expression yields (Fab & IgG1) 

� Aggregation propensity 

� Apparent Tm determination 

� Physical stress testing 

� Serum stability 

IgG1 (20 HC x 20 LC = 400 combinations) 

Single antibody cloning 

Human IgG1 expression vector 

(5) Expression yield 

(6) Serum stability 

Final master gene selection 

30-40 distinct HC/LC pairs. 



Screening for Fab Phage Display Levels 

Step 1: Gene synthesis of 20 HCs and 20 LCs (including gene and codon optimization) 

Step 2: Pool sub-cloning into CysDisplay and Fab expression vectors 

Step 3: Set-up of predictive screening assays for Fab display levels on phage, Fab expression 

levels and Fab thermostability from crude bacterial expression samples 

Step 4: ELISA-based screening of HC/LC pairs; sequence analysis for identification of ≥ 75% of 

HC/LC pairs (> 50% at least double determination) 

Example: Determination of Fab phage display levels in ELISA format 

cultivation of E. coli 

carrying phagemid; 

helper phage infection 

and antibody phage 

production in 96-well 

format 

phage capture 

via anti-g8p 

phage capture 

via anti-Fab 

anti-g8p 

detection 

calculation of relative 

Fab display rates 

(g8p vs. Fab-specific 

signal) using 

reference phage 

prep 



Screening for Fab Phage Display Levels 

VH1 

VH3 

VH4,5,6 

kappa 

lambda 

Framework combinations are selected for high display levels in CysDisplay format. 

HCs showing low display are excluded (VH1-2, VH3-73, VH4-31, -39). 



Screening for Thermostability 

VH1 

VH3 

VH4,5,6 

kappa 

lambda 

Framework combinations are selected for apparent Fab thermostability. 

Instable HCs and LCs are excluded (e.g., VH1-2, VK1-17, VK2-30). 



… 

Screening for IgG1 Expression Levels 

Step 5: Set-up of sandwich ELISA for IgG1 quantification from mammalian cell culture supernatants 

Step 6: Pool subcloning of HC & LC into mammalian hIgG1 expression vectors (“2-vector system”) 

Step 7: Cross-transfection of 20 HC with 20 LC into HEK.EBNA cells and IgG1 expression 

Step 8: IgG1 quantification in 384-well format 

Example: Determination of IgG1 levels from cell culture supernatants 

LC 

cross-transfection 

of 20 heavy chain 

with 20 light chain 

plasmids into 

HEK.EBNA 

HC 

HEK.EBNA 

cultivation and 

antibody 

production of 

400 pairs 

mouse antihuman 

IgG1 

capture from 

cell culture 

supernatants 

detection via 

anti-human IgG1 

specific 

biotinylated 

antibody (not 

cross-reactive 

with mouse Ig) 

calculation of 

relative IgG1 

expression levels 

via signal 

obtained with 

reference 

antibody 



Screening for IgG1 Expression Levels 

VH1 

VH3 

VH4,5,6 

kappa 

lambda 

Framework combinations are selected for human IgG1 expression levels 

and low expressing HCs are excluded (e.g., VH1_2, VH4_31). 



Combination of all Screening Parameters 

low Fab display 

low Fab expression level 

low Fab thermostability 

moderate hIgG1 expression 

high Fab display 

moderate Fab expression level 

high Fab thermostability 

high hIgG1 expression 

Selection of ~100 HC/LC pairs with favorable properties for research scale expression, 

purification and biophysical testing. 



In Depth Biophysical Characterization of 

Purified HC/LC Pairs 

� 100 HC/LC pairs pre-selected by ELISA-based screenings were 

expressed and purified in mg amounts, both in Fab and IgG1 formats: 

� Quantification of expression yields 

� Determination of monomeric content by analytical SEC 

� Apparent Tm by Thermofluor 

� Serum stability testing 

� Stress testing (turbidity/particle formation upon physical & pH stress) 

Ylanthia frameworks: 36 HC/LC pairs with optimal biophysical properties, 

plus featuring a diverse set of canonical CDR conformations for 

broad epitope coverage. 



Ylanthia CDRs: Slonomics Inside 



CDR-H3: JH Gene Contribution 

� Predominantly 

used HC joining 

region in 

rearranged human 

antibodies: JH4 

YFDY… 

� Long CDR-H3s: 

JH6 usage 

YYYYYGMDV… 

increases with 

length 



Ylanthia CDR-H3 Design 

� Lengths 6 to 17 with JH4 based design 

� JH4: dominant joining region in short to medium length human CDR-H3s 

� Lengths 12 to 17 have additional JH6 based design 

� JH6 gains importance with increasing CDR-H3 length 

� In CDR-H3 Asn (N) is completely omitted; Asp (D) and Met (M) are decreased to abolish or 

reduce occurrence of critical PTM sites 



CDR-H3 Length Distribution 

� Twelve different CDR- 

H3 lengths ranging 

from 6 to 17 amino 

acids 

� Covering 80% of the 

natural human CDR-H3 

diversity (according to 

Zemlin et al., 2003) 

� Lengths 6 to 17: 

JH4 specific design 

� Lengths 12 to 17: 

additional JH6 specific 

design 



AGENDA 






Library Size and Correctness 

� Sub-library sizes vary between 5E+8 and 4E+9 

clones. 

� Overall correctness: ~85%. 

Total library size: 

1.3E+11 independent clones with more than 

hundred billion fully correct human antibodies! 



Library QC by Next Generation Sequencing: 

Redundancy 

� 454 sequencing of VH 

of the unselected 

library (performed using 

pan-selective primers 

recognizing all HCs; 

kappa and lambda 

libraries „tagged“) 

� Proprietary software for 

data evaluation: 

More than 151.000 

Ylanthia VH sequences 

analyzed 

Number of replicates: 

High sequence diversity: From more than 151.000 sequences 94% were CDR- 

H3 unique (< 6% sequences found in duplicate). 



Library QC by Next Generation Sequencing: 

CDR-H3 Length Distribution 

� Analysis of more than 151.000 unselected Ylanthia VH sequences: 

Unselected CDR-H3 length distribution matches design. 



Library Features – Correctness 

� QC of final library: 

� Sequence analysis of VL 

and corresponding VH of at 

least 48 clones per sub- 

library; in total almost 4000 

antibodies sequenced and 

evaluated. 

Overall library correctness: 

~85% 

failed 

false 

TRIM TRIM Slonomics 

kappa lambda LC HC 

total sequences 2367 1482 3849 3849 

mixed or bad sequence 4.3% 4.4% 4.4% 4.8% 

seq break 

before KpnI or XhoI 

0% 0% 0% 3.6% 

analyzed sequences 2265 1416 3681 3523 

frameshifts 6.2% 8.8% 7.2% 3.0% 

mixed in AND after 

CDR3 

0.6% 0.8% 0.7% 2.2% 

stop mutation 

(w/o frameshift) 

0.3% 0.1% 0.2% 0% 

multiple inserts 0% 0% 0% 4.0% 

functional 

clones 

93% 90% 92% 91% 

undesired CDR3 length 0.6% 0.2% 0.5% 0.2% 

undesired aa 0.7% 0.5% 0.6% 0.4% 

correctness 92% 90% 91% 90% 



AGENDA 






Antibody Selections – Snapshot* 

*beta-testing in progress 

� So far, antibodies from 

27/36 tested VH/VL 

pairs identified 

� Different targets favor 

different VH/VL pairs 

� A minimum of 94 

antibodies identified 

per target from a 

limited number of 

selections and 

analyzed sequences of 

the germline version 

only (only 36 VH/VL 

pairs of 72 tested) 



Biophysical Properties of Selected Antibodies 

Isoelectric Point (pI) 

The calculated pI values of the selected antibodies are mostly above 8.5, 

even for lambda antibodies. 

� The pI values were 

calculated using VNTI 

software. 

� Rational CDR3 design 

results in an increase of pI 

values in most selected 

antibodies (as compared 

to the initial VH/VL pair; 

or ) . 




Thermostability 

� IgG1 Melting Temperature 

(Tm) was determined by 

Thermofluor. 

� The median thermal 

stability for each family 

equals the parental 

antibody ( or ) that 

was initially used for 

selecting the VH/VL pairs. 

The Melting Temperature of the selected antibodies clusters around the apparent 

Tm of the initial HC/LC pairs. 90% of tested antibodies show Tm >68°C. 




IgG1 Production Yields 

More than 95% of the antibodies express more than 10 mg IgG1 per liter in a three 

day transient mammalian expression culture as determined after purification. 

Average production yields: ~25 mg/L. 

� Human IgG1 antibodies 

were transiently expressed 

for 3 days in HKB11 cells 

in shake flasks and 

purified via Protein A. 




Aggregation Propensity 

90% of the IgGs show

Ylanthia ® – Moving Further Towards 

Quality by Design 

� New antibody library platform* 

� 36 fixed HC/LC combinations: 

� High structural diversity 

� HC/LC pairs pre-selected for optimal 

biophysical properties 

� More than hundred billion fully human and 

entirely correct antibodies 

� Full flexibility for engineering; 

rapid Ig conversion 

� Slonomics inside 

� Applies Slonomics for tailored antibody 

optimization (“arYla ® ”) 

*beta-testing in progress 

Ailanthus spec: Tree of the Gods, 

Tree of Heaven 



www.morphosys.com 

HuCAL Page 33 2011 Antibody Engineering 

© MorphoSys AG 

® , HuCAL GOLD ® , HuCAL PLATINUM ® , CysDisplay ® , RapMAT ® , AutoCAL ® , arYla ® and Ylanthia ® are registered trademarks of MorphoSys AG.

Antibody Engineering Conference, San Diego - MorphoSys

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