a new antibody library concept (ab engineering, san diego ......fully germline human antibodies...

© MorphoSys AG 2011 Antibody Engineering

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

Stefanie Urlinger, Director R&D, 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:

© MorphoSys AG 2011 Antibody Engineering © MorphoSys AG

AGENDA

1. Features of Ylanthia

2. Realization of the Concept

3. Quality Control

4. Library Performance


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.

VH

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 Immunity (2007); Mietzner B. PNAS (2008); Kofer J. et al: unpublished/ personal communication;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

V

Ranking according to natural

occurrence.


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)

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

In vitro testing of 400 HC/LC pairs to

select for pairs with most favorable

biophysical properties


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



3. Quality Control



Screening 400 HC/LC Pairs for Biophysical

Properties

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

Ranking; Exclusion of unfavorable HC/LC pairs or frameworks

Human IgG1 expression vector

(5) Expression yield

(6) Serum stability

Fab (20 HC x 20 LC = 400 combinations)

Pool cloning

IgG1 (20 HC x 20 LC = 400 combinations)

Single antibody cloning

Fab display vector Fab expression vector

(1) Phage display (2) Expression yield

(3) Thermal stability

(4) 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

anti-g8p

detection

calculation of relative

Fab display rates

(g8p vs. Fab-specific

signal) using

reference phage

prep

phage capture

via anti-Fab


Screening for Fab Phage Display Levels

lambda kappa

VH1

VH3

VH4,5,6

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

Framework combinations are selected for apparent Fab thermostability.

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

lambda kappa

VH1

VH3

VH4,5,6


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

cross-transfection

of 20 heavy chain

with 20 light chain

plasmids into

HEK.EBNA

mouse anti-

human 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

HEK.EBNA

cultivation and

antibody

production of

400 pairs

HC

LC


Screening for IgG1 Expression Levels

Framework combinations are selected for human IgG1 expression levels

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

lambda kappa

VH1

VH3

VH4,5,6


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



3. Quality Control



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

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

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

Number of replicates:


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%

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 XhoI0% 0% 0% 3.6%

analyzed sequences 2265 1416 3681 3523

frameshifts 6.2% 8.8% 7.2% 3.0%

mixed in AND after

CDR30.6% 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

clones93% 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%

TRIM

fail

ed

fals

e


AGENDA



3. Quality Control



Antibody Selections – Snapshot*

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)

*beta-testing in progress


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

Monomeric content of

IgG1 antibodies was

quantified via analytical

size exclusion

chromatography

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

a new antibody library concept (ab engineering, san diego ......fully germline human antibodies...

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